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RNA architecture and reactivity
Publications
2021
Saeb Sepideh, Ravanshad Mehrdad, Pourkarim Mahmoud Reza, Daouad Fadoua, Baesi Kazem, Rohr Olivier, Wallet Clémentine, Schwartz Christian
Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy Journal Article
In: Virol J, vol. 18, no. 1, pp. 107, 2021, ISSN: 1743-422X.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid34059075,
title = {Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy},
author = {Sepideh Saeb and Mehrdad Ravanshad and Mahmoud Reza Pourkarim and Fadoua Daouad and Kazem Baesi and Olivier Rohr and Clémentine Wallet and Christian Schwartz},
doi = {10.1186/s12985-021-01584-2},
issn = {1743-422X},
year = {2021},
date = {2021-05-01},
urldate = {2021-05-01},
journal = {Virol J},
volume = {18},
number = {1},
pages = {107},
abstract = {Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.
Vicens Q, Bochler A, Jobe A, Frank J, Hashem Y
Interaction Networks of Ribosomal Expansion Segments in Kinetoplastids Book Chapter
In: vol. 96, pp. 433-450, Subcellular Biochemistry, 2021.
Abstract | Links | BibTeX | Tags: Eukaryotic translation, Expansion segment, Kinetoplastid parasite, Ribosomal RNA, Ribosome structure, Unité ARN
@inbook{Vicens2021,
title = {Interaction Networks of Ribosomal Expansion Segments in Kinetoplastids },
author = {Q Vicens and A Bochler and A Jobe and J Frank and Y Hashem },
url = {https://pubmed.ncbi.nlm.nih.gov/33252739/},
doi = {10.1007/978-3-030-58971-4_13 },
year = {2021},
date = {2021-01-01},
volume = {96},
pages = {433-450},
publisher = {Subcellular Biochemistry},
abstract = {Expansion segments (ES) are insertions of a few to hundreds of nucleotides at discrete locations on eukaryotic ribosomal RNA (rRNA) chains. Some cluster around ‘hot spots’ involved in translation regulation and some may participate in biogenesis. Whether ES play the same roles in different organisms is currently unclear, especially since their size may vary dramatically from one species to another and very little is known about their functions. Most likely, ES variation is linked to adaptation to a particular environment. In this chapter, we compare the interaction networks of ES from four kinetoplastid parasites, which have evolved in diverse insect vectors and mammalian hosts: Trypanosoma cruzi, Trypanosoma brucei, Leishmania donovani and Leishmania major. Here, we comparatively analyze ribosome structures from these representative kinetoplastids and ascertain meaningful structural differences from mammalian ribosomes. We base our analysis on sequence alignments and three-dimensional structures of 80S ribosomes solved by cryo-electron microscopy (cryo-EM). Striking differences in size are observed between ribosomes of different parasites, indicating that not all ES are expanded equally. Larger ES are not always matched by large surrounding ES or proteins extensions in their vicinity, a particularity that may lead to clues about their biological function. ES display different species-specific patterns of conservation, which underscore the density of their interaction network at the surface of the ribosome. Making sense of the conservation patterns of ES is part of a global effort to lay the basis for functional studies aimed at discovering unique kinetoplastid-specific sites suitable for therapeutic applications against these human and often animal pathogens.},
keywords = {Eukaryotic translation, Expansion segment, Kinetoplastid parasite, Ribosomal RNA, Ribosome structure, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Expansion segments (ES) are insertions of a few to hundreds of nucleotides at discrete locations on eukaryotic ribosomal RNA (rRNA) chains. Some cluster around ‘hot spots’ involved in translation regulation and some may participate in biogenesis. Whether ES play the same roles in different organisms is currently unclear, especially since their size may vary dramatically from one species to another and very little is known about their functions. Most likely, ES variation is linked to adaptation to a particular environment. In this chapter, we compare the interaction networks of ES from four kinetoplastid parasites, which have evolved in diverse insect vectors and mammalian hosts: Trypanosoma cruzi, Trypanosoma brucei, Leishmania donovani and Leishmania major. Here, we comparatively analyze ribosome structures from these representative kinetoplastids and ascertain meaningful structural differences from mammalian ribosomes. We base our analysis on sequence alignments and three-dimensional structures of 80S ribosomes solved by cryo-electron microscopy (cryo-EM). Striking differences in size are observed between ribosomes of different parasites, indicating that not all ES are expanded equally. Larger ES are not always matched by large surrounding ES or proteins extensions in their vicinity, a particularity that may lead to clues about their biological function. ES display different species-specific patterns of conservation, which underscore the density of their interaction network at the surface of the ribosome. Making sense of the conservation patterns of ES is part of a global effort to lay the basis for functional studies aimed at discovering unique kinetoplastid-specific sites suitable for therapeutic applications against these human and often animal pathogens.
Bec G, Ennifar E
switchSENSE Technology for Analysis of DNA Polymerase Kinetics Book Chapter
In: Rederstorff, M (Ed.): vol. 2247, pp. 145-153, Springer Protocols, Humana Press, New York, NY, Small Non-Coding RNAs, 2021.
Abstract | BibTeX | Tags: Biosensor, DNA polymerase, ENNIFAR, HIV reverse transcriptase, Kinetics, switchSENSE technology, Unité ARN
@inbook{Bec2021,
title = {switchSENSE Technology for Analysis of DNA Polymerase Kinetics },
author = {G Bec and E Ennifar
},
editor = {M Rederstorff},
year = {2021},
date = {2021-01-01},
journal = {Methods in Molecular Biology },
volume = {2247},
pages = {145-153},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
edition = {Small Non-Coding RNAs},
series = {Methods in Molecular Biology},
abstract = {The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme. },
keywords = {Biosensor, DNA polymerase, ENNIFAR, HIV reverse transcriptase, Kinetics, switchSENSE technology, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme.
Lalaouna D, Fochesato S, Harir M, Ortet P, Schmitt-Kopplin P, Heulin T, Achouak W
In: Microorganisms, vol. 9, no. 2, pp. 250, 2021.
Abstract | Links | BibTeX | Tags: GacA-dependent, nutritional stress, Pseudomonas brassicacearum, ROMBY, Rsm sRNAs, sRNAs stability, Unité ARN
@article{,
title = {Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments},
author = {D Lalaouna and S Fochesato and M Harir and P Ortet and P Schmitt-Kopplin and T Heulin and W Achouak},
url = {https://www.mdpi.com/2076-2607/9/2/250},
doi = {10.3390/microorganisms9020250},
year = {2021},
date = {2021-01-01},
journal = {Microorganisms},
volume = {9},
number = {2},
pages = {250},
abstract = {In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.},
keywords = {GacA-dependent, nutritional stress, Pseudomonas brassicacearum, ROMBY, Rsm sRNAs, sRNAs stability, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.
Li G, Eriani G, Wang E D, Zhou X L
Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease Journal Article
In: Sci China Life Sci, vol. 64, no. 10, pp. 1645-1660, 2021, ISBN: 33515434, (1869-1889 (Electronic) 1674-7305 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: aminoacyl-tRNA synthetase (aaRS), central nervous system (CNS), ERIANI, Protein Biosynthesis, translation initiation, tRNA, Unité ARN
@article{,
title = {Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease},
author = {G Li and G Eriani and E D Wang and X L Zhou},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33515434},
doi = {10.1007/s11427-020-1838-2},
isbn = {33515434},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Sci China Life Sci},
volume = {64},
number = {10},
pages = {1645-1660},
abstract = {Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5A>G, p.D2G; c.1367C>T, p.S456L; c.1535G>A, p.R512Q and c.1846_1847del, p. Y616Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-tRNA synthetase (hArgRS), are linked to Pelizaeus-Merzbacher-like disease (PMLD) with unclear pathogenesis. Among these mutations, c.5A>G is the most extensively reported mutation, leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS. Here, we showed the detrimental effects of R512Q substitution and DeltaC mutations on the structure and function of hArgRS, while the most frequent mutation c.5A>G, p.D2G acted in a different manner without impairing hArgRS activity. The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.},
note = {1869-1889 (Electronic)
1674-7305 (Linking)
Journal Article},
keywords = {aminoacyl-tRNA synthetase (aaRS), central nervous system (CNS), ERIANI, Protein Biosynthesis, translation initiation, tRNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mutations of the genes encoding aminoacyl-tRNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5A>G, p.D2G; c.1367C>T, p.S456L; c.1535G>A, p.R512Q and c.1846_1847del, p. Y616Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-tRNA synthetase (hArgRS), are linked to Pelizaeus-Merzbacher-like disease (PMLD) with unclear pathogenesis. Among these mutations, c.5A>G is the most extensively reported mutation, leading to a p.D2G mutation in the N-terminal extension of the long-form hArgRS. Here, we showed the detrimental effects of R512Q substitution and DeltaC mutations on the structure and function of hArgRS, while the most frequent mutation c.5A>G, p.D2G acted in a different manner without impairing hArgRS activity. The nucleotide substitution c.5A>G reduced translation of hArgRS mRNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.
Mercier N, Prévost K, Massé E, Romby P, Caldelari I, Lalaouna D
MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria Journal Article
In: J Vis Exp, pp. e61731, 2021.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{Mercier2021,
title = {MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria},
author = {N Mercier and K Prévost and E Massé and P Romby and I Caldelari and D Lalaouna},
url = {https://www.jove.com/t/61731/ms2-affinity-purification-coupled-with-rna-sequencing-gram-positive},
doi = {10.3791/61731},
year = {2021},
date = {2021-01-01},
journal = {J Vis Exp},
pages = {e61731},
abstract = {Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5’ extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5’ extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.
Catalan-Moreno A, Cela M, Menendez-Gil P, Irurzun N, Caballero C J, Caldelari I, Toledo-Arana A
RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures Journal Article
In: Nucleic Acids Res, pp. on press, 2021, ISBN: 33660769, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{Catalan-Moreno2021,
title = {RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures},
author = {A Catalan-Moreno and M Cela and P Menendez-Gil and N Irurzun and C J Caballero and I Caldelari and A Toledo-Arana},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33660769},
doi = {0.1093/nar/gkab117},
isbn = {33660769},
year = {2021},
date = {2021-01-01},
journal = {Nucleic Acids Res},
pages = {on press},
abstract = {Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5'UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22 degrees C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37 degrees C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37 degrees C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5'UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22 degrees C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37 degrees C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37 degrees C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.
