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2023
Zheng Wen-Qiang, Zhang Jian-Hui, Li Zi-Han, Liu Xiuxiu, Zhang Yong, Huang Shuo, Li Jinsong, Zhou Bin, Eriani Gilbert, Wang En-Duo, Zhou Xiao-Long
Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy Journal Article
In: Proc Natl Acad Sci U S A, vol. 120, no. 37, pp. e2309714120, 2023, ISSN: 1091-6490.
Abstract | Links | BibTeX | Tags: ERIANI, Unité ARN
@article{pmid37669377,
title = {Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy},
author = {Wen-Qiang Zheng and Jian-Hui Zhang and Zi-Han Li and Xiuxiu Liu and Yong Zhang and Shuo Huang and Jinsong Li and Bin Zhou and Gilbert Eriani and En-Duo Wang and Xiao-Long Zhou},
doi = {10.1073/pnas.2309714120},
issn = {1091-6490},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {Proc Natl Acad Sci U S A},
volume = {120},
number = {37},
pages = {e2309714120},
abstract = {Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by ), disruption of which accumulated Ser-tRNA and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.},
keywords = {ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by ), disruption of which accumulated Ser-tRNA and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.
Dufossez Robin, Krafft Marie-Pierre, Ursuegui Sylvain, Mosser Michel, Mouftakhir Safae, Pernod Ketty, Chaubet Guilhem, Ryckelynck Michael, Wagner Alain
Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface Journal Article
In: ACS Appl Mater Interfaces, 2023, ISSN: 1944-8252.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid37704020,
title = {Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface},
author = {Robin Dufossez and Marie-Pierre Krafft and Sylvain Ursuegui and Michel Mosser and Safae Mouftakhir and Ketty Pernod and Guilhem Chaubet and Michael Ryckelynck and Alain Wagner},
doi = {10.1021/acsami.3c10655},
issn = {1944-8252},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {ACS Appl Mater Interfaces},
abstract = {Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.
Tidu Antonin, Martin Franck
The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes Journal Article
In: Biochimie, 2023, ISSN: 1638-6183.
Abstract | Links | BibTeX | Tags: MARTIN, Unité ARN
@article{pmid37741547,
title = {The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes},
author = {Antonin Tidu and Franck Martin},
doi = {10.1016/j.biochi.2023.09.017},
issn = {1638-6183},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {Biochimie},
abstract = {Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.},
keywords = {MARTIN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.
Wright Duncan E, Krol Alain
FEBS fellowships: supporting excellent science for over four decades Miscellaneous
2023, ISSN: 2211-5463.
Abstract | Links | BibTeX | Tags: KROL, Unité ARN
@misc{pmid37394995,
title = {FEBS fellowships: supporting excellent science for over four decades},
author = {Duncan E Wright and Alain Krol},
doi = {10.1002/2211-5463.13659},
issn = {2211-5463},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {FEBS Open Bio},
volume = {13},
number = {7},
pages = {1138--1139},
abstract = {The Federation of European Biochemical Societies (FEBS) awarded FEBS Long-Term Fellowships from 1979 until 2020, at which time the scheme was replaced with the FEBS Excellence Award. Over four decades, FEBS awarded a huge number of Long-Term Fellowships, helping to support and promote the careers of excellent young researchers across Europe. To celebrate the exciting work performed by the FEBS Long-Term Fellows, we present here a special 'In the Limelight' issue of FEBS Open Bio, containing four Mini-reviews and four Research Protocols authored by the fellows themselves. The four Review articles provide timely updates on the respective research fields, while the Research Protocols describe how to perform challenging experimental methods in detail. We hope this issue will be a valuable resource for the community, and a celebration of the high-quality work done by young scientists.},
keywords = {KROL, Unité ARN},
pubstate = {published},
tppubtype = {misc}
}
The Federation of European Biochemical Societies (FEBS) awarded FEBS Long-Term Fellowships from 1979 until 2020, at which time the scheme was replaced with the FEBS Excellence Award. Over four decades, FEBS awarded a huge number of Long-Term Fellowships, helping to support and promote the careers of excellent young researchers across Europe. To celebrate the exciting work performed by the FEBS Long-Term Fellows, we present here a special 'In the Limelight' issue of FEBS Open Bio, containing four Mini-reviews and four Research Protocols authored by the fellows themselves. The four Review articles provide timely updates on the respective research fields, while the Research Protocols describe how to perform challenging experimental methods in detail. We hope this issue will be a valuable resource for the community, and a celebration of the high-quality work done by young scientists.
Pitolli Martina, Cela Marta, Paulus Caroline, Rudinger-Thirion Joëlle, Frugier Magali
RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium Journal Article
In: Biochimie, 2023, ISSN: 1638-6183.
Abstract | Links | BibTeX | Tags: FRUGIER, Unité ARN
@article{pmid37414209,
title = {RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium},
author = {Martina Pitolli and Marta Cela and Caroline Paulus and Joëlle Rudinger-Thirion and Magali Frugier},
doi = {10.1016/j.biochi.2023.06.011},
issn = {1638-6183},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {Biochimie},
abstract = {Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.},
keywords = {FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.
Tardivat Yann, Sosnowski Piotr, Tidu Antonin, Westhof Eric, Eriani Gilbert, Martin Franck
SARS-CoV-2 NSP1 induces mRNA cleavages on the ribosome Journal Article
In: Nucleic Acids Res, 2023, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ERIANI, MARTIN, Unité ARN
@article{pmid37503833b,
title = {SARS-CoV-2 NSP1 induces mRNA cleavages on the ribosome},
author = {Yann Tardivat and Piotr Sosnowski and Antonin Tidu and Eric Westhof and Gilbert Eriani and Franck Martin},
doi = {10.1093/nar/gkad627},
issn = {1362-4962},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {Nucleic Acids Res},
abstract = {In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the non-structural protein NSP1 inhibits translation of host mRNAs by binding to the mRNA entry channel of the ribosome and, together with the 5'-untranslated region (UTR) of the viral mRNAs, allows the evasion of that inhibition. Here, we show that NSP1 mediates endonucleolytic cleavages of both host and viral mRNAs in the 5'UTR, but with different cleavage patterns. The first pattern is observed in host mRNAs with cleavages interspersed regularly and close to the 5' cap (6-11 nt downstream of the cap). Those cleavage positions depend more on the position relative to the 5' cap than on the sequence itself. The second cleavage pattern occurs at high NSP1 concentrations and only in SARS-CoV-2 RNAs, with the cleavages clustered at positions 45, 46 and 49. Both patterns of cleavage occur with the mRNA and NSP1 bound to the ribosome, with the SL1 hairpin at the 5' end sufficient to protect from NSP1-mediated degradation at low NSP1 concentrations. We show further that the N-terminal domain of NSP1 is necessary and sufficient for efficient cleavage. We suggest that in the ribosome-bound NSP1 protein the catalytic residues of the N-terminal domain are unmasked by the remodelling of the α1- and α2-helices of the C-terminal domain.},
keywords = {ERIANI, MARTIN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the non-structural protein NSP1 inhibits translation of host mRNAs by binding to the mRNA entry channel of the ribosome and, together with the 5'-untranslated region (UTR) of the viral mRNAs, allows the evasion of that inhibition. Here, we show that NSP1 mediates endonucleolytic cleavages of both host and viral mRNAs in the 5'UTR, but with different cleavage patterns. The first pattern is observed in host mRNAs with cleavages interspersed regularly and close to the 5' cap (6-11 nt downstream of the cap). Those cleavage positions depend more on the position relative to the 5' cap than on the sequence itself. The second cleavage pattern occurs at high NSP1 concentrations and only in SARS-CoV-2 RNAs, with the cleavages clustered at positions 45, 46 and 49. Both patterns of cleavage occur with the mRNA and NSP1 bound to the ribosome, with the SL1 hairpin at the 5' end sufficient to protect from NSP1-mediated degradation at low NSP1 concentrations. We show further that the N-terminal domain of NSP1 is necessary and sufficient for efficient cleavage. We suggest that in the ribosome-bound NSP1 protein the catalytic residues of the N-terminal domain are unmasked by the remodelling of the α1- and α2-helices of the C-terminal domain.
Gaucherand Lea, Iyer Amrita, Gilabert Isabel, Rycroft Chris H, Gaglia Marta M
Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs Journal Article
In: Nat Microbiol, 2023, ISSN: 2058-5276.
Abstract | Links | BibTeX | Tags: PFEFFER, Unité ARN
@article{pmid37349586,
title = {Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs},
author = {Lea Gaucherand and Amrita Iyer and Isabel Gilabert and Chris H Rycroft and Marta M Gaglia},
doi = {10.1038/s41564-023-01409-8},
issn = {2058-5276},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Nat Microbiol},
abstract = {Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.},
keywords = {PFEFFER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.
Pivard Mariane, Caldelari Isabelle, Brun Virginie, Croisier Delphine, Jaquinod Michel, Anzala Nelson, Gilquin Benoît, Teixeira Chloé, Benito Yvonne, Couzon Florence, Romby Pascale, Moreau Karen, Vandenesch François
Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence Journal Article
In: Microbiol Spectr, pp. e0107323, 2023, ISSN: 2165-0497.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid37347186,
title = {Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence},
author = {Mariane Pivard and Isabelle Caldelari and Virginie Brun and Delphine Croisier and Michel Jaquinod and Nelson Anzala and Benoît Gilquin and Chloé Teixeira and Yvonne Benito and Florence Couzon and Pascale Romby and Karen Moreau and François Vandenesch},
doi = {10.1128/spectrum.01073-23},
issn = {2165-0497},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Microbiol Spectr},
pages = {e0107323},
abstract = {Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.
Jakob Celia, Lovate Gabriel L, Desirò Daniel, Gießler Lara, Smyth Redmond P, Marquet Roland, Lamkiewicz Kevin, Marz Manja, Schwemmle Martin, Bolte Hardin
Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging Journal Article
In: Nucleic Acids Res, 2023, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: MARQUET, Unité ARN
@article{pmid37224537,
title = {Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging},
author = {Celia Jakob and Gabriel L Lovate and Daniel Desirò and Lara Gießler and Redmond P Smyth and Roland Marquet and Kevin Lamkiewicz and Manja Marz and Martin Schwemmle and Hardin Bolte},
doi = {10.1093/nar/gkad442},
issn = {1362-4962},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Nucleic Acids Res},
abstract = {A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.},
keywords = {MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.
Lavie Julie, Lalou Claude, Mahfouf Walid, Dupuy Jean-William, Lacaule Aurélie, Cywinska Agata Ars, Lacombe Didier, Duchêne Anne-Marie, Raymond Anne-Aurélie, Rezvani Hamid Reza, Ngondo Richard Patryk, Bénard Giovanni
The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins Journal Article
In: Cell Rep, vol. 42, no. 6, pp. 112579, 2023, ISSN: 2211-1247.
