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2024
Holvec Samuel, Barchet Charles, Lechner Antony, Fréchin Léo, Silva S Nimali T De, Hazemann Isabelle, Wolff Philippe, von Loeffelholz Ottilie, Klaholz Bruno P
The structure of the human 80S ribosome at 1.9 Å resolution reveals the molecular role of chemical modifications and ions in RNA Article de journal
Dans: Nat Struct Mol Biol, 2024, ISSN: 1545-9985.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, Unité ARN
@article{pmid38844527,
title = {The structure of the human 80S ribosome at 1.9 Å resolution reveals the molecular role of chemical modifications and ions in RNA},
author = {Samuel Holvec and Charles Barchet and Antony Lechner and Léo Fréchin and S Nimali T De Silva and Isabelle Hazemann and Philippe Wolff and Ottilie von Loeffelholz and Bruno P Klaholz},
doi = {10.1038/s41594-024-01274-x},
issn = {1545-9985},
year = {2024},
date = {2024-06-01},
urldate = {2024-06-01},
journal = {Nat Struct Mol Biol},
abstract = {The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn, K and Mg, including their associated individual water molecules. The 2'-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U-A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.},
keywords = {ARN-MS, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn, K and Mg, including their associated individual water molecules. The 2'-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U-A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.
Urzhumtseva Ludmila, Barchet Charles, Klaholz Bruno P, Urzhumtsev Alexandre G
Program : analysing distributions of cryo-EM projections using uniform spherical grids Article de journal
Dans: J Appl Crystallogr, vol. 57, no. Pt 3, p. 865–876, 2024, ISSN: 0021-8898.
Résumé | Liens | BibTeX | Étiquettes: Unité ARN
@article{pmid38846771,
title = {Program : analysing distributions of cryo-EM projections using uniform spherical grids},
author = {Ludmila Urzhumtseva and Charles Barchet and Bruno P Klaholz and Alexandre G Urzhumtsev},
doi = {10.1107/S1600576724002383},
issn = {0021-8898},
year = {2024},
date = {2024-06-01},
urldate = {2024-06-01},
journal = {J Appl Crystallogr},
volume = {57},
number = {Pt 3},
pages = {865--876},
abstract = {Three-dimensional cryo electron microscopy reconstructions are obtained by extracting information from a large number of projections of the object. These projections correspond to different 'views' or 'orientations', directions in which these projections show the reconstructed object. Uneven distribution of these views and the presence of dominating preferred orientations may distort the reconstructed spatial images. This work describes the program (views on uniform grids for cryo electron microscopy), designed to study such distributions. Its algorithms, based on uniform virtual grids on a sphere, allow an easy calculation and accurate quantitative analysis of the frequency distribution of the views. The key computational element is the Lambert azimuthal equal-area projection of a spherical uniform grid onto a disc. This projection keeps the surface area constant and represents the frequency distribution with no visual bias. Since it has multiple tunable parameters, the program is easily adaptable to individual needs, and to the features of a particular project or of the figure to be produced. It can help identify problems related to an uneven distribution of views. Optionally, it can modify the list of projections, distributing the views more uniformly. The program can also be used as a teaching tool.},
keywords = {Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Three-dimensional cryo electron microscopy reconstructions are obtained by extracting information from a large number of projections of the object. These projections correspond to different 'views' or 'orientations', directions in which these projections show the reconstructed object. Uneven distribution of these views and the presence of dominating preferred orientations may distort the reconstructed spatial images. This work describes the program (views on uniform grids for cryo electron microscopy), designed to study such distributions. Its algorithms, based on uniform virtual grids on a sphere, allow an easy calculation and accurate quantitative analysis of the frequency distribution of the views. The key computational element is the Lambert azimuthal equal-area projection of a spherical uniform grid onto a disc. This projection keeps the surface area constant and represents the frequency distribution with no visual bias. Since it has multiple tunable parameters, the program is easily adaptable to individual needs, and to the features of a particular project or of the figure to be produced. It can help identify problems related to an uneven distribution of views. Optionally, it can modify the list of projections, distributing the views more uniformly. The program can also be used as a teaching tool.
Lim Shey-Li, Liu Jinhong, Dupouy Gilles, Singh Gaurav, Baudrey Stéphanie, Yang Lang, Zhong Jia Yi, Chabouté Marie-Edith, Lim Boon Leong
In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors Article de journal
Dans: Plant J, 2024, ISSN: 1365-313X.
Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN
@article{pmid38761168,
title = {In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors},
author = {Shey-Li Lim and Jinhong Liu and Gilles Dupouy and Gaurav Singh and Stéphanie Baudrey and Lang Yang and Jia Yi Zhong and Marie-Edith Chabouté and Boon Leong Lim},
doi = {10.1111/tpj.16796},
issn = {1365-313X},
year = {2024},
date = {2024-05-01},
urldate = {2024-05-01},
journal = {Plant J},
abstract = {Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.
Omar Reine El, Abdellaoui Naoill, Coulibaly Safiatou T, Fontenille Laura, Lanza François, Gachet Christian, Freund Jean-Noel, Negroni Matteo, Kissa Karima, Tavian Manuela
Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo Article de journal
Dans: Cell Death Dis, vol. 15, no. 5, p. 305, 2024, ISSN: 2041-4889.