Pitchai F, Chameettachal A, Vivet-Boudou V, Ali L M, Pillai V N, Krishnan A, Bernacchi S, Mustafa F, R Marquet, Rizvi T A
Identification of Pr78Gag binding sites on the Mason-Pfizer monkey virus genomic RNA packaging determinants Journal Article
In: J Mol Biol, vol. 433, no. 10, pp. 166923, 2021.
Abstract | Links | BibTeX | Tags: MARQUET, Retroviruses Gag-RNA interactions Purines Footprinting hSHAPE, Unité ARN
@article{F2021,
title = {Identification of Pr78Gag binding sites on the Mason-Pfizer monkey virus genomic RNA packaging determinants},
author = {F Pitchai and A Chameettachal and V Vivet-Boudou and L M Ali and V N Pillai and A Krishnan and S Bernacchi and F Mustafa and Marquet R and T A Rizvi},
url = {https://www.sciencedirect.com/science/article/pii/S0022283621001224?via%3Dihub},
doi = {10.1016/j.jmb.2021.166923},
year = {2021},
date = {2021-01-01},
journal = {J Mol Biol},
volume = {433},
number = {10},
pages = {166923},
abstract = {How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.},
keywords = {MARQUET, Retroviruses Gag-RNA interactions Purines Footprinting hSHAPE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.
Busienne C, Marquet R, Paillart J C, Bernacchi S
Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role Journal Article
In: Int. J. Mol. Sci., vol. 22, no. 6, pp. 2871, 2021.
Abstract | Links | BibTeX | Tags: HIV-1, MARQUET, PAILLART, post-translational modifications, Pr55Gag precursor, retroviral Gag precursors, retroviral life cycle, Unité ARN
@article{C2021b,
title = {Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role},
author = {C Busienne and R Marquet and J C Paillart and S Bernacchi},
url = {https://www.mdpi.com/1422-0067/22/6/2871},
doi = {10.3390/ijms22062871},
year = {2021},
date = {2021-01-01},
journal = {Int. J. Mol. Sci.},
volume = {22},
number = {6},
pages = {2871},
abstract = {Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.},
keywords = {HIV-1, MARQUET, PAILLART, post-translational modifications, Pr55Gag precursor, retroviral Gag precursors, retroviral life cycle, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Protein post-translational modifications (PTMs) play key roles in eukaryotes since they finely regulate numerous mechanisms used to diversify the protein functions and to modulate their signaling networks. Besides, these chemical modifications also take part in the viral hijacking of the host, and also contribute to the cellular response to viral infections. All domains of the human immunodeficiency virus type 1 (HIV-1) Gag precursor of 55-kDa (Pr55Gag), which is the central actor for viral RNA specific recruitment and genome packaging, are post-translationally modified. In this review, we summarize the current knowledge about HIV-1 Pr55Gag PTMs such as myristoylation, phosphorylation, ubiquitination, sumoylation, methylation, and ISGylation in order to figure out how these modifications affect the precursor functions and viral replication. Indeed, in HIV-1, PTMs regulate the precursor trafficking between cell compartments and its anchoring at the plasma membrane, where viral assembly occurs. Interestingly, PTMs also allow Pr55Gag to hijack the cell machinery to achieve viral budding as they drive recognition between viral proteins or cellular components such as the ESCRT machinery. Finally, we will describe and compare PTMs of several other retroviral Gag proteins to give a global overview of their role in the retroviral life cycle.
Chameettachal A, Vivet-Boudou V, Pitchai F N, N Pillai V, Ali L M, Krishnan A, Bernacchi S, Mustafa F, Marquet R, Rizvi T A
A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag Journal Article
In: Nucleic Acids Res, vol. 49, no. 8, pp. 4668-4688, 2021.
BibTeX | Tags: MARQUET, PAILLART, Unité ARN
@article{Chameettachal2021,
title = {A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag},
author = {A Chameettachal and V Vivet-Boudou and F N Pitchai and Pillai V N and L M Ali and A Krishnan and S Bernacchi and F Mustafa and R Marquet and T A Rizvi},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {8},
pages = {4668-4688},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Westhof E, Leontis N B
An RNA-centric historical narrative around the Protein Data Bank Journal Article
In: J Biol Chem, vol. 296, pp. 100555, 2021, ISBN: 33744291, (1083-351X (Electronic) 0021-9258 (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: Computational Biology, Databases, modelling, Protein Data Bank, RNA, Structural biology, Unité ARN, WESTHOF
@article{Westhof2021,
title = {An RNA-centric historical narrative around the Protein Data Bank},
author = {E Westhof and N B Leontis},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33744291},
doi = {10.1016/j.jbc.2021.100555},
isbn = {33744291},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {J Biol Chem},
volume = {296},
pages = {100555},
abstract = {Some of the amazing contributions brought to the scientific community by the PDB are described. The focus is on nucleic acid structures with a bias towards RNA. The evolution and key roles in science of the PDB and other structural databases for nucleic acids illustrate how small initial ideas can become huge and indispensable resources with the unflinching willingness of scientists to cooperate globally. The progress in the understanding of the molecular interactions driving RNA architectures followed the rapid increase in RNA structures in the PDB. That increase was consecutive to improvements in chemical synthesis and purification of RNA molecules, as well as in biophysical methods for structure determination and computer technology. The RNA modeling efforts from the early beginnings are also described together with their links to the state of structural knowledge and technological development. Structures of RNA and of its assemblies are physical objects which, together with genomic data, allow us to integrate present-day biological functions and the historical evolution in all living species on earth.},
note = {1083-351X (Electronic)
0021-9258 (Linking)
Journal Article
Review},
keywords = {Computational Biology, Databases, modelling, Protein Data Bank, RNA, Structural biology, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Some of the amazing contributions brought to the scientific community by the PDB are described. The focus is on nucleic acid structures with a bias towards RNA. The evolution and key roles in science of the PDB and other structural databases for nucleic acids illustrate how small initial ideas can become huge and indispensable resources with the unflinching willingness of scientists to cooperate globally. The progress in the understanding of the molecular interactions driving RNA architectures followed the rapid increase in RNA structures in the PDB. That increase was consecutive to improvements in chemical synthesis and purification of RNA molecules, as well as in biophysical methods for structure determination and computer technology. The RNA modeling efforts from the early beginnings are also described together with their links to the state of structural knowledge and technological development. Structures of RNA and of its assemblies are physical objects which, together with genomic data, allow us to integrate present-day biological functions and the historical evolution in all living species on earth.
Husser C, Dentz N, Ryckelynck M
Structure-Switching RNAs: From Gene Expression Regulation to Small Molecule Detection Journal Article
In: Small Structures, vol. 2, no. 4, pp. 2000132, 2021.
Abstract | Links | BibTeX | Tags: biosensing, Gene Expression Regulation, riboswitches, RNA aptamers, RYCKELYNCK, Synthetic Biology, Unité ARN
@article{C2021c,
title = {Structure-Switching RNAs: From Gene Expression Regulation to Small Molecule Detection},
author = {C Husser and N Dentz and M Ryckelynck},
url = {https://doi.org/10.1002/sstr.202000132},
doi = {10.1002/sstr.202000132},
year = {2021},
date = {2021-01-01},
journal = {Small Structures},
volume = {2},
number = {4},
pages = {2000132},
abstract = {RNA is instrumental to cell life in many aspects, especially gene expression regulation. Among the various known regulatory RNAs, riboswitches are particularly interesting cis‐acting molecules as they do not need cellular factor to achieve their function and are therefore highly portable from one organism to the other. These molecules usually found in the 5′ untranslated region of bacterial messenger RNAs are able to specifically sense a target ligand via an aptamer domain prior to transmitting this recognition event to an expression platform that turns on, or off, the expression of downstream genes. In addition to their obvious scientific interest, these modular molecules can also serve for the development of synthetic RNA devices with applications ranging from the control of transgene expression in gene therapy to the specific biosensing of small molecules. The engineering of such nanomachines is greatly facilitated by the proper understanding of their structure as well as the introduction of new technologies. Herein, a general overview of the current knowledge on natural riboswitches prior to explaining the main strategies used to develop new synthetic structure‐switching molecules (riboswitches or biosensors) controlled by small molecules is given.},
keywords = {biosensing, Gene Expression Regulation, riboswitches, RNA aptamers, RYCKELYNCK, Synthetic Biology, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA is instrumental to cell life in many aspects, especially gene expression regulation. Among the various known regulatory RNAs, riboswitches are particularly interesting cis‐acting molecules as they do not need cellular factor to achieve their function and are therefore highly portable from one organism to the other. These molecules usually found in the 5′ untranslated region of bacterial messenger RNAs are able to specifically sense a target ligand via an aptamer domain prior to transmitting this recognition event to an expression platform that turns on, or off, the expression of downstream genes. In addition to their obvious scientific interest, these modular molecules can also serve for the development of synthetic RNA devices with applications ranging from the control of transgene expression in gene therapy to the specific biosensing of small molecules. The engineering of such nanomachines is greatly facilitated by the proper understanding of their structure as well as the introduction of new technologies. Herein, a general overview of the current knowledge on natural riboswitches prior to explaining the main strategies used to develop new synthetic structure‐switching molecules (riboswitches or biosensors) controlled by small molecules is given.
Stupfler B, Verriez C, Gallois-Montbrun S, Marquet R, Paillart J C
Degradation-independent inhibition of APOBEC3G by HIV-1 Vif protein Journal Article
In: Viruses, vol. 13, no. 4, pp. 617, 2021.
Abstract | Links | BibTeX | Tags: APOBEC3G, deamination, encapsidation, HIV, MARQUET, PAILLART, proteasome, RNP granules, Translation, ubiquitin, Unité ARN, vif
@article{Stupfler2021,
title = {Degradation-independent inhibition of APOBEC3G by HIV-1 Vif protein},
author = {B Stupfler and C Verriez and S Gallois-Montbrun and R Marquet and J C Paillart},
url = {https://www.mdpi.com/1999-4915/13/4/617},
doi = {10.3390/v13040617},
year = {2021},
date = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {4},
pages = {617},
abstract = {The ubiquitinproteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein.},
keywords = {APOBEC3G, deamination, encapsidation, HIV, MARQUET, PAILLART, proteasome, RNP granules, Translation, ubiquitin, Unité ARN, vif},
pubstate = {published},
tppubtype = {article}
}
The ubiquitinproteasome system plays an important role in the cell under normal physiological conditions but also during viral infections. Indeed, many auxiliary proteins from the (HIV-1) divert this system to its own advantage, notably to induce the degradation of cellular restriction factors. For instance, the HIV-1 viral infectivity factor (Vif) has been shown to specifically counteract several cellular deaminases belonging to the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 or A3) family (A3A to A3H) by recruiting an E3-ubiquitin ligase complex and inducing their polyubiquitination and degradation through the proteasome. Although this pathway has been extensively characterized so far, Vif has also been shown to impede A3s through degradation-independent processes, but research on this matter remains limited. In this review, we describe our current knowledge regarding the degradation-independent inhibition of A3s, and A3G in particular, by the HIV-1 Vif protein, the molecular mechanisms involved, and highlight important properties of this small viral protein.