Abstract | Links | BibTeX | Tags: MARTEYN, Unité ARN
@article{pmid37267103,
title = {The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins},
author = {Julie Lavie and Claude Lalou and Walid Mahfouf and Jean-William Dupuy and Aurélie Lacaule and Agata Ars Cywinska and Didier Lacombe and Anne-Marie Duchêne and Anne-Aurélie Raymond and Hamid Reza Rezvani and Richard Patryk Ngondo and Giovanni Bénard},
doi = {10.1016/j.celrep.2023.112579},
issn = {2211-1247},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Cell Rep},
volume = {42},
number = {6},
pages = {112579},
abstract = {In mammals, about 99% of mitochondrial proteins are synthesized in the cytosol as precursors that are subsequently imported into the organelle. The mitochondrial health and functions rely on an accurate quality control of these imported proteins. Here, we show that the E3 ubiquitin ligase F box/leucine-rich-repeat protein 6 (FBXL6) regulates the quality of cytosolically translated mitochondrial proteins. Indeed, we found that FBXL6 substrates are newly synthesized mitochondrial ribosomal proteins. This E3 binds to chaperones involved in the folding and trafficking of newly synthesized peptide and to ribosomal-associated quality control proteins. Deletion of these interacting partners is sufficient to hamper interactions between FBXL6 and its substrate. Furthermore, we show that cells lacking FBXL6 fail to degrade specifically mistranslated mitochondrial ribosomal proteins. Finally, showing the role of FBXL6-dependent mechanism, FBXL6-knockout (KO) cells display mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism, and inhibited cell cycle in oxidative conditions.},
keywords = {MARTEYN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In mammals, about 99% of mitochondrial proteins are synthesized in the cytosol as precursors that are subsequently imported into the organelle. The mitochondrial health and functions rely on an accurate quality control of these imported proteins. Here, we show that the E3 ubiquitin ligase F box/leucine-rich-repeat protein 6 (FBXL6) regulates the quality of cytosolically translated mitochondrial proteins. Indeed, we found that FBXL6 substrates are newly synthesized mitochondrial ribosomal proteins. This E3 binds to chaperones involved in the folding and trafficking of newly synthesized peptide and to ribosomal-associated quality control proteins. Deletion of these interacting partners is sufficient to hamper interactions between FBXL6 and its substrate. Furthermore, we show that cells lacking FBXL6 fail to degrade specifically mistranslated mitochondrial ribosomal proteins. Finally, showing the role of FBXL6-dependent mechanism, FBXL6-knockout (KO) cells display mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism, and inhibited cell cycle in oxidative conditions.
Vindry Caroline, Guillin Olivia, Wolff Philippe, Marie Paul, Mortreux Franck, Mangeot Philippe E, Ohlmann Théophile, Chavatte Laurent
A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium Journal Article
In: Nucleic Acids Res, 2023, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ARN-MS, Unité ARN
@article{pmid37254812,
title = {A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium},
author = {Caroline Vindry and Olivia Guillin and Philippe Wolff and Paul Marie and Franck Mortreux and Philippe E Mangeot and Théophile Ohlmann and Laurent Chavatte},
doi = {10.1093/nar/gkad482},
issn = {1362-4962},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Nucleic Acids Res},
abstract = {The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression.},
keywords = {ARN-MS, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression.
Lista María José, Jousset Anne-Caroline, Cheng Mingpan, Saint-André Violaine, Perrot Elouan, Rodrigues Melissa, Primo Carmelo Di, Gadelle Danielle, Toccafondi Elenia, Segeral Emmanuel, Berlioz-Torrent Clarisse, Emiliani Stéphane, Mergny Jean-Louis, Lavigne Marc
DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence Journal Article
In: Retrovirology, vol. 20, no. 1, pp. 10, 2023, ISSN: 1742-4690.
Abstract | Links | BibTeX | Tags: MARQUET, NEGRONI, PAILLART, Unité ARN
@article{pmid37254203,
title = {DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence},
author = {María José Lista and Anne-Caroline Jousset and Mingpan Cheng and Violaine Saint-André and Elouan Perrot and Melissa Rodrigues and Carmelo Di Primo and Danielle Gadelle and Elenia Toccafondi and Emmanuel Segeral and Clarisse Berlioz-Torrent and Stéphane Emiliani and Jean-Louis Mergny and Marc Lavigne},
doi = {10.1186/s12977-023-00627-6},
issn = {1742-4690},
year = {2023},
date = {2023-05-01},
urldate = {2023-05-01},
journal = {Retrovirology},
volume = {20},
number = {1},
pages = {10},
abstract = {BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus.nnRESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes.nnCONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.},
keywords = {MARQUET, NEGRONI, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus.nnRESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes.nnCONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.
Husser Claire, Vuilleumier Stéphane, Ryckelynck Michael
FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride Journal Article
In: Small, vol. 19, iss. 13, pp. e2205232, 2023, ISSN: 1613-6829.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid36436882,
title = {FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride},
author = {Claire Husser and Stéphane Vuilleumier and Michael Ryckelynck},
doi = {10.1002/smll.202205232},
issn = {1613-6829},
year = {2023},
date = {2023-03-29},
urldate = {2023-03-01},
journal = {Small},
volume = {19},
issue = {13},
pages = {e2205232},
abstract = {Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.
Toccafondi Elenia, Kanja Marine, Winter Flore, Lener Daniela, Negroni Matteo
A snapshot on HIV-1 evolution through the identification of phylogenetic-specific properties of HIV-1 integrases M/O Journal Article
In: PLoS Pathog, vol. 19, no. 3, pp. e1011207, 2023, ISSN: 1553-7374.
Abstract | Links | BibTeX | Tags: NEGRONI, Unité ARN
@article{pmid36996029,
title = {A snapshot on HIV-1 evolution through the identification of phylogenetic-specific properties of HIV-1 integrases M/O},
author = {Elenia Toccafondi and Marine Kanja and Flore Winter and Daniela Lener and Matteo Negroni},
doi = {10.1371/journal.ppat.1011207},
issn = {1553-7374},
year = {2023},
date = {2023-03-01},
urldate = {2023-03-01},
journal = {PLoS Pathog},
volume = {19},
number = {3},
pages = {e1011207},
abstract = {Transmissions of simian viruses to humans has originated the different groups of HIV-1. We recently identified a functional motif (CLA), in the C-terminal domain of the integrase, essential for integration in HIV-1 group M. Here, we found that the motif is instead dispensable in group O isolates, because of the presence, in the N-terminal domain of HIV-1 O of a specific sequence, Q7G27P41H44, that we define as the NOG motif. Alterations of reverse transcription and of 3' processing observed by mutating the CLA motif of IN M are fully rescued to wt levels by inserting the sequence of the NOG motif in the N-ter of the protein. These results indicate that the two motifs (CLA and NOG) functionally complement each other and a working model accounting for these observations is proposed. The establishment of these two alternative motifs seems to be due to the different phylogenetic origin and history of these two groups. Indeed, the NOG motif is already present in the ancestor of group O (SIVgor) while it is absent from SIVcpzPtt, the ancestor of group M. The CLA motif, instead, seems to have emerged after SIVcpzPtt has been transferred to humans, since no conservation is found at the same positions in these simian viruses. These results show the existence of two-group specific motifs in HIV-1 M and O integrases. In each group, only one of the motifs is functional, potentially leading the other motif to diverge from its original function and, in an evolutionary perspective, assist other functions of the protein, further increasing HIV genetic diversity.},
keywords = {NEGRONI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Transmissions of simian viruses to humans has originated the different groups of HIV-1. We recently identified a functional motif (CLA), in the C-terminal domain of the integrase, essential for integration in HIV-1 group M. Here, we found that the motif is instead dispensable in group O isolates, because of the presence, in the N-terminal domain of HIV-1 O of a specific sequence, Q7G27P41H44, that we define as the NOG motif. Alterations of reverse transcription and of 3' processing observed by mutating the CLA motif of IN M are fully rescued to wt levels by inserting the sequence of the NOG motif in the N-ter of the protein. These results indicate that the two motifs (CLA and NOG) functionally complement each other and a working model accounting for these observations is proposed. The establishment of these two alternative motifs seems to be due to the different phylogenetic origin and history of these two groups. Indeed, the NOG motif is already present in the ancestor of group O (SIVgor) while it is absent from SIVcpzPtt, the ancestor of group M. The CLA motif, instead, seems to have emerged after SIVcpzPtt has been transferred to humans, since no conservation is found at the same positions in these simian viruses. These results show the existence of two-group specific motifs in HIV-1 M and O integrases. In each group, only one of the motifs is functional, potentially leading the other motif to diverge from its original function and, in an evolutionary perspective, assist other functions of the protein, further increasing HIV genetic diversity.
Dufossez Robin, Ursuegui Sylvain, Baudrey Stephanie, Pernod Ketty, Mouftakhir Safae, Oulad-Abdelghani Mustapha, Mosser Michel, Chaubet Guilhem, Ryckelynck Michael, Wagner Alain
Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level Journal Article
In: Anal Chem, vol. 95, iss. 9, pp. 4470-4478, 2023, ISSN: 1520-6882.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid36821722,
title = {Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level},
author = {Robin Dufossez and Sylvain Ursuegui and Stephanie Baudrey and Ketty Pernod and Safae Mouftakhir and Mustapha Oulad-Abdelghani and Michel Mosser and Guilhem Chaubet and Michael Ryckelynck and Alain Wagner},
doi = {10.1021/acs.analchem.2c05168},
issn = {1520-6882},
year = {2023},
date = {2023-02-01},
urldate = {2023-02-01},
journal = {Anal Chem},
volume = {95},
issue = {9},
pages = {4470-4478},
abstract = {Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.
Lee Seungjae, Jee David, Srivastava Sid, Yang Acong, Ramidi Abhinav, Shang Renfu, Bortolamiol-Becet Diane, Pfeffer Sébastien, Gu Shuo, Wen Jiayu, Lai Eric C
Promiscuous splicing-derived hairpins are dominant substrates of tailing-mediated defense of miRNA biogenesis in mammals Journal Article
In: Cell Rep, vol. 42, no. 2, pp. 112111, 2023, ISSN: 2211-1247.
Abstract | Links | BibTeX | Tags: PFEFFER, Unité ARN
@article{pmid36800291,
title = {Promiscuous splicing-derived hairpins are dominant substrates of tailing-mediated defense of miRNA biogenesis in mammals},
author = {Seungjae Lee and David Jee and Sid Srivastava and Acong Yang and Abhinav Ramidi and Renfu Shang and Diane Bortolamiol-Becet and Sébastien Pfeffer and Shuo Gu and Jiayu Wen and Eric C Lai},
doi = {10.1016/j.celrep.2023.112111},
issn = {2211-1247},
year = {2023},
date = {2023-02-01},
urldate = {2023-02-01},
journal = {Cell Rep},
volume = {42},
number = {2},
pages = {112111},
abstract = {Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data. Since we originally defined mirtrons from small RNA duplexes, we term this larger set as structured splicing-derived RNAs (ssdRNAs). These associate with Dicer and/or Ago complexes, but generally accumulate modestly and are poorly conserved. We propose they contaminate the canonical miRNA pathway, which consequently requires defense against the siege of splicing-derived substrates. Accordingly, ssdRNAs/mirtrons comprise dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. Overall, the rampant proliferation of young mammalian mirtrons/ssdRNAs, coupled with an inhibitory molecular defense, comprises a Red Queen's race of intragenomic conflict.},
keywords = {PFEFFER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data. Since we originally defined mirtrons from small RNA duplexes, we term this larger set as structured splicing-derived RNAs (ssdRNAs). These associate with Dicer and/or Ago complexes, but generally accumulate modestly and are poorly conserved. We propose they contaminate the canonical miRNA pathway, which consequently requires defense against the siege of splicing-derived substrates. Accordingly, ssdRNAs/mirtrons comprise dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. Overall, the rampant proliferation of young mammalian mirtrons/ssdRNAs, coupled with an inhibitory molecular defense, comprises a Red Queen's race of intragenomic conflict.