Chagot M E, Quinternet M, Manival X, Lebars I
Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs Journal Article
In: Methods Mol Biol, vol. 2300, pp. 251-266, 2021, ISBN: 33792884, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: 3D structure, dynamics, ENNIFAR, NMR, RNA, Unité ARN
@article{Chagot2021,
title = {Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs},
author = {M E Chagot and M Quinternet and X Manival and I Lebars},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792884},
doi = {10.1007/978-1-0716-1386-3_19},
isbn = {33792884},
year = {2021},
date = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2300},
pages = {251-266},
abstract = {Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {3D structure, dynamics, ENNIFAR, NMR, RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.
Andre A C, Debande L, Marteyn B S
The selective advantage of facultative anaerobes relies on their unique ability to cope with changing oxygen levels during infection Journal Article
In: Cell Microbiol, vol. 23, no. 8, pp. e13338, 2021, ISBN: 33813807, (1462-5822 (Electronic) 1462-5814 (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: MARTEYN, Unité ARN
@article{Andre2021,
title = {The selective advantage of facultative anaerobes relies on their unique ability to cope with changing oxygen levels during infection},
author = {A C Andre and L Debande and B S Marteyn},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33813807},
doi = {10.1111/cmi.13338},
isbn = {33813807},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Cell Microbiol},
volume = {23},
number = {8},
pages = {e13338},
abstract = {Bacteria, including those that are pathogenic, have been generally classified according to their ability to survive and grow in the presence or absence of oxygen: aerobic and anaerobic bacteria, respectively. Strict aerobes require oxygen to grow (e.g. Neisseria), and strict anaerobes grow exclusively without, and do not survive oxygen exposure (e.g. Clostridia); aerotolerant bacteria (e.g. Lactobacilli) are insensitive to oxygen exposure. Facultative anaerobes (e.g. E. coli) have the unique ability to grow in the presence or in the absence of oxygen. and are thus well-adapted to these changing conditions which may constitute an underestimated selective advantage for infection. In the WHO antibiotic-resistant "priority pathogens" list, facultative anaerobes are overrepresented (8 among 12 listed pathogens), consistent with clinical studies performed in populations particularly susceptible to infectious diseases. Bacteria aerobic respiratory chain plays a central role in oxygen consumption, leading to the formation of hypoxic infectious sites (infectious hypoxia). Facultative anaerobes have developed a wide diversity of aerotolerance and anaerotolerance strategies in vivo. However, at a single cell level, the modulation of the intracellular oxygen level in host infected cells remains elusive and will be discussed in this review. In conclusion, the ability of facultative bacteria to evolve in the presence or the absence of oxygen is essential for their virulence strategy and constitute a selective advantage. This article is protected by copyright. All rights reserved.},
note = {1462-5822 (Electronic)
1462-5814 (Linking)
Journal Article
Review},
keywords = {MARTEYN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Bacteria, including those that are pathogenic, have been generally classified according to their ability to survive and grow in the presence or absence of oxygen: aerobic and anaerobic bacteria, respectively. Strict aerobes require oxygen to grow (e.g. Neisseria), and strict anaerobes grow exclusively without, and do not survive oxygen exposure (e.g. Clostridia); aerotolerant bacteria (e.g. Lactobacilli) are insensitive to oxygen exposure. Facultative anaerobes (e.g. E. coli) have the unique ability to grow in the presence or in the absence of oxygen. and are thus well-adapted to these changing conditions which may constitute an underestimated selective advantage for infection. In the WHO antibiotic-resistant "priority pathogens" list, facultative anaerobes are overrepresented (8 among 12 listed pathogens), consistent with clinical studies performed in populations particularly susceptible to infectious diseases. Bacteria aerobic respiratory chain plays a central role in oxygen consumption, leading to the formation of hypoxic infectious sites (infectious hypoxia). Facultative anaerobes have developed a wide diversity of aerotolerance and anaerotolerance strategies in vivo. However, at a single cell level, the modulation of the intracellular oxygen level in host infected cells remains elusive and will be discussed in this review. In conclusion, the ability of facultative bacteria to evolve in the presence or the absence of oxygen is essential for their virulence strategy and constitute a selective advantage. This article is protected by copyright. All rights reserved.
de Wijn R, Rollet K, Olieric V, Hennig O, Thome N, Nous C, Paulus C, Lorber B, Betat H, Morl M, Sauter C
Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip Journal Article
In: J Vis Exp, no. 169, 2021, ISBN: 33818565, (1940-087X (Electronic) 1940-087X (Linking) Journal Article Video-Audio Media).
Abstract | Links | BibTeX | Tags: SAUTER, Unité ARN
@article{deWijn2021,
title = {Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip},
author = {R de Wijn and K Rollet and V Olieric and O Hennig and N Thome and C Nous and C Paulus and B Lorber and H Betat and M Morl and C Sauter},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33818565},
doi = {10.3791/61972},
isbn = {33818565},
year = {2021},
date = {2021-01-01},
journal = {J Vis Exp},
number = {169},
abstract = {The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.},
note = {1940-087X (Electronic)
1940-087X (Linking)
Journal Article
Video-Audio Media},
keywords = {SAUTER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.
Injarabian L, Skerniskyte J, Gianetto Q G, Witko-Sarsat V, Marteyn B S
Reducing neutrophil exposure to oxygen allows their basal state maintenance Journal Article
In: Immunol Cell Biol, vol. 99, no. 7, pp. 782-789, 2021, ISBN: 33811670, (1440-1711 (Electronic) 0818-9641 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Activation, anoxia, hyperoxia, MARTEYN, neutrophils, Unité ARN, viability
@article{Injarabian2021,
title = {Reducing neutrophil exposure to oxygen allows their basal state maintenance},
author = {L Injarabian and J Skerniskyte and Q G Gianetto and V Witko-Sarsat and B S Marteyn},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33811670},
doi = {10.1111/imcb.12458},
isbn = {33811670},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Immunol Cell Biol},
volume = {99},
number = {7},
pages = {782-789},
abstract = {Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1-4% O2), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic, and susceptible to oxygen exposure, which induces their activation and death, through mechanisms, which remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper-activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared to neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11b(high), CD54(high), CD62L(neg)) or viability (DAPI(+)). We identified two clusters of proteins belonging to the cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and prevent the alteration cytosolic protein composition.},
note = {1440-1711 (Electronic)
0818-9641 (Linking)
Journal Article},
keywords = {Activation, anoxia, hyperoxia, MARTEYN, neutrophils, Unité ARN, viability},
pubstate = {published},
tppubtype = {article}
}
Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1-4% O2), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic, and susceptible to oxygen exposure, which induces their activation and death, through mechanisms, which remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper-activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared to neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11b(high), CD54(high), CD62L(neg)) or viability (DAPI(+)). We identified two clusters of proteins belonging to the cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and prevent the alteration cytosolic protein composition.
Velours C, Aumont-Nicaise M, Uebel S, England P, Velazquez-Campoy A, Stroebel D, Bec G, Soule P, Quetard C, Ebel C, Roussel A, Charbonnier J B, Varela P F
Macromolecular interactions in vitro, comparing classical and novel approaches Journal Article
In: Eur Biophys J, vol. 50, no. 3-4, pp. 313-330, 2021, ISBN: 33792745, (1432-1017 (Electronic) 0175-7571 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Artificial binders, Double-stranded DNA breaks repair factors, ENNIFAR, Macromolecular interactions, Molecular scale biophysics, Unité ARN
@article{,
title = {Macromolecular interactions in vitro, comparing classical and novel approaches},
author = {C Velours and M Aumont-Nicaise and S Uebel and P England and A Velazquez-Campoy and D Stroebel and G Bec and P Soule and C Quetard and C Ebel and A Roussel and J B Charbonnier and P F Varela},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792745},
doi = {10.1007/s00249-021-01517-5},
isbn = {33792745},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Eur Biophys J},
volume = {50},
number = {3-4},
pages = {313-330},
abstract = {Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.},
note = {1432-1017 (Electronic)
0175-7571 (Linking)
Journal Article},
keywords = {Artificial binders, Double-stranded DNA breaks repair factors, ENNIFAR, Macromolecular interactions, Molecular scale biophysics, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.
Alghoul F, Eriani G, Martin F
RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT Book Chapter
In: Rederstorff, M (Ed.): Methods Mol Biol, vol. 2300, pp. 241-250, Springer Protocols, Humana Press, New York, NY, 2021, ISBN: 978-1-0716-1385-6/ISSN, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Capillary electrophoresis, chemical probing, CMCT, DMS, ERIANI, Primer extension, QuSHAPE, RNA secondary structure, Unité ARN
@inbook{Alghoul2021,
title = {RNA Secondary Structure Study by Chemical Probing Methods Using DMS and CMCT},
author = {F Alghoul and G Eriani and F Martin},
editor = {M Rederstorff},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792883},
doi = {10.1007/978-1-0716-1386-3_18},
isbn = {978-1-0716-1385-6/ISSN},
year = {2021},
date = {2021-01-01},
booktitle = {Methods Mol Biol},
volume = {2300},
pages = {241-250},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {RNA folds into secondary structures that can serve in understanding various RNA functions (Weeks KM. Curr Opin Struct Biol 20(3):295-304, 2010). Chemical probing is a method that enables the characterization of RNA secondary structures using chemical reagents that specifically modify RNA nucleotides that are located in single-stranded areas. In our protocol, we used Dimethyl Sulfate (DMS) and Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluene sulfonate (CMCT) that are both base-specific modifying reagents (Behm-Ansmant I, et al. J Nucleic Acids 2011:408053, 2011). These modifications are mapped by primer extension arrests using 5' fluorescently labeled primers. In this protocol, we show a comprehensive method to identify RNA secondary structures in vitro using fluorescently labeled oligos. To demonstrate the efficiency of the method, we give an example of a structure we have designed which corresponds to a part of the 5'-UTR regulatory element called Translation Inhibitory Element (TIE) from Hox a3 mRNA (Xue S, et al. Nature 517(7532):33-38, 2015).},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {Capillary electrophoresis, chemical probing, CMCT, DMS, ERIANI, Primer extension, QuSHAPE, RNA secondary structure, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
RNA folds into secondary structures that can serve in understanding various RNA functions (Weeks KM. Curr Opin Struct Biol 20(3):295-304, 2010). Chemical probing is a method that enables the characterization of RNA secondary structures using chemical reagents that specifically modify RNA nucleotides that are located in single-stranded areas. In our protocol, we used Dimethyl Sulfate (DMS) and Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluene sulfonate (CMCT) that are both base-specific modifying reagents (Behm-Ansmant I, et al. J Nucleic Acids 2011:408053, 2011). These modifications are mapped by primer extension arrests using 5' fluorescently labeled primers. In this protocol, we show a comprehensive method to identify RNA secondary structures in vitro using fluorescently labeled oligos. To demonstrate the efficiency of the method, we give an example of a structure we have designed which corresponds to a part of the 5'-UTR regulatory element called Translation Inhibitory Element (TIE) from Hox a3 mRNA (Xue S, et al. Nature 517(7532):33-38, 2015).