Giegé Richard, Eriani Gilbert
The tRNA identity landscape for aminoacylation and beyond Journal Article
In: Nucleic Acids Res, vol. 51, no. 4, pp. 1528–1570, 2023, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ERIANI, GIEGE, Unité ARN
@article{pmid36744444,
title = {The tRNA identity landscape for aminoacylation and beyond},
author = {Richard Giegé and Gilbert Eriani},
doi = {10.1093/nar/gkad007},
issn = {1362-4962},
year = {2023},
date = {2023-02-01},
urldate = {2023-02-01},
journal = {Nucleic Acids Res},
volume = {51},
number = {4},
pages = {1528--1570},
abstract = {tRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.},
keywords = {ERIANI, GIEGE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
tRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.
Husser Claire, Baudrey Stéphanie, Ryckelynck Michael
High-Throughput Development and Optimization of RNA-Based Fluorogenic Biosensors of Small Molecules Using Droplet-Based Microfluidics Journal Article
In: Methods Mol Biol, vol. 2570, pp. 243–269, 2023, ISSN: 1940-6029.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid36156788,
title = {High-Throughput Development and Optimization of RNA-Based Fluorogenic Biosensors of Small Molecules Using Droplet-Based Microfluidics},
author = {Claire Husser and Stéphanie Baudrey and Michael Ryckelynck},
doi = {10.1007/978-1-0716-2695-5_19},
issn = {1940-6029},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Methods Mol Biol},
volume = {2570},
pages = {243--269},
abstract = {Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.
Gosset-Erard Clarisse, Didierjean Mévie, Pansanel Jérome, Lechner Antony, Wolff Philippe, Kuhn Lauriane, Aubriet Frédéric, Leize-Wagner Emmanuelle, Chaimbault Patrick, François Yannis-Nicolas
In: Anal Chem, vol. 95, iss. 2, pp. 1608-1617, 2023, ISSN: 1520-6882.
Abstract | Links | BibTeX | Tags: ARN-MS, PPSE, Unité ARN
@article{pmid36598775,
title = {Nucleos'ID: A New Search Engine Enabling the Untargeted Identification of RNA Post-transcriptional Modifications from Tandem Mass Spectrometry Analyses of Nucleosides},
author = {Clarisse Gosset-Erard and Mévie Didierjean and Jérome Pansanel and Antony Lechner and Philippe Wolff and Lauriane Kuhn and Frédéric Aubriet and Emmanuelle Leize-Wagner and Patrick Chaimbault and Yannis-Nicolas François},
doi = {10.1021/acs.analchem.2c04722},
issn = {1520-6882},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Anal Chem},
volume = {95},
issue = {2},
pages = {1608-1617},
abstract = {As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described transfer RNA and total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.},
keywords = {ARN-MS, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described transfer RNA and total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.
Ponce José R Jaramillo, Théobald-Dietrich Anne, Bénas Philippe, Paulus Caroline, Sauter Claude, Frugier Magali
Solution x-ray scattering highlights discrepancies in Plasmodium multi-aminoacyl-tRNA synthetase complexes Journal Article
In: Protein Sci, pp. e4564, 2023, ISSN: 1469-896X.
Abstract | Links | BibTeX | Tags: FRUGIER, Unité ARN
@article{pmid36606712,
title = {Solution x-ray scattering highlights discrepancies in Plasmodium multi-aminoacyl-tRNA synthetase complexes},
author = {José R Jaramillo Ponce and Anne Théobald-Dietrich and Philippe Bénas and Caroline Paulus and Claude Sauter and Magali Frugier},
doi = {10.1002/pro.4564},
issn = {1469-896X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Protein Sci},
pages = {e4564},
abstract = {tRip is a tRNA import protein specific to Plasmodium, the causative agent of malaria. In addition to its membrane localization and tRNA trafficking properties, tRip has the capacity to associate with three aminoacyl-tRNA synthetases (aaRS), the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases. In eukaryotes, such multi-aaRSs complexes (MSC) regulate the moonlighting activities of aaRSs. In Plasmodium, tRip and the three aaRSs all contain an N-terminal GST-like domain involved in the assembly of two independent complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS) with a 2:2:2 stoichiometry and in which the association of the GST-like domains of tRip and ERS (tRip-N:ERS-N) is central. In this study, the crystal structure of the N-terminal GST-like domain of ERS was solved and made possible further investigation of the solution architecture of the Q- and M-complexes by small-angle X-ray scattering (SAXS). This strategy relied on the engineering of a tRip-N-ERS-N chimeric protein to study the structural scaffold of both Plasmodium MSCs and confirm the unique homodimerization pattern of tRip in solution. The biological impact of these structural arrangements is discussed. This article is protected by copyright. All rights reserved.},
keywords = {FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
tRip is a tRNA import protein specific to Plasmodium, the causative agent of malaria. In addition to its membrane localization and tRNA trafficking properties, tRip has the capacity to associate with three aminoacyl-tRNA synthetases (aaRS), the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases. In eukaryotes, such multi-aaRSs complexes (MSC) regulate the moonlighting activities of aaRSs. In Plasmodium, tRip and the three aaRSs all contain an N-terminal GST-like domain involved in the assembly of two independent complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS) with a 2:2:2 stoichiometry and in which the association of the GST-like domains of tRip and ERS (tRip-N:ERS-N) is central. In this study, the crystal structure of the N-terminal GST-like domain of ERS was solved and made possible further investigation of the solution architecture of the Q- and M-complexes by small-angle X-ray scattering (SAXS). This strategy relied on the engineering of a tRip-N-ERS-N chimeric protein to study the structural scaffold of both Plasmodium MSCs and confirm the unique homodimerization pattern of tRip in solution. The biological impact of these structural arrangements is discussed. This article is protected by copyright. All rights reserved.
L. Urzhumtseva, V. Lunin, A. Urzhumtsev
Algorithms and programs for the shell decomposition of oscillating functions in space Journal Article
In: J Appl Cryst, vol. 56, no. 1, pp. 302-311, 2023.
Abstract | Links | BibTeX | Tags: Unité ARN, Urzhumtseva
@article{nokey,
title = {Algorithms and programs for the shell decomposition of oscillating functions in space},
author = {Urzhumtseva L. and Lunin V. and Urzhumtsev A.},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1107/S160057672201144X},
doi = {10.1107/S160057672201144X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {J Appl Cryst},
volume = {56},
number = {1},
pages = {302-311},
abstract = {Real-space refinement of atomic models in macromolecular crystallography and cryo-electron microscopy fits a model to a map obtained with experimental data. To do so, the atomic model is converted into a map of limited resolution and then this map is compared quantitatively with the experimental one. For an appropriate comparison, the atomic contributions comprising the model map should reflect the resolution of the experimental map and the atomic displacement parameter (ADP) values. Such contributions are spherically symmetric oscillating functions, different for chemically different kinds of atoms, different ADPs and different resolution values, and their derivatives with respect to atomic parameters rule the model refinement. For given parameter values, every contribution may be calculated numerically using two Fourier transforms, which is highly time consuming and makes calculation of the respective derivatives problematic. Alternatively, for an atom of each required type its contribution can be expressed in an analytical form as a sum of specially designed terms. Each term is different from zero essentially inside a spherical shell, and changing the ADP value does not change its form but rather changes the value of one of its arguments. In general, these terms become a convenient tool for the decomposition of oscillating spherically symmetric functions. This work describes the algorithms and respective software, named dec3D, to carry out such a shell decomposition for density contributions of different kinds of atoms and ions.},
keywords = {Unité ARN, Urzhumtseva},
pubstate = {published},
tppubtype = {article}
}
Real-space refinement of atomic models in macromolecular crystallography and cryo-electron microscopy fits a model to a map obtained with experimental data. To do so, the atomic model is converted into a map of limited resolution and then this map is compared quantitatively with the experimental one. For an appropriate comparison, the atomic contributions comprising the model map should reflect the resolution of the experimental map and the atomic displacement parameter (ADP) values. Such contributions are spherically symmetric oscillating functions, different for chemically different kinds of atoms, different ADPs and different resolution values, and their derivatives with respect to atomic parameters rule the model refinement. For given parameter values, every contribution may be calculated numerically using two Fourier transforms, which is highly time consuming and makes calculation of the respective derivatives problematic. Alternatively, for an atom of each required type its contribution can be expressed in an analytical form as a sum of specially designed terms. Each term is different from zero essentially inside a spherical shell, and changing the ADP value does not change its form but rather changes the value of one of its arguments. In general, these terms become a convenient tool for the decomposition of oscillating spherically symmetric functions. This work describes the algorithms and respective software, named dec3D, to carry out such a shell decomposition for density contributions of different kinds of atoms and ions.
Giuliodori Anna Maria, Belardinelli Riccardo, Duval Melodie, Garofalo Raffaella, Schenckbecher Emma, Hauryliuk Vasili, Ennifar Eric, Marzi Stefano
CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression Journal Article
In: Front Microbiol, vol. 14, pp. 1118329, 2023, ISSN: 1664-302X.
Abstract | Links | BibTeX | Tags: ENNIFAR, ROMBY, Unité ARN
@article{pmid36846801b,
title = { CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression},
author = {Anna Maria Giuliodori and Riccardo Belardinelli and Melodie Duval and Raffaella Garofalo and Emma Schenckbecher and Vasili Hauryliuk and Eric Ennifar and Stefano Marzi},
doi = {10.3389/fmicb.2023.1118329},
issn = {1664-302X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Front Microbiol},
volume = {14},
pages = {1118329},
abstract = { CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.},
keywords = {ENNIFAR, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.
Giuliodori Anna Maria, Londei Paola, Marzi Stefano
2023, ISSN: 1664-302X.
Links | BibTeX | Tags: MARZI, ROMBY, Unité ARN
@misc{pmid37383636,
title = {Editorial: Interview with the translational apparatus: stories of intriguing circuits and mechanisms to regulate translation in bacteria, volume II},
author = {Anna Maria Giuliodori and Paola Londei and Stefano Marzi},
doi = {10.3389/fmicb.2023.1195257},
issn = {1664-302X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Front Microbiol},
volume = {14},
pages = {1195257},
keywords = {MARZI, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {misc}
}
2022
Westhof E
Data, data, burning deep, in the forests of the net Journal Article
In: Biochem Biophys Res Commun, vol. 633, pp. 42-44, 2022, ISSN: 1090-2104.
Abstract | Links | BibTeX | Tags: Unité ARN, WESTHOF
@article{pmid36344159,
title = {Data, data, burning deep, in the forests of the net},
author = {E Westhof},
doi = {10.1016/j.bbrc.2022.09.030},
issn = {1090-2104},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {Biochem Biophys Res Commun},
volume = {633},
pages = {42-44},
abstract = {Continuous and imaginative technological developments are leading to a massive accumulation of various types of data in all areas of biological research. As a result, the central importance of databases is increasing. Databases related to biology must not only be structured using controlled vocabularies, but also be fully integrated into the whole biological domain. To achieve this goal, they must be systematically grounded in biological evolution and exploit the available tools of evolutionary systematics to contribute to our understanding of life processes.},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Continuous and imaginative technological developments are leading to a massive accumulation of various types of data in all areas of biological research. As a result, the central importance of databases is increasing. Databases related to biology must not only be structured using controlled vocabularies, but also be fully integrated into the whole biological domain. To achieve this goal, they must be systematically grounded in biological evolution and exploit the available tools of evolutionary systematics to contribute to our understanding of life processes.
Urzhumtsev Alexandre G, Urzhumtseva Ludmila M, Lunin Vladimir Y
Direct calculation of cryo-EM and crystallographic model maps for real-space refinement Journal Article
In: Acta Crystallogr D Struct Biol, vol. 78, no. Pt 12, pp. 1451–1468, 2022, ISSN: 2059-7983.
Abstract | Links | BibTeX | Tags: Unité ARN
@article{pmid36458616,
title = {Direct calculation of cryo-EM and crystallographic model maps for real-space refinement},
author = {Alexandre G Urzhumtsev and Ludmila M Urzhumtseva and Vladimir Y Lunin},
doi = {10.1107/S2059798322010907},
issn = {2059-7983},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {Acta Crystallogr D Struct Biol},
volume = {78},
number = {Pt 12},
pages = {1451--1468},
abstract = {This work addresses the problem of the calculation of limited-resolution maps from an atomic model in cryo-electron microscopy and in X-ray and neutron crystallography, including cases where the resolution varies from one molecular region to another. Such maps are necessary in real-space refinement for comparison with the experimental maps. For an appropriate numeric comparison, the calculated maps should reproduce not only the structural features contained in the experimental maps but also the principal map distortions. These model maps can be obtained with no use of Fourier transforms but, similar to density distributions, as a sum of individual atomic contributions. Such contributions, referred to as atomic density images, are atomic densities morphed to reflect distortions of the experimental map, in particular the loss of resolution. They are described by functions composed of a central peak surrounded by Fourier ripples. For practical calculations, atomic images should be cut at some distance. It is shown that to reach a reasonable accuracy such a distance should be significantly larger than the distance customarily applied when calculating density distributions. This is a consequence of the slow rate with which the amplitude of the Fourier ripples decreases. Such a large distance means that at least a few ripples should be included in calculations in order to obtain a map that is sufficiently accurate. Oscillating functions describing these atomic contributions depend, for a given atomic type, on the resolution and on the atomic displacement parameter values. To express both the central peak and the Fourier ripples of the atomic images, these functions are represented by the sums of especially designed terms, each concentrated in a spherical shell and depending analytically on the atomic parameters. In this work, the strength of the dependence of the accuracy of resulting map on the accuracy of the atomic displacement parameters and on the truncation distance, i.e. the number of ripples included in atomic density images, is analyzed. This analysis is completed by practical aspects of the calculation of maps of inhomogeneous resolution. Tests show that the calculation of limited-resolution maps from an atomic model as a sum of atomic contributions requires a large truncation radius extending beyond the central peak of an atomic image and the first Fourier ripples. The article discusses the practical details of such calculations expressing atomic contributions as analytic functions of the atomic coordinates, the atomic displacement parameters and the local resolution.},
keywords = {Unité ARN},
pubstate = {published},
tppubtype = {article}
}
This work addresses the problem of the calculation of limited-resolution maps from an atomic model in cryo-electron microscopy and in X-ray and neutron crystallography, including cases where the resolution varies from one molecular region to another. Such maps are necessary in real-space refinement for comparison with the experimental maps. For an appropriate numeric comparison, the calculated maps should reproduce not only the structural features contained in the experimental maps but also the principal map distortions. These model maps can be obtained with no use of Fourier transforms but, similar to density distributions, as a sum of individual atomic contributions. Such contributions, referred to as atomic density images, are atomic densities morphed to reflect distortions of the experimental map, in particular the loss of resolution. They are described by functions composed of a central peak surrounded by Fourier ripples. For practical calculations, atomic images should be cut at some distance. It is shown that to reach a reasonable accuracy such a distance should be significantly larger than the distance customarily applied when calculating density distributions. This is a consequence of the slow rate with which the amplitude of the Fourier ripples decreases. Such a large distance means that at least a few ripples should be included in calculations in order to obtain a map that is sufficiently accurate. Oscillating functions describing these atomic contributions depend, for a given atomic type, on the resolution and on the atomic displacement parameter values. To express both the central peak and the Fourier ripples of the atomic images, these functions are represented by the sums of especially designed terms, each concentrated in a spherical shell and depending analytically on the atomic parameters. In this work, the strength of the dependence of the accuracy of resulting map on the accuracy of the atomic displacement parameters and on the truncation distance, i.e. the number of ripples included in atomic density images, is analyzed. This analysis is completed by practical aspects of the calculation of maps of inhomogeneous resolution. Tests show that the calculation of limited-resolution maps from an atomic model as a sum of atomic contributions requires a large truncation radius extending beyond the central peak of an atomic image and the first Fourier ripples. The article discusses the practical details of such calculations expressing atomic contributions as analytic functions of the atomic coordinates, the atomic displacement parameters and the local resolution.
Neyroud Anne Sophie, Rudinger-Thirion Joëlle, Frugier Magali, Riley Lisa G, Bidet Maud, Akloul Linda, Simpson Andrea, Gilot David, Christodoulou John, Ravel Célia, Sinclair Andrew H, Belaud-Rotureau Marc-Antoine, Tucker Elena J, Jaillard Sylvie
LARS2 variants can present as premature ovarian insufficiency in the absence of overt hearing loss Journal Article
In: Eur J Hum Genet, vol. 31, iss. 4, pp. 453-460, 2022, ISSN: 1476-5438.
Abstract | Links | BibTeX | Tags: FRUGIER, Unité ARN
@article{pmid36450801,
title = {LARS2 variants can present as premature ovarian insufficiency in the absence of overt hearing loss},
author = {Anne Sophie Neyroud and Joëlle Rudinger-Thirion and Magali Frugier and Lisa G Riley and Maud Bidet and Linda Akloul and Andrea Simpson and David Gilot and John Christodoulou and Célia Ravel and Andrew H Sinclair and Marc-Antoine Belaud-Rotureau and Elena J Tucker and Sylvie Jaillard},
doi = {10.1038/s41431-022-01252-1},
issn = {1476-5438},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {Eur J Hum Genet},
volume = {31},
issue = {4},
pages = {453-460},
abstract = {Premature ovarian insufficiency (POI) affects 1 in 100 women and is a leading cause of female infertility. There are over 80 genes in which variants can cause POI, with these explaining only a minority of cases. Whole exome sequencing (WES) can be a useful tool for POI patient management, allowing clinical care to be personalized to underlying cause. We performed WES to investigate two French sisters, whose only clinical complaint was POI. Surprisingly, they shared one known and one novel likely pathogenic variant in the Perrault syndrome gene, LARS2. Using amino-acylation studies, we established that the novel missense variant significantly impairs LARS2 function. Perrault syndrome is characterized by sensorineural hearing loss in addition to POI. This molecular diagnosis alerted the sisters to the significance of their difficulty in following conversation. Subsequent audiology assessment revealed a mild bilateral hearing loss. We describe the first cases presenting with perceived isolated POI and causative variants in a Perrault syndrome gene. Our study expands the phenotypic spectrum associated with LARS2 variants and highlights the clinical benefit of having a genetic diagnosis, with prediction of potential co-morbidity and prompt and appropriate medical care, in this case by an audiologist for early detection of hearing loss.},
keywords = {FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Premature ovarian insufficiency (POI) affects 1 in 100 women and is a leading cause of female infertility. There are over 80 genes in which variants can cause POI, with these explaining only a minority of cases. Whole exome sequencing (WES) can be a useful tool for POI patient management, allowing clinical care to be personalized to underlying cause. We performed WES to investigate two French sisters, whose only clinical complaint was POI. Surprisingly, they shared one known and one novel likely pathogenic variant in the Perrault syndrome gene, LARS2. Using amino-acylation studies, we established that the novel missense variant significantly impairs LARS2 function. Perrault syndrome is characterized by sensorineural hearing loss in addition to POI. This molecular diagnosis alerted the sisters to the significance of their difficulty in following conversation. Subsequent audiology assessment revealed a mild bilateral hearing loss. We describe the first cases presenting with perceived isolated POI and causative variants in a Perrault syndrome gene. Our study expands the phenotypic spectrum associated with LARS2 variants and highlights the clinical benefit of having a genetic diagnosis, with prediction of potential co-morbidity and prompt and appropriate medical care, in this case by an audiologist for early detection of hearing loss.
Mráziková Klaudia, Kruse Holger, Mlýnský Vojtěch, Auffinger Pascal, Šponer Jiří
Multiscale Modeling of Phosphate···π Contacts in RNA U-Turns Exposes Differences between Quantum-Chemical and AMBER Force Field Descriptions Journal Article
In: J Chem Inf Model, vol. 62, iss. 23, pp. 6182-6200, 2022, ISSN: 1549-960X.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{pmid36454943,
title = {Multiscale Modeling of Phosphate···π Contacts in RNA U-Turns Exposes Differences between Quantum-Chemical and AMBER Force Field Descriptions},
author = {Klaudia Mráziková and Holger Kruse and Vojtěch Mlýnský and Pascal Auffinger and Jiří Šponer},
doi = {10.1021/acs.jcim.2c01064},
issn = {1549-960X},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {J Chem Inf Model},
volume = {62},
issue = {23},
pages = {6182-6200},
abstract = {Phosphate···π, also called anion···π, contacts occur between nucleobases and anionic phosphate oxygens (OP2) in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). These contacts were investigated using state-of-the-art quantum-chemical methods (QM) to characterize their physicochemical properties and to serve as a reference to evaluate AMBER force field (AFF) performance. We found that phosphate···π interaction energies calculated with the AFF for dimethyl phosphate···nucleobase model systems are less stabilizing in comparison with double-hybrid DFT and that minimum contact distances are larger for all nucleobases. These distance stretches are also observed in large-scale AFF vs QM/MM computations and classical molecular dynamics (MD) simulations on several r(gcGNRAgc) tetraloop hairpins when compared to experimental data extracted from X-ray/cryo-EM structures (res. ≤ 2.5 Å) using the WebFR3D bioinformatic tool. MD simulations further revealed shifted OP2/nucleobase positions. We propose that discrepancies between the QM and AFF result from a combination of missing polarization in the AFF combined with too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms in addition to an exaggerated short-range repulsion of the LJ repulsive term. We compared these results with earlier data gathered on lone pair···π contacts in CpG Z-steps occurring in r(UNCG) tetraloops. In both instances, charge transfer calculations do not support any significant → π* donation effects. We also investigated thiophosphate···π contacts that showed reduced stabilizing interaction energies when compared to phosphate···π contacts. Thus, we challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be explained by larger London dispersion.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Phosphate···π, also called anion···π, contacts occur between nucleobases and anionic phosphate oxygens (OP2) in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). These contacts were investigated using state-of-the-art quantum-chemical methods (QM) to characterize their physicochemical properties and to serve as a reference to evaluate AMBER force field (AFF) performance. We found that phosphate···π interaction energies calculated with the AFF for dimethyl phosphate···nucleobase model systems are less stabilizing in comparison with double-hybrid DFT and that minimum contact distances are larger for all nucleobases. These distance stretches are also observed in large-scale AFF vs QM/MM computations and classical molecular dynamics (MD) simulations on several r(gcGNRAgc) tetraloop hairpins when compared to experimental data extracted from X-ray/cryo-EM structures (res. ≤ 2.5 Å) using the WebFR3D bioinformatic tool. MD simulations further revealed shifted OP2/nucleobase positions. We propose that discrepancies between the QM and AFF result from a combination of missing polarization in the AFF combined with too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms in addition to an exaggerated short-range repulsion of the LJ repulsive term. We compared these results with earlier data gathered on lone pair···π contacts in CpG Z-steps occurring in r(UNCG) tetraloops. In both instances, charge transfer calculations do not support any significant → π* donation effects. We also investigated thiophosphate···π contacts that showed reduced stabilizing interaction energies when compared to phosphate···π contacts. Thus, we challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be explained by larger London dispersion.