Bouhedda F, Cubi R, Baudrey S, Ryckelynck M
microIVC-Seq: A Method for Ultrahigh-Throughput Development and Functional Characterization of Small RNAs Book Chapter
In: Rederstorff, M (Ed.): Small Non-Coding RNAs, vol. 2300, pp. 203-237, Springer Protocols, Humana Press, New York, NY, Small Non-Coding RNAs, 2021, ISBN: 978-1-0716-1385-6/ISSN, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Artificial RNAs, directed evolution, Droplet microfluidics, Functional screening, In vitro selection, RNA, RYCKELYNCK, RYCKELYNCK Artificial RNAs, Unité ARN
@inbook{Bouhedda2021,
title = {microIVC-Seq: A Method for Ultrahigh-Throughput Development and Functional Characterization of Small RNAs},
author = {F Bouhedda and R Cubi and S Baudrey and M Ryckelynck},
editor = {M Rederstorff},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792882},
doi = {10.1007/978-1-0716-1386-3_17},
isbn = {978-1-0716-1385-6/ISSN},
year = {2021},
date = {2021-01-01},
booktitle = {Small Non-Coding RNAs},
volume = {2300},
pages = {203-237},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
edition = {Small Non-Coding RNAs},
series = {Methods in Molecular Biology},
abstract = {For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (muIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting muIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {Artificial RNAs, directed evolution, Droplet microfluidics, Functional screening, In vitro selection, RNA, RYCKELYNCK, RYCKELYNCK Artificial RNAs, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (muIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting muIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.
Skerniskyte J, Karazijaite E, Luciunaite A, Suziedeliene E
OmpA Protein-Deficient Acinetobacter baumannii Outer Membrane Vesicles Trigger Reduced Inflammatory Response Journal Article
In: Pathogens, vol. 10, no. 4, pp. 407, 2021, ISBN: 33807410, (2076-0817 (Print) 2076-0817 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Acinetobacter baumannii, inflammasome, inflammation, Macrophages, MARTEYN, outer membrane vesicles, Unité ARN
@article{Skerniskyte2021,
title = {OmpA Protein-Deficient Acinetobacter baumannii Outer Membrane Vesicles Trigger Reduced Inflammatory Response},
author = {J Skerniskyte and E Karazijaite and A Luciunaite and E Suziedeliene},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33807410},
doi = {10.3390/pathogens10040407},
isbn = {33807410},
year = {2021},
date = {2021-01-01},
journal = {Pathogens},
volume = {10},
number = {4},
pages = {407},
abstract = {Multidrug resistant Acinetobacter baumannii shows a growing number of nosocomial infections worldwide during the last decade. The outer membrane vesicles (OMVs) produced by this bacterium draw increasing attention as a possible treatment target. OMVs have been implicated in the reduction of antibiotic level in the surrounding environment, transfer of virulence factors into the host cells, and induction of inflammatory response. Although the evidence on the involvement of OMVs in A. baumannii pathogenesis is currently growing, their role during inflammation is insufficiently explored. It is likely that bacteria, by secreting OMVs, can expand the area of their exposure and prepare surrounding matrix for infection. Here, we investigated the impact of A. baumannii OMVs on activation of macrophages in vitro. We show that OmpA protein present in A. baumannii OMVs substantially contributes to the proinflammatory response in J774 murine macrophages and to the cell death in both lung epithelium cells and macrophages. The loss of OmpA protein in OMVs, obtained from A. baumannii ompA mutant, resulted in the altered expression of genes coding for IL-6, NLRP3 and IL-1beta proinflammatory molecules in macrophages in vitro. These results imply that OmpA protein in bacterial OMVs could trigger a more intense proinflammatory response.},
note = {2076-0817 (Print)
2076-0817 (Linking)
Journal Article},
keywords = {Acinetobacter baumannii, inflammasome, inflammation, Macrophages, MARTEYN, outer membrane vesicles, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Multidrug resistant Acinetobacter baumannii shows a growing number of nosocomial infections worldwide during the last decade. The outer membrane vesicles (OMVs) produced by this bacterium draw increasing attention as a possible treatment target. OMVs have been implicated in the reduction of antibiotic level in the surrounding environment, transfer of virulence factors into the host cells, and induction of inflammatory response. Although the evidence on the involvement of OMVs in A. baumannii pathogenesis is currently growing, their role during inflammation is insufficiently explored. It is likely that bacteria, by secreting OMVs, can expand the area of their exposure and prepare surrounding matrix for infection. Here, we investigated the impact of A. baumannii OMVs on activation of macrophages in vitro. We show that OmpA protein present in A. baumannii OMVs substantially contributes to the proinflammatory response in J774 murine macrophages and to the cell death in both lung epithelium cells and macrophages. The loss of OmpA protein in OMVs, obtained from A. baumannii ompA mutant, resulted in the altered expression of genes coding for IL-6, NLRP3 and IL-1beta proinflammatory molecules in macrophages in vitro. These results imply that OmpA protein in bacterial OMVs could trigger a more intense proinflammatory response.
Bereiter R, Himmelstoss M, Renard E, Mairhofer E, Egger M, Breuker K, Kreutz C, Ennifar E, Micura R
Impact of 3-deazapurine nucleobases on RNA properties Journal Article
In: Nucleic Acids Res, vol. 49, no. 8, pp. 4281-4293, 2021, ISBN: 33856457, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Impact of 3-deazapurine nucleobases on RNA properties},
author = {R Bereiter and M Himmelstoss and E Renard and E Mairhofer and M Egger and K Breuker and C Kreutz and E Ennifar and R Micura},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33856457},
isbn = {33856457},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {8},
pages = {4281-4293},
abstract = {Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.
Omar R El, Julien E, Biasch K, Guffroy B, Lioure B, Vallat L, Gross I, Domon-Dell C, Lanza F, Gachet C, Negroni M, Freund J N, Tavian M
CDX2 regulates ACE expression in blood development and leukemia cells Journal Article
In: Blood Adv, vol. 5, no. 7, pp. 2012-2016, 2021, ISBN: 33843985, (2473-9537 (Electronic) 2473-9529 (Linking) Journal Article).
Links | BibTeX | Tags: NEGRONI, Unité ARN
@article{,
title = {CDX2 regulates ACE expression in blood development and leukemia cells},
author = {R El Omar and E Julien and K Biasch and B Guffroy and B Lioure and L Vallat and I Gross and C Domon-Dell and F Lanza and C Gachet and M Negroni and J N Freund and M Tavian},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33843985},
doi = {10.1182/bloodadvances.2020003563},
isbn = {33843985},
year = {2021},
date = {2021-01-01},
journal = {Blood Adv},
volume = {5},
number = {7},
pages = {2012-2016},
note = {2473-9537 (Electronic)
2473-9529 (Linking)
Journal Article},
keywords = {NEGRONI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Schenckbecher E, Bec G, Sakamoto T, Meyer B, Ennifar E
Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE Journal Article
In: Methods Mol Biol, vol. 2263, pp. 341-350, 2021, ISBN: 33877606, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Biophysics, ENNIFAR, Kinetics, Ribosome, RNA, switchSENSE, Unité ARN
@article{,
title = {Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE},
author = {E Schenckbecher and G Bec and T Sakamoto and B Meyer and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33877606},
doi = {10.1007/978-1-0716-1197-5_15},
isbn = {33877606},
year = {2021},
date = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2263},
pages = {341-350},
abstract = {Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome, initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE((R)) technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome. This study provides a proof of concept for the application of this method on large complexes.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {Biophysics, ENNIFAR, Kinetics, Ribosome, RNA, switchSENSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome, initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE((R)) technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome. This study provides a proof of concept for the application of this method on large complexes.
Toccafondi E, Lener D, Negroni M
HIV-1 Capsid Core: A Bullet to the Heart of the Target Cell Journal Article
In: Front Microbiol, vol. 12, pp. 652486, 2021, ISBN: 33868211, (1664-302X (Print) 1664-302X (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: NEGRONI, Unité ARN
@article{,
title = {HIV-1 Capsid Core: A Bullet to the Heart of the Target Cell},
author = {E Toccafondi and D Lener and M Negroni},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33868211},
doi = {10.3389/fmicb.2021.652486},
isbn = {33868211},
year = {2021},
date = {2021-01-01},
journal = {Front Microbiol},
volume = {12},
pages = {652486},
abstract = {The first step of the intracellular phase of retroviral infection is the release of the viral capsid core in the cytoplasm. This structure contains the viral genetic material that will be reverse transcribed and integrated into the genome of infected cells. Up to recent times, the role of the capsid core was considered essentially to protect this genetic material during the earlier phases of this process. However, increasing evidence demonstrates that the permanence inside the cell of the capsid as an intact, or almost intact, structure is longer than thought. This suggests its involvement in more aspects of the infectious cycle than previously foreseen, particularly in the steps of viral genomic material translocation into the nucleus and in the phases preceding integration. During the trip across the infected cell, many host factors are brought to interact with the capsid, some possessing antiviral properties, others, serving as viral cofactors. All these interactions rely on the properties of the unique component of the capsid core, the capsid protein CA. Likely, the drawback of ensuring these multiple functions is the extreme genetic fragility that has been shown to characterize this protein. Here, we recapitulate the busy agenda of an HIV-1 capsid in the infectious process, in particular in the light of the most recent findings.},
note = {1664-302X (Print)
1664-302X (Linking)
Journal Article
Review},
keywords = {NEGRONI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The first step of the intracellular phase of retroviral infection is the release of the viral capsid core in the cytoplasm. This structure contains the viral genetic material that will be reverse transcribed and integrated into the genome of infected cells. Up to recent times, the role of the capsid core was considered essentially to protect this genetic material during the earlier phases of this process. However, increasing evidence demonstrates that the permanence inside the cell of the capsid as an intact, or almost intact, structure is longer than thought. This suggests its involvement in more aspects of the infectious cycle than previously foreseen, particularly in the steps of viral genomic material translocation into the nucleus and in the phases preceding integration. During the trip across the infected cell, many host factors are brought to interact with the capsid, some possessing antiviral properties, others, serving as viral cofactors. All these interactions rely on the properties of the unique component of the capsid core, the capsid protein CA. Likely, the drawback of ensuring these multiple functions is the extreme genetic fragility that has been shown to characterize this protein. Here, we recapitulate the busy agenda of an HIV-1 capsid in the infectious process, in particular in the light of the most recent findings.