Girardi Erika, Messmer Melanie, Lopez Paula, Fender Aurelie, Chicher Johana, Chane-Woon-Ming Beatrice, Hammann Philippe, Pfeffer Sebastien
Proteomics-based determination of double stranded RNA interactome reveals known and new factors involved in Sindbis virus infection Journal Article
In: RNA, vol. 29, iss. 3, pp. 361-375, 2022, ISSN: 1469-9001.
Abstract | Links | BibTeX | Tags: PFEFFER, PPSE, Unité ARN
@article{pmid36617674,
title = {Proteomics-based determination of double stranded RNA interactome reveals known and new factors involved in Sindbis virus infection},
author = {Erika Girardi and Melanie Messmer and Paula Lopez and Aurelie Fender and Johana Chicher and Beatrice Chane-Woon-Ming and Philippe Hammann and Sebastien Pfeffer},
doi = {10.1261/rna.079270.122},
issn = {1469-9001},
year = {2022},
date = {2022-12-01},
urldate = {2022-12-01},
journal = {RNA},
volume = {29},
issue = {3},
pages = {361-375},
abstract = {Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated with viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry analysis to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human cells. Among the identified proteins, we characterized SFPQ (Splicing factor, proline-glutamine rich) as a new dsRNA-associated proviral factor upon SINV infection. We showed that SFPQ depletion reduces SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ enhances viral production. We demonstrated that the cytoplasmic fraction of SFPQ partially colocalizes with dsRNA upon SINV infection. In agreement, we proved by RNA-IP that SFPQ can bind dsRNA and viral RNA. Furthermore, we showed that overexpression of a wild type, but not an RNA binding mutant SFPQ, increased viral infection, suggesting that RNA binding is essential for its positive effect on the virus. Overall, this study provides the community with a compendium of dsRNA-associated factors during viral infection and identifies SFPQ as a new proviral dsRNA binding protein.},
keywords = {PFEFFER, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated with viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry analysis to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human cells. Among the identified proteins, we characterized SFPQ (Splicing factor, proline-glutamine rich) as a new dsRNA-associated proviral factor upon SINV infection. We showed that SFPQ depletion reduces SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ enhances viral production. We demonstrated that the cytoplasmic fraction of SFPQ partially colocalizes with dsRNA upon SINV infection. In agreement, we proved by RNA-IP that SFPQ can bind dsRNA and viral RNA. Furthermore, we showed that overexpression of a wild type, but not an RNA binding mutant SFPQ, increased viral infection, suggesting that RNA binding is essential for its positive effect on the virus. Overall, this study provides the community with a compendium of dsRNA-associated factors during viral infection and identifies SFPQ as a new proviral dsRNA binding protein.
Xu Rui, Lou Yanyan, Tidu Antonin, Bulet Philippe, Heinekamp Thorsten, Martin Franck, Brakhage Axel, Li Zi, Liégeois Samuel, Ferrandon Dominique
The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins Journal Article
In: EMBO Rep, pp. e56036, 2022, ISSN: 1469-3178.
Abstract | Links | BibTeX | Tags: ERIANI, ferrandon, M3i, MARTIN, Unité ARN
@article{pmid36322050,
title = {The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins},
author = {Rui Xu and Yanyan Lou and Antonin Tidu and Philippe Bulet and Thorsten Heinekamp and Franck Martin and Axel Brakhage and Zi Li and Samuel Liégeois and Dominique Ferrandon},
url = {https://pubmed.ncbi.nlm.nih.gov/36322050/},
doi = {10.15252/embr.202256036},
issn = {1469-3178},
year = {2022},
date = {2022-11-01},
urldate = {2022-11-01},
journal = {EMBO Rep},
pages = {e56036},
abstract = {Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.},
keywords = {ERIANI, ferrandon, M3i, MARTIN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.
Belinite Margarita, Khusainov Iskander, Marzi Stefano
30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis Journal Article
In: Bio Protoc, vol. 12, no. 20, 2022, ISSN: 2331-8325.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid36353712,
title = { 30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis},
author = {Margarita Belinite and Iskander Khusainov and Stefano Marzi},
doi = {10.21769/BioProtoc.4532},
issn = {2331-8325},
year = {2022},
date = {2022-10-01},
urldate = {2022-10-01},
journal = {Bio Protoc},
volume = {12},
number = {20},
abstract = {The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K and Mg , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K and Mg , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.
Belinite Margarita, Khusainov Iskander, Marzi Stefano
30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis Journal Article
In: Bio Protoc, vol. 12, no. 20, 2022, ISSN: 2331-8325.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid36353712b,
title = { 30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis},
author = {Margarita Belinite and Iskander Khusainov and Stefano Marzi},
doi = {10.21769/BioProtoc.4532},
issn = {2331-8325},
year = {2022},
date = {2022-10-01},
urldate = {2022-10-01},
journal = {Bio Protoc},
volume = {12},
number = {20},
abstract = {The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K and Mg , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K and Mg , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.
Bonaventure Boris, Rebendenne Antoine, Valadão Ana Luiza Chaves, Arnaud-Arnould Mary, Gracias Ségolène, de Gracia Francisco Garcia, McKellar Joe, Labaronne Emmanuel, Tauziet Marine, Vivet-Boudou Valérie, Bernard Eric, Briant Laurence, Gros Nathalie, Djilli Wassila, Courgnaud Valérie, Parrinello Hugues, Rialle Stéphanie, Blaise Mickaël, Lacroix Laurent, Lavigne Marc, Paillart Jean-Christophe, Ricci Emiliano P, Schulz Reiner, Jouvenet Nolwenn, Moncorgé Olivier, Goujon Caroline
The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses Journal Article
In: EMBO Rep, pp. e54061, 2022, ISSN: 1469-3178.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{pmid36161446,
title = {The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses},
author = {Boris Bonaventure and Antoine Rebendenne and Ana Luiza Chaves Valadão and Mary Arnaud-Arnould and Ségolène Gracias and Francisco Garcia de Gracia and Joe McKellar and Emmanuel Labaronne and Marine Tauziet and Valérie Vivet-Boudou and Eric Bernard and Laurence Briant and Nathalie Gros and Wassila Djilli and Valérie Courgnaud and Hugues Parrinello and Stéphanie Rialle and Mickaël Blaise and Laurent Lacroix and Marc Lavigne and Jean-Christophe Paillart and Emiliano P Ricci and Reiner Schulz and Nolwenn Jouvenet and Olivier Moncorgé and Caroline Goujon},
url = {https://pubmed.ncbi.nlm.nih.gov/36161446/},
doi = {10.15252/embr.202154061},
issn = {1469-3178},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {EMBO Rep},
pages = {e54061},
abstract = {Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.
Tong Xiaoling, Han Min-Jin, Lu Kunpeng, Tai Shuaishuai, Liang Shubo, Liu Yucheng, Hu Hai, Shen Jianghong, Long Anxing, Zhan Chengyu, Ding Xin, Liu Shuo, Gao Qiang, Zhang Bili, Zhou Linli, Tan Duan, Yuan Yajie, Guo Nangkuo, Li Yan-Hong, Wu Zhangyan, Liu Lulu, Li Chunlin, Lu Yaru, Gai Tingting, Zhang Yahui, Yang Renkui, Qian Heying, Liu Yanqun, Luo Jiangwen, Zheng Lu, Lou Jinghou, Peng Yunwu, Zuo Weidong, Song Jiangbo, He Songzhen, Wu Songyuan, Zou Yunlong, Zhou Lei, Cheng Lan, Tang Yuxia, Cheng Guotao, Yuan Lianwei, He Weiming, Xu Jiabao, Fu Tao, Xiao Yang, Lei Ting, Xu Anying, Yin Ye, Wang Jian, Monteiro Antónia, Westhof E, Lu Cheng, Tian Zhixi, Wang Wen, Xiang Zhonghuai, Dai Fangyin
High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation Journal Article
In: Nat Commun, vol. 13, no. 1, pp. 5619, 2022, ISSN: 2041-1723.
Abstract | Links | BibTeX | Tags: Unité ARN, WESTHOF
@article{pmid36153338,
title = {High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation},
author = {Xiaoling Tong and Min-Jin Han and Kunpeng Lu and Shuaishuai Tai and Shubo Liang and Yucheng Liu and Hai Hu and Jianghong Shen and Anxing Long and Chengyu Zhan and Xin Ding and Shuo Liu and Qiang Gao and Bili Zhang and Linli Zhou and Duan Tan and Yajie Yuan and Nangkuo Guo and Yan-Hong Li and Zhangyan Wu and Lulu Liu and Chunlin Li and Yaru Lu and Tingting Gai and Yahui Zhang and Renkui Yang and Heying Qian and Yanqun Liu and Jiangwen Luo and Lu Zheng and Jinghou Lou and Yunwu Peng and Weidong Zuo and Jiangbo Song and Songzhen He and Songyuan Wu and Yunlong Zou and Lei Zhou and Lan Cheng and Yuxia Tang and Guotao Cheng and Lianwei Yuan and Weiming He and Jiabao Xu and Tao Fu and Yang Xiao and Ting Lei and Anying Xu and Ye Yin and Jian Wang and Antónia Monteiro and E Westhof and Cheng Lu and Zhixi Tian and Wen Wang and Zhonghuai Xiang and Fangyin Dai},
doi = {10.1038/s41467-022-33366-x},
issn = {2041-1723},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {Nat Commun},
volume = {13},
number = {1},
pages = {5619},
abstract = {The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.
Hayek Hassan, Eriani Gilbert, Allmang Christine
eIF3 Interacts with Selenoprotein mRNAs Journal Article
In: Biomolecules, vol. 12, no. 9, 2022, ISSN: 2218-273X.
Abstract | Links | BibTeX | Tags: ERIANI, Unité ARN
@article{pmid36139107,
title = {eIF3 Interacts with Selenoprotein mRNAs},
author = {Hassan Hayek and Gilbert Eriani and Christine Allmang},
doi = {10.3390/biom12091268},
issn = {2218-273X},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {Biomolecules},
volume = {12},
number = {9},
abstract = {The synthesis of selenoproteins requires the co-translational recoding of an in-frame UGASec codon. Interactions between the Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2) in the 3'untranslated region (3'UTR) of selenoprotein mRNAs enable the recruitment of the selenocysteine insertion machinery. Several selenoprotein mRNAs undergo unusual cap hypermethylation and are not recognized by the translation initiation factor 4E (eIF4E) but nevertheless translated. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), can selectively recruit several cellular mRNAs and plays roles in specialized translation initiation. Here, we analyzed the ability of eIF3 to interact with selenoprotein mRNAs. By combining ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro with cross-linking experiments, we found interactions between eIF3 and a subgroup of selenoprotein mRNAs. We showed that eIF3 preferentially interacts with hypermethylated capped selenoprotein mRNAs rather than mG-capped mRNAs. We identified direct contacts between GPx1 mRNA and eIF3 c, d, and e subunits and showed the existence of common interaction patterns for all hypermethylated capped selenoprotein mRNAs. Differential interactions of eIF3 with selenoprotein mRNAs may trigger specific translation pathways independent of eIF4E. eIF3 could represent a new player in the translation regulation and hierarchy of selenoprotein expression.},
keywords = {ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The synthesis of selenoproteins requires the co-translational recoding of an in-frame UGASec codon. Interactions between the Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2) in the 3'untranslated region (3'UTR) of selenoprotein mRNAs enable the recruitment of the selenocysteine insertion machinery. Several selenoprotein mRNAs undergo unusual cap hypermethylation and are not recognized by the translation initiation factor 4E (eIF4E) but nevertheless translated. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), can selectively recruit several cellular mRNAs and plays roles in specialized translation initiation. Here, we analyzed the ability of eIF3 to interact with selenoprotein mRNAs. By combining ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro with cross-linking experiments, we found interactions between eIF3 and a subgroup of selenoprotein mRNAs. We showed that eIF3 preferentially interacts with hypermethylated capped selenoprotein mRNAs rather than mG-capped mRNAs. We identified direct contacts between GPx1 mRNA and eIF3 c, d, and e subunits and showed the existence of common interaction patterns for all hypermethylated capped selenoprotein mRNAs. Differential interactions of eIF3 with selenoprotein mRNAs may trigger specific translation pathways independent of eIF4E. eIF3 could represent a new player in the translation regulation and hierarchy of selenoprotein expression.
Diallo Idrissa, Ho Jeffrey, Lambert Marine, Benmoussa Abderrahim, Husseini Zeinab, Lalaouna David, Massé Eric, Provost Patrick
A tRNA-derived fragment present in E. coli OMVs regulates host cell gene expression and proliferation Journal Article
In: PLoS Pathog, vol. 18, no. 9, pp. e1010827, 2022, ISSN: 1553-7374.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid36108089,
title = {A tRNA-derived fragment present in E. coli OMVs regulates host cell gene expression and proliferation},
author = {Idrissa Diallo and Jeffrey Ho and Marine Lambert and Abderrahim Benmoussa and Zeinab Husseini and David Lalaouna and Eric Massé and Patrick Provost},
doi = {10.1371/journal.ppat.1010827},
issn = {1553-7374},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {PLoS Pathog},
volume = {18},
number = {9},
pages = {e1010827},
abstract = {RNA-sequencing has led to a spectacular increase in the repertoire of bacterial sRNAs and improved our understanding of their biological functions. Bacterial sRNAs have also been found in outer membrane vesicles (OMVs), raising questions about their potential involvement in bacteria-host relationship, but few studies have documented this issue. Recent RNA-Sequencing analyses of bacterial RNA unveiled the existence of abundant very small RNAs (vsRNAs) shorter than 16 nt. These especially include tRNA fragments (tRFs) that are selectively loaded in OMVs and are predicted to target host mRNAs. Here, in Escherichia coli (E. coli), we report the existence of an abundant vsRNA, Ile-tRF-5X, which is selectively modulated by environmental stress, while remaining unaffected by inhibition of transcription or translation. Ile-tRF-5X is released through OMVs and can be transferred to human HCT116 cells, where it promoted MAP3K4 expression. Our findings provide a novel perspective and paradigm on the existing symbiosis between bacteria and human cells.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA-sequencing has led to a spectacular increase in the repertoire of bacterial sRNAs and improved our understanding of their biological functions. Bacterial sRNAs have also been found in outer membrane vesicles (OMVs), raising questions about their potential involvement in bacteria-host relationship, but few studies have documented this issue. Recent RNA-Sequencing analyses of bacterial RNA unveiled the existence of abundant very small RNAs (vsRNAs) shorter than 16 nt. These especially include tRNA fragments (tRFs) that are selectively loaded in OMVs and are predicted to target host mRNAs. Here, in Escherichia coli (E. coli), we report the existence of an abundant vsRNA, Ile-tRF-5X, which is selectively modulated by environmental stress, while remaining unaffected by inhibition of transcription or translation. Ile-tRF-5X is released through OMVs and can be transferred to human HCT116 cells, where it promoted MAP3K4 expression. Our findings provide a novel perspective and paradigm on the existing symbiosis between bacteria and human cells.
Danne Camille, Michaudel Chloé, Skerniskyte Jurate, Planchais Julien, Magniez Aurélie, Agus Allison, Michel Marie-Laure, Lamas Bruno, Costa Gregory Da, Spatz Madeleine, Oeuvray Cyriane, Galbert Chloé, Poirier Maxime, Wang Yazhou, Lapière Alexia, Rolhion Nathalie, Ledent Tatiana, Pionneau Cédric, Chardonnet Solenne, Bellvert Floriant, Cahoreau Edern, Rocher Amandine, Arguello Rafael Rose, Peyssonnaux Carole, Louis Sabine, Richard Mathias L, Langella Philippe, El-Benna Jamel, Marteyn Benoit, Sokol Harry
CARD9 in neutrophils protects from colitis and controls mitochondrial metabolism and cell survival Journal Article
In: Gut, vol. 72, iss. 6, pp. 1081-1092, 2022, ISSN: 1468-3288.
Abstract | Links | BibTeX | Tags: MARTEYN, Unité ARN
@article{pmid36167663,
title = {CARD9 in neutrophils protects from colitis and controls mitochondrial metabolism and cell survival},
author = {Camille Danne and Chloé Michaudel and Jurate Skerniskyte and Julien Planchais and Aurélie Magniez and Allison Agus and Marie-Laure Michel and Bruno Lamas and Gregory Da Costa and Madeleine Spatz and Cyriane Oeuvray and Chloé Galbert and Maxime Poirier and Yazhou Wang and Alexia Lapière and Nathalie Rolhion and Tatiana Ledent and Cédric Pionneau and Solenne Chardonnet and Floriant Bellvert and Edern Cahoreau and Amandine Rocher and Rafael Rose Arguello and Carole Peyssonnaux and Sabine Louis and Mathias L Richard and Philippe Langella and Jamel El-Benna and Benoit Marteyn and Harry Sokol},
doi = {10.1136/gutjnl-2022-326917},
issn = {1468-3288},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {Gut},
volume = {72},
issue = {6},
pages = {1081-1092},
abstract = {OBJECTIVES: Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 (), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown.
DESIGN: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse).
RESULTS: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation.
CONCLUSION: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.},
keywords = {MARTEYN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVES: Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 (), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown.
DESIGN: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse).
RESULTS: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation.
CONCLUSION: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.
DESIGN: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse).
RESULTS: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation.
CONCLUSION: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.
Jakob Celia, Paul-Stansilaus Rithu, Schwemmle Martin, Marquet Roland, Bolte Hardin
The influenza A virus genome packaging network - complex, flexible and yet unsolved Journal Article
In: Nucleic Acids Res, vol. 50, iss. 16, pp. 9023-9038, 2022, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: MARQUET, Unité ARN
@article{pmid35993811,
title = {The influenza A virus genome packaging network - complex, flexible and yet unsolved},
author = {Celia Jakob and Rithu Paul-Stansilaus and Martin Schwemmle and Roland Marquet and Hardin Bolte},
doi = {10.1093/nar/gkac688},
issn = {1362-4962},
year = {2022},
date = {2022-08-01},
urldate = {2022-08-01},
journal = {Nucleic Acids Res},
volume = {50},
issue = {16},
pages = {9023-9038},
abstract = {The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA-RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA-RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA-RNA interactions. We speculate that identifying such functional intersegmental RNA-RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.},
keywords = {MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA-RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA-RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA-RNA interactions. We speculate that identifying such functional intersegmental RNA-RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.
Mair Stefan, Erharter Kevin, Renard Eva, Brillet Karl, Brunner Melanie, Lusser Alexandra, Kreutz Christoph, Ennifar Eric, Micura Ronald
Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA Journal Article
In: Nucleic Acids Res, vol. 50, iss. 11, pp. 6038-6051, 2022, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{pmid35687141,
title = {Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA},
author = {Stefan Mair and Kevin Erharter and Eva Renard and Karl Brillet and Melanie Brunner and Alexandra Lusser and Christoph Kreutz and Eric Ennifar and Ronald Micura},
doi = {10.1093/nar/gkac477},
issn = {1362-4962},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Nucleic Acids Res},
volume = {50},
issue = {11},
pages = {6038-6051},
abstract = {Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.
Vilimova Monika, Pfeffer Sébastien
Post-transcriptional regulation of polycistronic microRNAs Journal Article
In: Wiley Interdiscip Rev RNA, vol. 14, iss. 2, pp. e1749, 2022, ISSN: 1757-7012.
Abstract | Links | BibTeX | Tags: PFEFFER, Unité ARN
@article{pmid35702737,
title = {Post-transcriptional regulation of polycistronic microRNAs},
author = {Monika Vilimova and Sébastien Pfeffer},
doi = {10.1002/wrna.1749},
issn = {1757-7012},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Wiley Interdiscip Rev RNA},
volume = {14},
issue = {2},
pages = {e1749},
abstract = {An important proportion of microRNA (miRNA) genes tend to lie close to each other within animal genomes. Such genomic organization is generally referred to as miRNA clusters. Even though many miRNA clusters have been greatly studied, most attention has been usually focused on functional impacts of clustered miRNA co-expression. However, there is also another compelling aspect about these miRNA clusters, their polycistronic nature. Being transcribed on a single RNA precursor, polycistronic miRNAs benefit from common transcriptional regulation allowing their coordinated expression. And yet, numerous reports have revealed striking discrepancies in the accumulation of mature miRNAs produced from the same cluster. Indeed, the larger polycistronic transcripts can act as platforms providing unforeseen post-transcriptional regulatory mechanisms controlling individual miRNA processing, thus leading to differential miRNA expression, and sometimes even challenging the general assumption that polycistronic miRNAs are co-expressed. In this review, we aim to address the current knowledge about how miRNA polycistrons are post-transcriptionally regulated. In particular, we will focus on the mechanisms occurring at the level of the primary transcript, which are highly relevant for individual miRNA processing and as such have a direct repercussion on miRNA function within the cell. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.},
keywords = {PFEFFER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
An important proportion of microRNA (miRNA) genes tend to lie close to each other within animal genomes. Such genomic organization is generally referred to as miRNA clusters. Even though many miRNA clusters have been greatly studied, most attention has been usually focused on functional impacts of clustered miRNA co-expression. However, there is also another compelling aspect about these miRNA clusters, their polycistronic nature. Being transcribed on a single RNA precursor, polycistronic miRNAs benefit from common transcriptional regulation allowing their coordinated expression. And yet, numerous reports have revealed striking discrepancies in the accumulation of mature miRNAs produced from the same cluster. Indeed, the larger polycistronic transcripts can act as platforms providing unforeseen post-transcriptional regulatory mechanisms controlling individual miRNA processing, thus leading to differential miRNA expression, and sometimes even challenging the general assumption that polycistronic miRNAs are co-expressed. In this review, we aim to address the current knowledge about how miRNA polycistrons are post-transcriptionally regulated. In particular, we will focus on the mechanisms occurring at the level of the primary transcript, which are highly relevant for individual miRNA processing and as such have a direct repercussion on miRNA function within the cell. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
McKellar Stuart W, Ivanova Ivayla, Arede Pedro, Zapf Rachel L, Mercier Noémie, Chu Liang-Cui, Mediati Daniel G, Pickering Amy C, Briaud Paul, Foster Robert G, Kudla Grzegorz, Fitzgerald J Ross, Caldelari Isabelle, Carroll Ronan K, Tree Jai J, Granneman Sander
RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression Journal Article
In: Nat Commun, vol. 13, no. 1, pp. 3560, 2022, ISSN: 2041-1723.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid35732654,
title = {RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression},
author = {Stuart W McKellar and Ivayla Ivanova and Pedro Arede and Rachel L Zapf and Noémie Mercier and Liang-Cui Chu and Daniel G Mediati and Amy C Pickering and Paul Briaud and Robert G Foster and Grzegorz Kudla and J Ross Fitzgerald and Isabelle Caldelari and Ronan K Carroll and Jai J Tree and Sander Granneman},
doi = {10.1038/s41467-022-31173-y},
issn = {2041-1723},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Nat Commun},
volume = {13},
number = {1},
pages = {3560},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.