Velazquez-Campoy A, Claro B, Abian O, Horing J, Bourlon L, Claveria-Gimeno R, Ennifar E, England P, Chaires J B, Wu D, Piszczek G, Brautigam C, Tso S C, Zhao H, Schuck P, Keller S, Bastos M
A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca(2+) and Mg(2+) binding to EDTA Journal Article
In: Eur Biophys J, vol. 50, no. 3-4, pp. 429-451, 2021, ISBN: 33864101, (1432-1017 (Electronic) 0175-7571 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Benchmark study, Data treatment, ENNIFAR, Isothermal Titration Calorimetry (ITC), Ligand-binding, Sample preparation, Standard reaction, Unité ARN
@article{,
title = {A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca(2+) and Mg(2+) binding to EDTA},
author = {A Velazquez-Campoy and B Claro and O Abian and J Horing and L Bourlon and R Claveria-Gimeno and E Ennifar and P England and J B Chaires and D Wu and G Piszczek and C Brautigam and S C Tso and H Zhao and P Schuck and S Keller and M Bastos},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33864101},
doi = {10.1007/s00249-021-01523-7},
isbn = {33864101},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Eur Biophys J},
volume = {50},
number = {3-4},
pages = {429-451},
abstract = {A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (DeltaH), and the so-called "stoichiometry" (n), a concentration-correction factor.},
note = {1432-1017 (Electronic)
0175-7571 (Linking)
Journal Article},
keywords = {Benchmark study, Data treatment, ENNIFAR, Isothermal Titration Calorimetry (ITC), Ligand-binding, Sample preparation, Standard reaction, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (DeltaH), and the so-called "stoichiometry" (n), a concentration-correction factor.
Goettsch W, Beerenwinkel N, Deng L, Dolken L, Dutilh B E, Erhard F, Kaderali L, von Kleist M, Marquet R, Matthijnssens J, McCallin S, McMahon D, Rattei T, Rij R P Van, Robertson D L, Schwemmle M, Stern-Ginossar N, Marz M
ITN-VIROINF: Understanding (Harmful) Virus-Host Interactions by Linking Virology and Bioinformatics Journal Article
In: Viruses, vol. 13, no. 5, 2021, ISBN: 33925452, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: MARQUET, Unité ARN
@article{Goettsch2021,
title = {ITN-VIROINF: Understanding (Harmful) Virus-Host Interactions by Linking Virology and Bioinformatics},
author = {W Goettsch and N Beerenwinkel and L Deng and L Dolken and B E Dutilh and F Erhard and L Kaderali and M von Kleist and R Marquet and J Matthijnssens and S McCallin and D McMahon and T Rattei and R P Van Rij and D L Robertson and M Schwemmle and N Stern-Ginossar and M Marz},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33925452},
doi = {10.3390/v13050766},
isbn = {33925452},
year = {2021},
date = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {5},
abstract = {Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Sklodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article},
keywords = {MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Sklodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals.
Cubi R, Bouhedda F, Collot M, Klymchenko A S, Ryckelynck M
In: Rna, vol. 27, no. 7, pp. 841-853, 2021, ISBN: 33952671, (1469-9001 (Electronic) 1355-8382 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Bioinformatics, droplet-based microfluidics, high-throughput screening, light-up RNA aptamer, RNA engineering, RYCKELYNCK, Unité ARN
@article{,
title = {microIVC-Useq: a microfluidic-assisted high-throughput functionnal screening in tandem with next generation sequencing and artificial neural network to rapidly characterize RNA molecules},
author = {R Cubi and F Bouhedda and M Collot and A S Klymchenko and M Ryckelynck},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33952671},
doi = {10.1261/rna.077586.120},
isbn = {33952671},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Rna},
volume = {27},
number = {7},
pages = {841-853},
abstract = {The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (microIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved microIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "microIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.},
note = {1469-9001 (Electronic)
1355-8382 (Linking)
Journal Article},
keywords = {Bioinformatics, droplet-based microfluidics, high-throughput screening, light-up RNA aptamer, RNA engineering, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (microIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved microIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "microIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.
Montavon T C, Baldaccini M, Lefevre M, Girardi E, Chane-Woon-Ming B, Messmer M, Hammann P, Chicher J, Pfeffer S
Human DICER helicase domain recruits PKR and modulates its antiviral activity Journal Article
In: PLoS Pathog, vol. 17, no. 5, pp. e1009549, 2021, ISBN: 33984068, (1553-7374 (Electronic) 1553-7366 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: PFEFFER, PPSE, Unité ARN
@article{Montavon2021,
title = {Human DICER helicase domain recruits PKR and modulates its antiviral activity},
author = {T C Montavon and M Baldaccini and M Lefevre and E Girardi and B Chane-Woon-Ming and M Messmer and P Hammann and J Chicher and S Pfeffer},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33984068},
doi = {0.1371/journal.ppat.1009549},
isbn = {33984068},
year = {2021},
date = {2021-01-01},
journal = {PLoS Pathog},
volume = {17},
number = {5},
pages = {e1009549},
abstract = {The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.},
note = {1553-7374 (Electronic)
1553-7366 (Linking)
Journal Article},
keywords = {PFEFFER, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.
Alghoul F, Laure S, Eriani G, Martin F
Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition Journal Article
In: Elife, vol. 10, 2021, ISBN: 34076576, (2050-084X (Electronic) 2050-084X (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ERIANI, Unité ARN
@article{Alghoul2021b,
title = {Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition},
author = {F Alghoul and S Laure and G Eriani and F Martin},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34076576},
doi = {10.7554/eLife.66369},
isbn = {34076576},
year = {2021},
date = {2021-01-01},
journal = {Elife},
volume = {10},
abstract = {During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5'UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a 'start-stop'. The 'start-stop' sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.},
note = {2050-084X (Electronic)
2050-084X (Linking)
Journal Article},
keywords = {ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5'UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a 'start-stop'. The 'start-stop' sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.
Ramos-Morales E, Bayam E, Del-Pozo-Rodriguez J, Salinas-Giege T, Marek M, Tilly P, Wolff P, Troesch E, Ennifar E, Drouard L, Godin J D, Romier C
The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination Journal Article
In: Nucleic Acids Res, vol. 49, no. 11, pp. 6529-6548, 2021, ISBN: 34057470, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, Unité ARN
@article{,
title = {The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination},
author = {E Ramos-Morales and E Bayam and J Del-Pozo-Rodriguez and T Salinas-Giege and M Marek and P Tilly and P Wolff and E Troesch and E Ennifar and L Drouard and J D Godin and C Romier},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34057470},
doi = {10.1093/nar/gkab436},
isbn = {34057470},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {11},
pages = {6529-6548},
abstract = {Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.
Ortet P, Fochesato S, Bitbol A F, Whitworth D E, Lalaouna D, Santaella C, Heulin T, Achouak W, Barakat M
Evolutionary history expands the range of signaling interactions in hybrid multikinase networks Journal Article
In: Sci Rep, vol. 11, no. 1, pp. 11763, 2021, ISBN: 34083699.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Evolutionary history expands the range of signaling interactions in hybrid multikinase networks},
author = {P Ortet and S Fochesato and A F Bitbol and D E Whitworth and D Lalaouna and C Santaella and T Heulin and W Achouak and M Barakat},
url = {https://pubmed.ncbi.nlm.nih.gov/34083699/},
doi = {10.1038/s41598-021-91260-w},
isbn = {34083699},
year = {2021},
date = {2021-01-01},
journal = {Sci Rep},
volume = {11},
number = {1},
pages = {11763},
abstract = {Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS-GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS-GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.
Himmelstoss M, Erharter K, Renard E, Ennifar E, Kreutz C, Micura R
2'-O-Trifluoromethylated RNA - a powerful modification for RNA chemistry and NMR spectroscopy Journal Article
In: Chem Sci, vol. 11, no. 41, pp. 11322-11330, 2021, ISBN: 34094374, (2041-6520 (Print) 2041-6520 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {2'-O-Trifluoromethylated RNA - a powerful modification for RNA chemistry and NMR spectroscopy},
author = {M Himmelstoss and K Erharter and E Renard and E Ennifar and C Kreutz and R Micura},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34094374},
doi = {10.1039/d0sc04520a},
isbn = {34094374},
year = {2021},
date = {2021-01-01},
journal = {Chem Sci},
volume = {11},
number = {41},
pages = {11322-11330},
abstract = {New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA-ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with (19)F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2'-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2'-SCF3 modification being the most prominent one. A major drawback of the 2'-SCF3 group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2'-OCF3 modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2'-OCF3 labeled RNA and to investigate the impact of this modification. We present the syntheses of 2'-OCF3 adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2'-OCF3 group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3'-endo conformation of the 2'-OCF3 ribose as reflected in the (3) J (H1'-H2') coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in (19)F-NMR analysis of RNA structure equilibria and of RNA-small molecule interactions. The study is complemented by a crystal structure at 0.9 A resolution of a 27 nt hairpin RNA containing a single 2'-OCF3 group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.},
note = {2041-6520 (Print)
2041-6520 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA-ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with (19)F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2'-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2'-SCF3 modification being the most prominent one. A major drawback of the 2'-SCF3 group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2'-OCF3 modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2'-OCF3 labeled RNA and to investigate the impact of this modification. We present the syntheses of 2'-OCF3 adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2'-OCF3 group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3'-endo conformation of the 2'-OCF3 ribose as reflected in the (3) J (H1'-H2') coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in (19)F-NMR analysis of RNA structure equilibria and of RNA-small molecule interactions. The study is complemented by a crystal structure at 0.9 A resolution of a 27 nt hairpin RNA containing a single 2'-OCF3 group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.