Roovers Martine L, Labar Geoffray, Wolff Philippe, Feller Andre, Elder Dany Van, Soin Romuald, Gueydan Cyril, Kruys Veronique, Droogmans Louis
In: RNA, vol. 28, iss. 9, pp. 1185-1196, 2022, ISSN: 1469-9001.
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{pmid35710145b,
title = {The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA},
author = {Martine L Roovers and Geoffray Labar and Philippe Wolff and Andre Feller and Dany Van Elder and Romuald Soin and Cyril Gueydan and Veronique Kruys and Louis Droogmans},
doi = {10.1261/rna.079131.122},
issn = {1469-9001},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {RNA},
volume = {28},
issue = {9},
pages = {1185-1196},
abstract = {A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) N-terminal domain.},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) N-terminal domain.
Libre C, Seissler T, Guerrero S, Batisse J, Verriez C, Stupfler B, Gilmer O, Cabrera-Rodriguez R, Weber M, Valenzuela-Fernandez A, Cimarelli A, Etienne L, Marquet R, Paillart J C
A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor Journal Article
In: Biomedicines, vol. 10, no. 1, pp. 13, 2022.
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{Libre2022,
title = {A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor},
author = {C Libre and T Seissler and S Guerrero and J Batisse and C Verriez and B Stupfler and O Gilmer and R Cabrera-Rodriguez and M Weber and A Valenzuela-Fernandez and A Cimarelli and L Etienne and R Marquet and J C Paillart},
url = {https://www.mdpi.com/2227-9059/10/1/13},
doi = {10.1101/2021.01.13.426487},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Biomedicines},
volume = {10},
number = {1},
pages = {13},
abstract = {The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular cytosine deaminases APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutations during reverse transcription. Vif counteracts A3G by several non-redundant mechanisms (transcription, translation and protein degradation) that concur in reducing the levels of A3G in cell and in preventing its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5-untranslated region (5-UTR) of A3G mRNA. Extensive mutagenesis of A3G 5-UTR, combined with an analysis of their translational effect in transfected cells, indicated that the uORF represses A3G translation and that A3G mRNA is translated through a dual leaky-scanning and re-initiation mechanism. Interestingly, the uORF is also mandatory for the Vif-mediated repression of A3G translation. Furthermore, we showed that the redirection of A3G mRNA into stress granules was dependent not only on Vif, but also on the uORF. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific motif to repress its translation.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular cytosine deaminases APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutations during reverse transcription. Vif counteracts A3G by several non-redundant mechanisms (transcription, translation and protein degradation) that concur in reducing the levels of A3G in cell and in preventing its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5-untranslated region (5-UTR) of A3G mRNA. Extensive mutagenesis of A3G 5-UTR, combined with an analysis of their translational effect in transfected cells, indicated that the uORF represses A3G translation and that A3G mRNA is translated through a dual leaky-scanning and re-initiation mechanism. Interestingly, the uORF is also mandatory for the Vif-mediated repression of A3G translation. Furthermore, we showed that the redirection of A3G mRNA into stress granules was dependent not only on Vif, but also on the uORF. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific motif to repress its translation.
Desgranges E., Barrientos L., Herrgott L., Marzi S., Toledo-Arana A., Moreau K., Vandenesch F., Romby P., Caldelari I.
In: Mol Microbiol, 2022, ISBN: 34783400, (1365-2958 (Electronic) 0950-382X (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{nokey,
title = {The 3'UTR-derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose-6-phosphate uptake in Staphylococcus aureus},
author = {E. Desgranges and L. Barrientos and L. Herrgott and S. Marzi and A. Toledo-Arana and K. Moreau and F. Vandenesch and P. Romby and I. Caldelari},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34783400},
doi = {10.1111/mmi.14845},
isbn = {34783400},
year = {2022},
date = {2022-01-01},
urldate = {2021-01-01},
journal = {Mol Microbiol},
abstract = {Staphylococcus aureus RsaG is a 3' untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5' to 3' end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5' end of RsaG, leading to its accumulation. RsaG together with uhpT are induced when bacteria are internalized into host cells or in presence of mucus-secreting cells. Using MS2 affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, to degrade, or to repress translation of its mRNA targets. While RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors a sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.},
note = {1365-2958 (Electronic)
0950-382X (Linking)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus RsaG is a 3' untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5' to 3' end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5' end of RsaG, leading to its accumulation. RsaG together with uhpT are induced when bacteria are internalized into host cells or in presence of mucus-secreting cells. Using MS2 affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, to degrade, or to repress translation of its mRNA targets. While RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors a sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.
Camara A., Lavanant A. C., Abe J., Desforges H. L., Alexandre Y. O., Girardi E., Igamberdieva Z., Asano K., Tanaka M., Hehlgans T., Pfeffer K., Pfeffer S., Mueller S. N., Stein J. V., Mueller C. G.
CD169(+) macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling Journal Article
In: Proc Natl Acad Sci U S A, vol. 119, no. 3, pp. e2108540119, 2022, ISBN: 35031565, (1091-6490 (Electronic) 0027-8424 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: PFEFFER, Team-Mueller, Unité ARN
@article{nokey,
title = {CD169(+) macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling},
author = {A. Camara and A. C. Lavanant and J. Abe and H. L. Desforges and Y. O. Alexandre and E. Girardi and Z. Igamberdieva and K. Asano and M. Tanaka and T. Hehlgans and K. Pfeffer and S. Pfeffer and S. N. Mueller and J. V. Stein and C. G. Mueller},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35031565},
isbn = {35031565},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {119},
number = {3},
pages = {e2108540119},
abstract = {CD169(+) macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169(+) macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTbeta) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTbetaR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTbetaR revealed that both receptors contribute equally to LN CD169(+) macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8(+) T cells. Taken together, the data provide evidence that CD169(+) macrophage differentiation in LN and spleen requires dual signals from LTbetaR and RANK with implications for the immune response.},
note = {1091-6490 (Electronic)
0027-8424 (Linking)
Journal Article},
keywords = {PFEFFER, Team-Mueller, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
CD169(+) macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169(+) macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTbeta) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTbetaR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTbetaR revealed that both receptors contribute equally to LN CD169(+) macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8(+) T cells. Taken together, the data provide evidence that CD169(+) macrophage differentiation in LN and spleen requires dual signals from LTbetaR and RANK with implications for the immune response.
Costa P. J. Da, Hamdane M., Buee L., Martin F.
Tau mRNA Metabolism in Neurodegenerative Diseases: A Tangle Journey Journal Article
In: Biomedicines, vol. 10, no. 2, pp. 241, 2022.
Abstract | Links | BibTeX | Tags: ERIANI, mRNA metabolism, Neurodegenerative Diseases, tau protein, Translation, Unité ARN
@article{nokey,
title = {Tau mRNA Metabolism in Neurodegenerative Diseases: A Tangle Journey},
author = {P. J. Da Costa and M. Hamdane and L. Buee and F. Martin},
url = {https://www.mdpi.com/2227-9059/10/2/241/htm},
doi = {10.3390/biomedicines10020241},
year = {2022},
date = {2022-01-01},
journal = {Biomedicines},
volume = {10},
number = {2},
pages = {241},
abstract = {Tau proteins are known to be mainly involved in regulation of microtubule dynamics. Besides this function, which is critical for axonal transport and signal transduction, tau proteins also have other roles in neurons. Moreover, tau proteins are turned into aggregates and consequently trigger many neurodegenerative diseases termed tauopathies, of which Alzheimerﾒs disease (AD) is the figurehead. Such pathological aggregation processes are critical for the onset of these diseases. Among the various causes of tau protein pathogenicity, abnormal tau mRNA metabolism, expression and dysregulation of tau post-translational modifications are critical steps. Moreover, the relevance of tau function to general mRNA metabolism has been highlighted recently in tauopathies. In this review, we mainly focus on how mRNA metabolism impacts the onset and development of tauopathies. Thus, we intend to portray how mRNA metabolism of, or mediated by, tau is associated with neurodegenerative diseases.},
keywords = {ERIANI, mRNA metabolism, Neurodegenerative Diseases, tau protein, Translation, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Tau proteins are known to be mainly involved in regulation of microtubule dynamics. Besides this function, which is critical for axonal transport and signal transduction, tau proteins also have other roles in neurons. Moreover, tau proteins are turned into aggregates and consequently trigger many neurodegenerative diseases termed tauopathies, of which Alzheimerﾒs disease (AD) is the figurehead. Such pathological aggregation processes are critical for the onset of these diseases. Among the various causes of tau protein pathogenicity, abnormal tau mRNA metabolism, expression and dysregulation of tau post-translational modifications are critical steps. Moreover, the relevance of tau function to general mRNA metabolism has been highlighted recently in tauopathies. In this review, we mainly focus on how mRNA metabolism impacts the onset and development of tauopathies. Thus, we intend to portray how mRNA metabolism of, or mediated by, tau is associated with neurodegenerative diseases.
Gilmer O., Mailler E., Paillart J. C., Mouhand A., Tisne C., Mak J., Smyth R. P., Marquet R., Vivet-Boudou V.
Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55(Gag) and its maturation products Journal Article
In: RNA Biol, vol. 19, no. 1, pp. 191-205, 2022, ISBN: 35067194, (1555-8584 (Electronic) 1547-6286 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{nokey,
title = {Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55(Gag) and its maturation products},
author = {O. Gilmer and E. Mailler and J. C. Paillart and A. Mouhand and C. Tisne and J. Mak and R. P. Smyth and R. Marquet and V. Vivet-Boudou},
url = {https://www.tandfonline.com/doi/full/10.1080/15476286.2021.2021677},
isbn = {35067194},
year = {2022},
date = {2022-01-01},
journal = {RNA Biol},
volume = {19},
number = {1},
pages = {191-205},
abstract = {Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55(Gag) precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55(Gag) or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55(Gag) and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55(Gag) maturation products on the gRNA structural maturation.},
note = {1555-8584 (Electronic)
1547-6286 (Linking)
Journal Article},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55(Gag) precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55(Gag) or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55(Gag) and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55(Gag) maturation products on the gRNA structural maturation.