Moya-Alvarez V, Koyembi J J, Kaye L M, Mbecko J R, Sanke-Waigana H, Djorie S G, Nyasenu Y T, Mad-Bondo D, Kongoma J B, Nakib S, Madec Y, Ulmann G, Neveux N, Sansonetti P J, Vray M, Marteyn B
Vitamin C levels in a Central-African mother-infant cohort: Does hypovitaminosis C increase the risk of enteric infections? Journal Article
In: Matern Child Nutr, pp. e13215, 2021, ISBN: 34137176, (1740-8709 (Electronic) 1740-8695 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: MARTEYN, Unité ARN
@article{,
title = {Vitamin C levels in a Central-African mother-infant cohort: Does hypovitaminosis C increase the risk of enteric infections?},
author = {V Moya-Alvarez and J J Koyembi and L M Kaye and J R Mbecko and H Sanke-Waigana and S G Djorie and Y T Nyasenu and D Mad-Bondo and J B Kongoma and S Nakib and Y Madec and G Ulmann and N Neveux and P J Sansonetti and M Vray and B Marteyn},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34137176},
doi = {10.1111/mcn.13215},
isbn = {34137176},
year = {2021},
date = {2021-01-01},
journal = {Matern Child Nutr},
pages = {e13215},
abstract = {In the MITICA (Mother-to-Infant TransmIssion of microbiota in Central-Africa) study, 48 mothers and their 50 infants were followed from delivery to 6 months between December 2017 and June 2019 in Bangui (Central-African Republic). Blood tests and stool analyses were performed in mothers at delivery, and their offspring at birth, 11 weeks and 25 weeks. Stool cultures were performed in specific growth media for Salmonella, Shigella, E. coli, Campylobacter, Enerobacter, Vibrio cholerae, Citrobacter and Klebsiella, as well as rotavirus, yeasts and parasitological exams. The median vitamin C levels in mothers at delivery were 15.3 mumol/L (inter-quartile-range [IQR] 6.2-27.8 mumol/L). In infants, the median vitamin C levels at birth were 35.2 mumol/L (IQR 16.5-63.9 mumol/L). At 11 and 25 weeks, the median vitamin C levels were 41.5 mumol/L (IQR 18.7-71.6 mumol/L) and 18.2 mumol/L (IQR 2.3-46.6 mumol/L), respectively. Hypovitaminosis C was defined as seric vitamin C levels <28 mumol/L and vitamin C deficiency was defined as vitamin C levels <11 mumol/L according to the WHO definition. In mothers, the prevalence of hypovitaminosis-C and vitamin C deficiency at delivery was 34/45 (75.6%) and 19/45 (42.2%), respectively. In infants, the prevalence of hypovitaminosis-C and vitamin C deficiency at 6 months was 18/33 (54.6%) and 11/33 (33.3%), respectively. Vitamin C levels in mothers and infants were correlated at birth (Spearman's rho = 0.5; P value = 0.002), and infants had significantly higher levels of vitamin C (median = 35.2 mumol/L; IQR 16.5-63.9 mumol/L), compared to mothers (median = 15.3 mumol/L; IQR 6.2-27.8 mumol/L; P value <0.001). The offspring of vitamin C-deficient mothers had significantly lower vitamin C levels at delivery (median = 18.7 mumol/L; IQR 13.3-30.7 mumol/L), compared to the offspring of non-deficient mothers (median = 62.2 mumol/L; IQR 34.6-89.2 mumol/L; P value <0.001). Infants with hypovitaminosis-C were at significantly higher risk of having a positive stool culture during the first 6 months of life (adjusted OR = 5.3, 95% CI 1.1; 26.1; P value = 0.038).},
note = {1740-8709 (Electronic)
1740-8695 (Linking)
Journal Article},
keywords = {MARTEYN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In the MITICA (Mother-to-Infant TransmIssion of microbiota in Central-Africa) study, 48 mothers and their 50 infants were followed from delivery to 6 months between December 2017 and June 2019 in Bangui (Central-African Republic). Blood tests and stool analyses were performed in mothers at delivery, and their offspring at birth, 11 weeks and 25 weeks. Stool cultures were performed in specific growth media for Salmonella, Shigella, E. coli, Campylobacter, Enerobacter, Vibrio cholerae, Citrobacter and Klebsiella, as well as rotavirus, yeasts and parasitological exams. The median vitamin C levels in mothers at delivery were 15.3 mumol/L (inter-quartile-range [IQR] 6.2-27.8 mumol/L). In infants, the median vitamin C levels at birth were 35.2 mumol/L (IQR 16.5-63.9 mumol/L). At 11 and 25 weeks, the median vitamin C levels were 41.5 mumol/L (IQR 18.7-71.6 mumol/L) and 18.2 mumol/L (IQR 2.3-46.6 mumol/L), respectively. Hypovitaminosis C was defined as seric vitamin C levels <28 mumol/L and vitamin C deficiency was defined as vitamin C levels <11 mumol/L according to the WHO definition. In mothers, the prevalence of hypovitaminosis-C and vitamin C deficiency at delivery was 34/45 (75.6%) and 19/45 (42.2%), respectively. In infants, the prevalence of hypovitaminosis-C and vitamin C deficiency at 6 months was 18/33 (54.6%) and 11/33 (33.3%), respectively. Vitamin C levels in mothers and infants were correlated at birth (Spearman's rho = 0.5; P value = 0.002), and infants had significantly higher levels of vitamin C (median = 35.2 mumol/L; IQR 16.5-63.9 mumol/L), compared to mothers (median = 15.3 mumol/L; IQR 6.2-27.8 mumol/L; P value <0.001). The offspring of vitamin C-deficient mothers had significantly lower vitamin C levels at delivery (median = 18.7 mumol/L; IQR 13.3-30.7 mumol/L), compared to the offspring of non-deficient mothers (median = 62.2 mumol/L; IQR 34.6-89.2 mumol/L; P value <0.001). Infants with hypovitaminosis-C were at significantly higher risk of having a positive stool culture during the first 6 months of life (adjusted OR = 5.3, 95% CI 1.1; 26.1; P value = 0.038).
Giuliodori A M, Marzi S
Editorial: Interview With the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria Journal Article
In: Front Microbiol, vol. 12, pp. 707354, 2021, ISBN: 34220790, (1664-302X (Print) 1664-302X (Linking) Editorial).
Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Editorial: Interview With the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria},
author = {A M Giuliodori and S Marzi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34220790},
doi = {10.3389/fmicb.2021.707354},
isbn = {34220790},
year = {2021},
date = {2021-01-01},
journal = {Front Microbiol},
volume = {12},
pages = {707354},
note = {1664-302X (Print)
1664-302X (Linking)
Editorial},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Marszalkowski M, Werner A, Feltens R, Helmecke D, Gossringer M, Westhof E, Hartmann R K
In: Rna, 2021, ISBN: 34266994, (1469-9001 (Electronic) 1355-8382 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Unité ARN, WESTHOF
@article{,
title = {Comparative study on tertiary contacts and folding of RNase P RNAs from a psychrophilic, a mesophilic/radiation-resistant and a thermophilic bacterium},
author = {M Marszalkowski and A Werner and R Feltens and D Helmecke and M Gossringer and E Westhof and R K Hartmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34266994},
doi = {10.1261/rna.078735.121},
isbn = {34266994},
year = {2021},
date = {2021-01-01},
journal = {Rna},
abstract = {In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8-P4 and L18-P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8-P4 and L18-P8 contacts. At 37 degrees C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9-P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.},
note = {1469-9001 (Electronic)
1355-8382 (Linking)
Journal Article},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8-P4 and L18-P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8-P4 and L18-P8 contacts. At 37 degrees C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9-P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.
Barrientos L, Mercier N, Lalaouna D, Caldelari I
Assembling the Current Pieces: The Puzzle of RNA-Mediated Regulation in Staphylococcus aureus Journal Article
In: Front Microbiol, vol. 12, pp. 706690, 2021, ISBN: 34367109, (1664-302X (Print) 1664-302X (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Assembling the Current Pieces: The Puzzle of RNA-Mediated Regulation in Staphylococcus aureus},
author = {L Barrientos and N Mercier and D Lalaouna and I Caldelari},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34367109},
doi = {10.3389/fmicb.2021.706690},
isbn = {34367109},
year = {2021},
date = {2021-01-01},
journal = {Front Microbiol},
volume = {12},
pages = {706690},
abstract = {The success of the major opportunistic human Staphylococcus aureus relies on the production of numerous virulence factors, which allow rapid colonization and dissemination in any tissues. Indeed, regulation of its virulence is multifactorial, and based on the production of transcriptional factors, two-component systems (TCS) and small regulatory RNAs (sRNAs). Advances in high-throughput sequencing technologies have unveiled the existence of hundreds of potential RNAs with regulatory functions, but only a fraction of which have been validated in vivo. These discoveries have modified our thinking and understanding of bacterial physiology and virulence fitness by placing sRNAs, alongside transcriptional regulators, at the center of complex and intertwined regulatory networks that allow S. aureus to rapidly adapt to the environmental cues present at infection sites. In this review, we describe the recently acquired knowledge of characterized regulatory RNAs in S. aureus that are associated with metal starvation, nutrient availability, stress responses and virulence. These findings highlight the importance of sRNAs for the comprehension of S. aureus infection processes while raising questions about the interplay between these key regulators and the pathways they control.},
note = {1664-302X (Print)
1664-302X (Linking)
Journal Article
Review},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The success of the major opportunistic human Staphylococcus aureus relies on the production of numerous virulence factors, which allow rapid colonization and dissemination in any tissues. Indeed, regulation of its virulence is multifactorial, and based on the production of transcriptional factors, two-component systems (TCS) and small regulatory RNAs (sRNAs). Advances in high-throughput sequencing technologies have unveiled the existence of hundreds of potential RNAs with regulatory functions, but only a fraction of which have been validated in vivo. These discoveries have modified our thinking and understanding of bacterial physiology and virulence fitness by placing sRNAs, alongside transcriptional regulators, at the center of complex and intertwined regulatory networks that allow S. aureus to rapidly adapt to the environmental cues present at infection sites. In this review, we describe the recently acquired knowledge of characterized regulatory RNAs in S. aureus that are associated with metal starvation, nutrient availability, stress responses and virulence. These findings highlight the importance of sRNAs for the comprehension of S. aureus infection processes while raising questions about the interplay between these key regulators and the pathways they control.