Chan D. L., Rudinger-Thirion J., Frugier M., Riley L. G., Ho G., Kothur K., Mohammad S. S.
A case of QARS1 associated epileptic encephalopathy and review of epilepsy in aminoacyl-tRNA synthetase disorders Journal Article
In: Brain Dev, vol. 44, no. 2, pp. 142-147, 2022, ISBN: 34774383, (1872-7131 (Electronic) 0387-7604 (Linking) Case Reports).
Abstract | Links | BibTeX | Tags: FRUGIER, Unité ARN
@article{nokey,
title = {A case of QARS1 associated epileptic encephalopathy and review of epilepsy in aminoacyl-tRNA synthetase disorders},
author = {D. L. Chan and J. Rudinger-Thirion and M. Frugier and L. G. Riley and G. Ho and K. Kothur and S. S. Mohammad},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34774383},
doi = {10.1016/j.braindev.2021.10.009},
isbn = {34774383},
year = {2022},
date = {2022-01-01},
journal = {Brain Dev},
volume = {44},
number = {2},
pages = {142-147},
abstract = {INTRODUCTION: Mutations in QARS1, which encodes human glutaminyl-tRNA synthetase, have been associated with epilepsy, developmental regression, progressive microcephaly and cerebral atrophy. Epilepsy caused by variants in QARS1 is usually drug-resistant and intractable. Childhood onset epilepsy is also reported in various aminoacyl-tRNA synthetase disorders. We describe a case with a milder neurological phenotype than previously reported with QARS1 variants and review the seizure associations with aminoacyl-tRNA synthetase disorders. CASE REPORT: The patient is a 4-year-old girl presenting at 6 weeks of age with orofacial dyskinesia and hand stereotypies. She developed focal seizures at 7 months of age. Serial electroencephalograms showed shifting focality. Her seizures were controlled after introduction of carbamazepine. Progress MRI showed very mild cortical volume loss without myelination abnormalities or cerebellar atrophy. She was found to have novel compound heterozygous variants in QARS1 (NM_005051.2): c.[1132C > T];[1574G > A], p.[(Arg378Cys)];[(Arg525Gln)] originally classified as "variants of uncertain significance" and later upgraded to "likely pathogenic" based on functional testing and updated variant database review. Functional testing showed reduced solubility of the corresponding QARS1 mutants in vitro, but only mild two-fold loss in catalytic efficiency with the c.1132C > T variant and no noted change in tRNA(Gln) aminoacylation with the c.1574G > A variant. CONCLUSION: We describe two QARS1 variants associated with overall conserved tRNA aminoacylation activity but characterized by significantly reduced QARS protein solubility, resulting in a milder clinical phenotype. 86% of previous patients reported with QARS1 had epilepsy and 79% were pharmaco-resistant. We also summarise literature regarding epilepsy in aminoacyl-tRNA synthetase disorders, which is also often early onset, severe and drug-refractory.},
note = {1872-7131 (Electronic)
0387-7604 (Linking)
Case Reports},
keywords = {FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
INTRODUCTION: Mutations in QARS1, which encodes human glutaminyl-tRNA synthetase, have been associated with epilepsy, developmental regression, progressive microcephaly and cerebral atrophy. Epilepsy caused by variants in QARS1 is usually drug-resistant and intractable. Childhood onset epilepsy is also reported in various aminoacyl-tRNA synthetase disorders. We describe a case with a milder neurological phenotype than previously reported with QARS1 variants and review the seizure associations with aminoacyl-tRNA synthetase disorders. CASE REPORT: The patient is a 4-year-old girl presenting at 6 weeks of age with orofacial dyskinesia and hand stereotypies. She developed focal seizures at 7 months of age. Serial electroencephalograms showed shifting focality. Her seizures were controlled after introduction of carbamazepine. Progress MRI showed very mild cortical volume loss without myelination abnormalities or cerebellar atrophy. She was found to have novel compound heterozygous variants in QARS1 (NM_005051.2): c.[1132C > T];[1574G > A], p.[(Arg378Cys)];[(Arg525Gln)] originally classified as "variants of uncertain significance" and later upgraded to "likely pathogenic" based on functional testing and updated variant database review. Functional testing showed reduced solubility of the corresponding QARS1 mutants in vitro, but only mild two-fold loss in catalytic efficiency with the c.1132C > T variant and no noted change in tRNA(Gln) aminoacylation with the c.1574G > A variant. CONCLUSION: We describe two QARS1 variants associated with overall conserved tRNA aminoacylation activity but characterized by significantly reduced QARS protein solubility, resulting in a milder clinical phenotype. 86% of previous patients reported with QARS1 had epilepsy and 79% were pharmaco-resistant. We also summarise literature regarding epilepsy in aminoacyl-tRNA synthetase disorders, which is also often early onset, severe and drug-refractory.
Andre A. C., Laborde M., Marteyn B. S.
The battle for oxygen during bacterial and fungal infections Journal Article
In: Trends Microbiol, vol. 30, iss. 7, pp. 643-653, 2022, ISBN: 35131160, (1878-4380 (Electronic) 0966-842X (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: MARTEYN, Unité ARN
@article{nokey,
title = {The battle for oxygen during bacterial and fungal infections},
author = {A. C. Andre and M. Laborde and B. S. Marteyn},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35131160},
doi = {10.1016/j.tim.2022.01.002},
isbn = {35131160},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Trends Microbiol},
volume = {30},
issue = {7},
pages = {643-653},
abstract = {Bacterial and fungal pathogens face various microenvironmental conditions during infection. In addition to acidosis, nutrient consumption, and hypercapnia, pathogen infections are associated with hypoxia, which is induced by bacterial and fungal respiration during the formation of foci of infection or biofilms. Consequently, the in vivo interaction between host immune cells and pathogens is anticipated to occur mainly under low-oxygen conditions. Various infectious disease models have reported that pathogens benefit from hypoxia, which dampens the oxygen-dependent antimicrobial activities of macrophages and neutrophils, such as the production of reactive oxygen species (ROS). Due to their dual respiration capacity (aerobic and anaerobic) or phenotypical adaptation (e.g., dormancy), pathogens have the capacity to survive and disseminate in the absence of oxygen. In addition, hypoxia modulates various mechanisms of pathogen virulence, promoting the dissemination of pathogens. Further investigations are still required to evaluate the relative importance of oxygen on the capacity of pathogens to invade and colonize host organs and to better understand alternative strategies developed by immune cells to circumvent pathogen dissemination in the absence of oxygen. Addressing this important and fundamental question in various models of infection may direct the development of innovative therapeutic strategies.},
note = {1878-4380 (Electronic)
0966-842X (Linking)
Journal Article
Review},
keywords = {MARTEYN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Bacterial and fungal pathogens face various microenvironmental conditions during infection. In addition to acidosis, nutrient consumption, and hypercapnia, pathogen infections are associated with hypoxia, which is induced by bacterial and fungal respiration during the formation of foci of infection or biofilms. Consequently, the in vivo interaction between host immune cells and pathogens is anticipated to occur mainly under low-oxygen conditions. Various infectious disease models have reported that pathogens benefit from hypoxia, which dampens the oxygen-dependent antimicrobial activities of macrophages and neutrophils, such as the production of reactive oxygen species (ROS). Due to their dual respiration capacity (aerobic and anaerobic) or phenotypical adaptation (e.g., dormancy), pathogens have the capacity to survive and disseminate in the absence of oxygen. In addition, hypoxia modulates various mechanisms of pathogen virulence, promoting the dissemination of pathogens. Further investigations are still required to evaluate the relative importance of oxygen on the capacity of pathogens to invade and colonize host organs and to better understand alternative strategies developed by immune cells to circumvent pathogen dissemination in the absence of oxygen. Addressing this important and fundamental question in various models of infection may direct the development of innovative therapeutic strategies.
Bernacchi S.
Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview Journal Article
In: Viruses, vol. 14, no. 2, 2022, ISBN: 35215917, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article Review Research Support, Non-U.S. Gov't).
Abstract | Links | BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{nokey,
title = {Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview},
author = {S. Bernacchi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35215917},
doi = {10.3390/v14020324},
isbn = {35215917},
year = {2022},
date = {2022-01-01},
journal = {Viruses},
volume = {14},
number = {2},
abstract = {Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article
Review
Research Support, Non-U.S. Gov't},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.
Sosnowski P, Tidu A, Eriani G, Westhof E, Martin F
Correlated sequence signatures are present within the genomic 5'UTR RNA and NSP1 protein in coronaviruses Journal Article
In: Rna, vol. 28, iss. 5, pp. 729-741, 2022, ISBN: 35236777, (1469-9001 (Electronic) 1355-8382 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ERIANI, MARTIN, Unité ARN, WESTHOF
@article{nokey,
title = {Correlated sequence signatures are present within the genomic 5'UTR RNA and NSP1 protein in coronaviruses},
author = {P Sosnowski and A Tidu and G Eriani and E Westhof and F Martin},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35236777},
doi = {10.1261/rna.078972.121},
isbn = {35236777},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Rna},
volume = {28},
issue = {5},
pages = {729-741},
abstract = {The 5'UTR part of coronavirus genomes plays key roles in the viral replication cycle and the translation of the viral mRNAs. The first 75-80 nucleotides, also called the leader sequence, are identical for the genomic mRNA and for the subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5'UTR segment and the non-structural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5'UTR RNA segment and the NSP1 protein have been done for several coronaviruses with special attention to the betacoronaviruses. The conclusions are (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures strongly correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein indicating a co-evolution of 5'UTR with NSP1 in betacoronaviruses. Experimental mutational data on 5'UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support those conclusions and show that the N-terminal half of the NSP1 protein contains conserved key residues that are essential for evasion to the inhibitory effect of NSP1 on translation.},
note = {1469-9001 (Electronic)
1355-8382 (Linking)
Journal Article},
keywords = {ERIANI, MARTIN, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
The 5'UTR part of coronavirus genomes plays key roles in the viral replication cycle and the translation of the viral mRNAs. The first 75-80 nucleotides, also called the leader sequence, are identical for the genomic mRNA and for the subgenomic mRNAs. Recently, it was shown that cooperative actions of a 5'UTR segment and the non-structural protein NSP1 are essential for both the inhibition of host mRNAs and for specific translation of viral mRNAs. Here, sequence analyses of both the 5'UTR RNA segment and the NSP1 protein have been done for several coronaviruses with special attention to the betacoronaviruses. The conclusions are (i) precise specific molecular signatures can be found in both the RNA and the NSP1 protein; (ii) both types of signatures strongly correlate between each other. Indeed, definite sequence motifs in the RNA correlate with sequence motifs in the protein indicating a co-evolution of 5'UTR with NSP1 in betacoronaviruses. Experimental mutational data on 5'UTR and NSP1 from SARS-CoV-2 using cell-free translation extracts support those conclusions and show that the N-terminal half of the NSP1 protein contains conserved key residues that are essential for evasion to the inhibitory effect of NSP1 on translation.