Mediati D G, Lalaouna D, Tree J J
Burning the Candle at Both Ends: Have Exoribonucleases Driven Divergence of Regulatory RNA Mechanisms in Bacteria? Journal Article
In: mBio, pp. e0104121, 2021, ISBN: 34372700, (2150-7511 (Electronic) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Burning the Candle at Both Ends: Have Exoribonucleases Driven Divergence of Regulatory RNA Mechanisms in Bacteria?},
author = {D G Mediati and D Lalaouna and J J Tree},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34372700},
doi = {10.1128/mBio.01041-21},
isbn = {34372700},
year = {2021},
date = {2021-01-01},
journal = {mBio},
pages = {e0104121},
abstract = {Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model Gammaproteobacteria Escherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling approximately 20 to 25% of the entire transcriptome. While the model Firmicutes Bacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3'-->5' exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3' UTR cleavage fragments that contain stabilizing 3' structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5' and 3' ends, efficiently degrading mRNA 3' UTR fragments. Here, we propose that the lack of 5'-->3' exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3' UTR ends as the "default" setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3' UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.},
note = {2150-7511 (Electronic)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model Gammaproteobacteria Escherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling approximately 20 to 25% of the entire transcriptome. While the model Firmicutes Bacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3'-->5' exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3' UTR cleavage fragments that contain stabilizing 3' structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5' and 3' ends, efficiently degrading mRNA 3' UTR fragments. Here, we propose that the lack of 5'-->3' exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3' UTR ends as the "default" setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3' UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.
Thepaut M, Campos-Silva R, Renard E, Barloy-Hubler F, Ennifar E, Boujard D, Gillet R
Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria Journal Article
In: Rna, 2021, ISBN: 34353925, (1469-9001 (Electronic) 1355-8382 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{,
title = {Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria},
author = {M Thepaut and R Campos-Silva and E Renard and F Barloy-Hubler and E Ennifar and D Boujard and R Gillet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34353925},
doi = {10.1261/rna.078773.121},
isbn = {34353925},
year = {2021},
date = {2021-01-01},
journal = {Rna},
abstract = {In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.},
note = {1469-9001 (Electronic)
1355-8382 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.
Vargas-Rodriguez O, Badran A H, Hoffman K S, Chen M, Crnkovic A, Ding Y, Krieger J R, Westhof E, Soll D, Melnikov S
Bacterial translation machinery for deliberate mistranslation of the genetic code Journal Article
In: Proc Natl Acad Sci U S A, vol. 118, no. 35, 2021, ISBN: 34413202, (1091-6490 (Electronic) 0027-8424 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Unité ARN
@article{,
title = {Bacterial translation machinery for deliberate mistranslation of the genetic code},
author = {O Vargas-Rodriguez and A H Badran and K S Hoffman and M Chen and A Crnkovic and Y Ding and J R Krieger and E Westhof and D Soll and S Melnikov},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34413202},
doi = {10.1073/pnas.2110797118},
isbn = {34413202},
year = {2021},
date = {2021-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {118},
number = {35},
abstract = {Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNA(ProA), that are predominately found in plant pathogens from Streptomyces species. We then show that tRNA(ProA) has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNA(ProA) can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala-->Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.},
note = {1091-6490 (Electronic)
0027-8424 (Linking)
Journal Article},
keywords = {Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNA(ProA), that are predominately found in plant pathogens from Streptomyces species. We then show that tRNA(ProA) has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNA(ProA) can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala-->Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.
Vilimova M, Contrant M, Randrianjafy R, Dumas P, Elbasani E, Ojala P M, Pfeffer S, Fender A
Cis regulation within a cluster of viral microRNAs Journal Article
In: Nucleic Acids Res, 2021, ISBN: 34417603, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: PFEFFER, Unité ARN
@article{Vilimova2021,
title = {Cis regulation within a cluster of viral microRNAs},
author = {M Vilimova and M Contrant and R Randrianjafy and P Dumas and E Elbasani and P M Ojala and S Pfeffer and A Fender},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34417603},
doi = {10.1093/nar/gkab806},
isbn = {34417603},
year = {2021},
date = {2021-01-01},
journal = {Nucleic Acids Res},
abstract = {MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {PFEFFER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.
Antoine L, Bahena-Ceron R, Bunwaree H Devi, Gobry M, Loegler V, Romby P, Marzi S
RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence Journal Article
In: Genes (Basel), vol. 12, no. 8, 2021, ISBN: 34440299, (2073-4425 (Electronic) 2073-4425 (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence},
author = {L Antoine and R Bahena-Ceron and H Devi Bunwaree and M Gobry and V Loegler and P Romby and S Marzi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34440299},
doi = {10.3390/genes12081125},
isbn = {34440299},
year = {2021},
date = {2021-01-01},
journal = {Genes (Basel)},
volume = {12},
number = {8},
abstract = {RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.},
note = {2073-4425 (Electronic)
2073-4425 (Linking)
Journal Article
Review},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.
Welker L, Paillart J C, Bernacchi S
Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses Journal Article
In: Viruses, vol. 13, no. 8, pp. 1559, 2021, ISBN: 34452424, (1999-4915 (Electronic) 1999-4915 (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: PAILLART, Unité ARN
@article{Welker2021,
title = {Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses},
author = {L Welker and J C Paillart and S Bernacchi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34452424},
doi = {10.3390/v13081559},
isbn = {34452424},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {8},
pages = {1559},
abstract = {Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.},
note = {1999-4915 (Electronic)
1999-4915 (Linking)
Journal Article
Review},
keywords = {PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.
Andre C, Veillard F, Wolff P, Lobstein A M, Compain G, Monsarrat C, Reichhart J M, Burnouf D Y, Guichard G, Wagner J E
Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome Journal Article
In: RSC Chem Biol, vol. 2, no. 4, pp. 1296, 2021, ISBN: 34459830, (2633-0679 (Electronic) 2633-0679 (Linking) Published Erratum).
Abstract | Links | BibTeX | Tags: ARN-MS, ENNIFAR, reichhart, Unité ARN
@article{,
title = {Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome},
author = {C Andre and F Veillard and P Wolff and A M Lobstein and G Compain and C Monsarrat and J M Reichhart and D Y Burnouf and G Guichard and J E Wagner},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34459830},
doi = {10.1039/d1cb90020j},
isbn = {34459830},
year = {2021},
date = {2021-01-01},
journal = {RSC Chem Biol},
volume = {2},
number = {4},
pages = {1296},
abstract = {[This corrects the article DOI: 10.1039/D0CB00060D.].},
note = {2633-0679 (Electronic)
2633-0679 (Linking)
Published Erratum},
keywords = {ARN-MS, ENNIFAR, reichhart, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
[This corrects the article DOI: 10.1039/D0CB00060D.].
Cela M, Theobald-Dietrich A, Rudinger-Thirion J, Wolff P, Geslain R, Frugier M
Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip Journal Article
In: Nucleic Acids Res, 2021, ISBN: 34530443, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ARN-MS, FRUGIER, Unité ARN
@article{,
title = {Identification of host tRNAs preferentially recognized by the Plasmodium surface protein tRip},
author = {M Cela and A Theobald-Dietrich and J Rudinger-Thirion and P Wolff and R Geslain and M Frugier},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34530443},
doi = {10.1093/nar/gkab769},
isbn = {34530443},
year = {2021},
date = {2021-01-01},
journal = {Nucleic Acids Res},
abstract = {Malaria is a life-threatening and devastating parasitic disease. Our previous work showed that parasite development requires the import of exogenous transfer RNAs (tRNAs), which represents a novel and unique form of host-pathogen interaction, as well as a potentially druggable target. This import is mediated by tRip (tRNA import protein), a membrane protein located on the parasite surface. tRip displays an extracellular domain homologous to the well-characterized OB-fold tRNA-binding domain, a structural motif known to indiscriminately interact with tRNAs. We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native Homo sapiens tRNAs and recombinant Plasmodium falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of affinities, suggesting that only a small subset of human tRNAs is preferentially imported into the parasite. In particular, we show with in vitro transcribed constructs that tRip does not bind specific tRNAs solely based on their primary sequence, hinting that post-transcriptional modifications modulate the formation of our host/parasite molecular complex. Finally, we discuss the potential utilization of the most efficient tRip ligands for the translation of the parasite's genetic information.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ARN-MS, FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Malaria is a life-threatening and devastating parasitic disease. Our previous work showed that parasite development requires the import of exogenous transfer RNAs (tRNAs), which represents a novel and unique form of host-pathogen interaction, as well as a potentially druggable target. This import is mediated by tRip (tRNA import protein), a membrane protein located on the parasite surface. tRip displays an extracellular domain homologous to the well-characterized OB-fold tRNA-binding domain, a structural motif known to indiscriminately interact with tRNAs. We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native Homo sapiens tRNAs and recombinant Plasmodium falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of affinities, suggesting that only a small subset of human tRNAs is preferentially imported into the parasite. In particular, we show with in vitro transcribed constructs that tRip does not bind specific tRNAs solely based on their primary sequence, hinting that post-transcriptional modifications modulate the formation of our host/parasite molecular complex. Finally, we discuss the potential utilization of the most efficient tRip ligands for the translation of the parasite's genetic information.
Gilmer O, Quignon E, Jousset A C, Paillart J C, Marquet R, Vivet-Boudou V
Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond Journal Article
In: Viruses, vol. 13, no. 10, pp. 1894, 2021.
Abstract | Links | BibTeX | Tags: Capillary electrophoresis, chemical probe, enzymatic probe, high-throughput sequencing, MARQUET, mutational profiling, PAILLART, RNA, SHAPE, structure, Unité ARN
@article{nokey,
title = {Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond},
author = {O Gilmer and E Quignon and A C Jousset and J C Paillart and R Marquet and V Vivet-Boudou},
url = {https://www.mdpi.com/1999-4915/13/10/1894},
doi = {v13101894},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Viruses},
volume = {13},
number = {10},
pages = {1894},
abstract = {RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.},
keywords = {Capillary electrophoresis, chemical probe, enzymatic probe, high-throughput sequencing, MARQUET, mutational profiling, PAILLART, RNA, SHAPE, structure, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.
Lalaouna D., Prévost K., Park S., Chénard T., Bouchard M - P., Caron M - P., Vanderpool C. K., Fei J., Massé E.
Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli Journal Article
In: Non-Coding RNA, vol. 7, no. 4, pp. 64, 2021.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{nokey,
title = {Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli},
author = {D. Lalaouna and K. Prévost and S. Park and T. Chénard and M - P. Bouchard and M - P. Caron and C. K. Vanderpool and J. Fei and E. Massé},
url = {https://www.mdpi.com/2311-553X/7/4/64},
doi = {10.3390/ncrna7040064},
year = {2021},
date = {2021-01-01},
journal = {Non-Coding RNA},
volume = {7},
number = {4},
pages = {64},
abstract = {Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.
Singh G., Pereira D., Baudrey S., Hoffmann E., Ryckelynck M., Asnacios A., Chaboute M. E.
Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach Journal Article
In: Plant J, vol. 108, iss. 2, pp. 303-313, 2021, ISBN: 34562320, (1365-313X (Electronic) 0960-7412 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{nokey,
title = {Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach},
author = {G. Singh and D. Pereira and S. Baudrey and E. Hoffmann and M. Ryckelynck and A. Asnacios and M. E. Chaboute},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34562320},
doi = {10.1111/tpj.15511},
isbn = {34562320},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Plant J},
volume = {108},
issue = {2},
pages = {303-313},
abstract = {Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RH shows a fast apical growth, constituting a unique single cell model system to analyse cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, but high-resolution imaging is still lacking to study accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) which enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, in order to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area, and aspect ratio of nuclei moving in growing and mature RH. Interestingly, we observed higher aspect ratios in the nuclei of mature RH, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.},
note = {1365-313X (Electronic)
0960-7412 (Linking)
Journal Article},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RH shows a fast apical growth, constituting a unique single cell model system to analyse cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, but high-resolution imaging is still lacking to study accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) which enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, in order to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area, and aspect ratio of nuclei moving in growing and mature RH. Interestingly, we observed higher aspect ratios in the nuclei of mature RH, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.
Wijn R. De, Rollet K., G.M.Ernst F., Wellner K., Betat H., Mörl M., Sauter M.
In: Computational and Structural Biotechnology Journal, vol. 19, pp. 5845-5855, 2021, ISBN: ISBN/2001-0370.
Abstract | Links | BibTeX | Tags: CCA-adding enzyme, Cold adaptation, Psychrophilic protein, Psychrophilic RNA polymerase, SAUTER, SAXS, tRNA, Unité ARN, X-ray crystallography
@article{nokey,
title = {CCA-addition in the cold: Structural characterization of the psychrophilic CCA-adding enzyme from the permafrost bacterium Planococcus halocryophilus},
author = {R. De Wijn and K. Rollet and F. G.M.Ernst and K. Wellner and H. Betat and M. Mörl and M. Sauter},
url = {https://www.sciencedirect.com/science/article/pii/S2001037021004402?via%3Dihub},
doi = {10.1016/j.csbj.2021.10.018},
isbn = {ISBN/2001-0370},
year = {2021},
date = {2021-01-01},
journal = {Computational and Structural Biotechnology Journal},
volume = {19},
pages = {5845-5855},
abstract = {CCA-adding enzymes are highly specific RNA polymerases that add and maintain the sequence C-C-A at tRNA 3ﾑ-ends. Recently, we could reveal that cold adaptation of such a polymerase is not only achieved at the expense of enzyme stability, but also at the cost of polymerization fidelity. Enzymes from psychrophilic organisms usually show an increased structural flexibility to enable catalysis at low temperatures. Here, polymerases face a dilemma, as there is a discrepancy between the need for a tightly controlled flexibility during polymerization and an increased flexibility as strategy for cold adaptation. Based on structural and biochemical analyses, we contribute to clarify the cold adaptation strategy of the psychrophilic CCA-adding enzyme from Planococcus halocryophilus, a gram-positive bacterium thriving in the arctic permafrost at low temperatures down to −15 °C. A comparison with the closely related enzyme from the thermophilic bacterium Geobacillus stearothermophilus reveals several features of cold adaptation - a significantly reduced amount of alpha-helical elements in the C-terminal tRNA-binding region and a structural adaptation in one of the highly conserved catalytic core motifs located in the N-terminal catalytic core of the enzyme},
keywords = {CCA-adding enzyme, Cold adaptation, Psychrophilic protein, Psychrophilic RNA polymerase, SAUTER, SAXS, tRNA, Unité ARN, X-ray crystallography},
pubstate = {published},
tppubtype = {article}
}
CCA-adding enzymes are highly specific RNA polymerases that add and maintain the sequence C-C-A at tRNA 3ﾑ-ends. Recently, we could reveal that cold adaptation of such a polymerase is not only achieved at the expense of enzyme stability, but also at the cost of polymerization fidelity. Enzymes from psychrophilic organisms usually show an increased structural flexibility to enable catalysis at low temperatures. Here, polymerases face a dilemma, as there is a discrepancy between the need for a tightly controlled flexibility during polymerization and an increased flexibility as strategy for cold adaptation. Based on structural and biochemical analyses, we contribute to clarify the cold adaptation strategy of the psychrophilic CCA-adding enzyme from Planococcus halocryophilus, a gram-positive bacterium thriving in the arctic permafrost at low temperatures down to −15 °C. A comparison with the closely related enzyme from the thermophilic bacterium Geobacillus stearothermophilus reveals several features of cold adaptation - a significantly reduced amount of alpha-helical elements in the C-terminal tRNA-binding region and a structural adaptation in one of the highly conserved catalytic core motifs located in the N-terminal catalytic core of the enzyme
Mrazikova K., Sponer J., Mlynsky V., Auffinger P., Kruse H.
Short-Range Imbalances in the AMBER Lennard-Jones Potential for (Deoxy)Ribose.Nucleobase Lone-Pair.pi Contacts in Nucleic Acids Journal Article
In: J Chem Inf Model, vol. 61, no. 11, pp. 5644-5657, 2021, ISBN: 34738826, (1549-960X (Electronic) 1549-9596 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ENNIFAR, Unité ARN
@article{nokey,
title = {Short-Range Imbalances in the AMBER Lennard-Jones Potential for (Deoxy)Ribose.Nucleobase Lone-Pair.pi Contacts in Nucleic Acids},
author = {K. Mrazikova and J. Sponer and V. Mlynsky and P. Auffinger and H. Kruse},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34738826},
doi = {10.1021/acs.jcim.1c01047},
isbn = {34738826},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {J Chem Inf Model},
volume = {61},
number = {11},
pages = {5644-5657},
abstract = {The lone-pair.pi (lp.pi) (deoxy)ribose.nucleobase stacking is a recurring interaction in Z-DNA and RNAs that is characterized by sub-van der Waals lp.pi contacts (<3.0 A). It is a part of the structural signature of CpG Z-step motifs in Z-DNA and r(UNCG) tetraloops that are known to behave poorly in molecular dynamics (MD) simulations. Although the exact origin of the MD simulation issues remains unclear, a significant part of the problem might be due to an imbalanced description of nonbonded interactions, including the characteristic lp.pi stacking. To gain insights into the links between lp.pi stacking and MD, we present an in-depth comparison between accurate large-basis-set double-hybrid Kohn-Sham density functional theory calculations DSD-BLYP-D3/ma-def2-QZVPP (DHDF-D3) and data obtained with the nonbonded potential of the AMBER force field (AFF) for NpN Z-steps (N = G, A, C, and U). Among other differences, we found that the AFF overestimates the DHDF-D3 lp.pi distances by approximately 0.1-0.2 A, while the deviation between the DHDF-D3 and AFF descriptions sharply increases in the short-range region of the interaction. Based on atom-in-molecule polarizabilities and symmetry-adapted perturbation theory analysis, we inferred that the DHDF-D3 versus AFF differences partly originate in identical nucleobase carbon atom Lennard-Jones (LJ) parameters despite the presence/absence of connected electron-withdrawing groups that lead to different effective volumes or vdW radii. Thus, to precisely model the very short CpG lp.pi contact distances, we recommend revision of the nucleobase atom LJ parameters. Additionally, we suggest that the large discrepancy between DHDF-D3 and AFF short-range repulsive part of the interaction energy potential may significantly contribute to the poor performances of MD simulations of nucleic acid systems containing Z-steps. Understanding where, and if possible why, the point-charge-type effective potentials reach their limits is vital for developing next-generation FFs and for addressing specific issues in contemporary MD simulations.},
note = {1549-960X (Electronic)
1549-9596 (Linking)
Journal Article},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The lone-pair.pi (lp.pi) (deoxy)ribose.nucleobase stacking is a recurring interaction in Z-DNA and RNAs that is characterized by sub-van der Waals lp.pi contacts (<3.0 A). It is a part of the structural signature of CpG Z-step motifs in Z-DNA and r(UNCG) tetraloops that are known to behave poorly in molecular dynamics (MD) simulations. Although the exact origin of the MD simulation issues remains unclear, a significant part of the problem might be due to an imbalanced description of nonbonded interactions, including the characteristic lp.pi stacking. To gain insights into the links between lp.pi stacking and MD, we present an in-depth comparison between accurate large-basis-set double-hybrid Kohn-Sham density functional theory calculations DSD-BLYP-D3/ma-def2-QZVPP (DHDF-D3) and data obtained with the nonbonded potential of the AMBER force field (AFF) for NpN Z-steps (N = G, A, C, and U). Among other differences, we found that the AFF overestimates the DHDF-D3 lp.pi distances by approximately 0.1-0.2 A, while the deviation between the DHDF-D3 and AFF descriptions sharply increases in the short-range region of the interaction. Based on atom-in-molecule polarizabilities and symmetry-adapted perturbation theory analysis, we inferred that the DHDF-D3 versus AFF differences partly originate in identical nucleobase carbon atom Lennard-Jones (LJ) parameters despite the presence/absence of connected electron-withdrawing groups that lead to different effective volumes or vdW radii. Thus, to precisely model the very short CpG lp.pi contact distances, we recommend revision of the nucleobase atom LJ parameters. Additionally, we suggest that the large discrepancy between DHDF-D3 and AFF short-range repulsive part of the interaction energy potential may significantly contribute to the poor performances of MD simulations of nucleic acid systems containing Z-steps. Understanding where, and if possible why, the point-charge-type effective potentials reach their limits is vital for developing next-generation FFs and for addressing specific issues in contemporary MD simulations.