Résumé | Liens | BibTeX | Étiquettes: NEGRONI, Unité ARN
@article{pmid38693109,
title = {Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo},
author = {Reine El Omar and Naoill Abdellaoui and Safiatou T Coulibaly and Laura Fontenille and François Lanza and Christian Gachet and Jean-Noel Freund and Matteo Negroni and Karima Kissa and Manuela Tavian},
doi = {10.1038/s41419-024-06682-x},
issn = {2041-4889},
year = {2024},
date = {2024-05-01},
urldate = {2024-05-01},
journal = {Cell Death Dis},
volume = {15},
number = {5},
pages = {305},
abstract = {Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.},
keywords = {NEGRONI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.
Niedner-Boblenz A., Monecke T., Hennig J., Klostermann M., Hofweber M., Gerber A. P., Anosova I., Mayer W., Müller M., Heym R., Janowski R., Paillart J. -C., Dormann D., Zarnack K., Sattler M., Niessing D.
Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation Article de journal
Dans: bioRxiv, 2024.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, PAILLART, Unité ARN
@article{A.2024,
title = {Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation},
author = {A. Niedner-Boblenz and T. Monecke and J. Hennig and M. Klostermann and M. Hofweber and A.P. Gerber and I. Anosova and W. Mayer and M. Müller and R. Heym and R. Janowski and J.-C. Paillart and D. Dormann and K. Zarnack and M. Sattler and D. Niessing},
url = {https://www.biorxiv.org/content/10.1101/2024.03.27.586925v2},
doi = {10.1101/2024.03.27.586925},
year = {2024},
date = {2024-03-29},
urldate = {2024-03-29},
journal = {bioRxiv},
abstract = {RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient “RNA world” PUN-like motifs may have supported the correct folding of early ribozymes.},
keywords = {MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient “RNA world” PUN-like motifs may have supported the correct folding of early ribozymes.
D'Agostino Mattia, Simonetti Angelita, Motta Stefano, Wolff Philippe, Romagnoli Alice, Piccinini Astra, Spinozzi Francesco, Marino Daniele Di, Teana Anna La, Ennifar Eric
Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism Article de journal
Dans: Structure, 2024, ISSN: 1878-4186.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ENNIFAR, Unité ARN
@article{pmid38582076,
title = {Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism},
author = {Mattia D'Agostino and Angelita Simonetti and Stefano Motta and Philippe Wolff and Alice Romagnoli and Astra Piccinini and Francesco Spinozzi and Daniele Di Marino and Anna La Teana and Eric Ennifar},
doi = {10.1016/j.str.2024.03.008},
issn = {1878-4186},
year = {2024},
date = {2024-03-01},
urldate = {2024-03-01},
journal = {Structure},
abstract = {The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.
Messmer Mélanie, Pierson Louison, Pasquier Charline, Djordjevic Nikola, Chicher Johana, Hammann Philippe, Pfeffer Sébastien, Girardi Erika
DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection Article de journal
Dans: Virol J, vol. 21, no. 1, p. 76, 2024, ISSN: 1743-422X.
Résumé | Liens | BibTeX | Étiquettes: PFEFFER, PPSE, Unité ARN
@article{pmid38553727,
title = {DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection},
author = {Mélanie Messmer and Louison Pierson and Charline Pasquier and Nikola Djordjevic and Johana Chicher and Philippe Hammann and Sébastien Pfeffer and Erika Girardi},
doi = {10.1186/s12985-024-02349-3},
issn = {1743-422X},
year = {2024},
date = {2024-03-01},
urldate = {2024-03-01},
journal = {Virol J},
volume = {21},
number = {1},
pages = {76},
abstract = {BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection.nnMETHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments.nnRESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV.nnCONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.},
keywords = {PFEFFER, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection.nnMETHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments.nnRESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV.nnCONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.
Pellequer Jean-Luc, Westhof Eric
Marc van Regenmortel, personal recollections on a forward-thinking editor Article de journal
Dans: J Mol Recognit, p. e3080, 2024, ISSN: 1099-1352.
Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF
@article{pmid38439188,
title = {Marc van Regenmortel, personal recollections on a forward-thinking editor},
author = {Jean-Luc Pellequer and Eric Westhof},
doi = {10.1002/jmr.3080},
issn = {1099-1352},
year = {2024},
date = {2024-03-01},
urldate = {2024-03-01},
journal = {J Mol Recognit},
pages = {e3080},
abstract = {Marc van Regenmortel was the Editor-in-Chief of the Journal of Molecular Recognition for the last 25 years. Without attempting to summarize Marc's exceptional career and achievements, we would like to tell the story of the tortuous and contingent path to the unravelling of a key molecular recognition process in antigenicity. Life is indeed full of contingencies and scientific life, full of meetings and random encounters, is prone to contingencies, a key element in discovery and innovation.},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Marc van Regenmortel was the Editor-in-Chief of the Journal of Molecular Recognition for the last 25 years. Without attempting to summarize Marc's exceptional career and achievements, we would like to tell the story of the tortuous and contingent path to the unravelling of a key molecular recognition process in antigenicity. Life is indeed full of contingencies and scientific life, full of meetings and random encounters, is prone to contingencies, a key element in discovery and innovation.
Compain Guillaume, Monsarrat Clément, Blagojevic Julie, Brillet Karl, Dumas Philippe, Hammann Philippe, Kuhn Lauriane, Martiel Isabelle, Engilberge Sylvain, Oliéric Vincent, Wolff Philippe, Burnouf Dominique Y, Wagner Jérôme, Guichard Gilles
Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp Article de journal
Dans: JACS Au, vol. 4, no. 2, p. 432–440, 2024, ISSN: 2691-3704.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ENNIFAR, PPSE, Unité ARN
@article{pmid38425897,
title = {Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp},
author = {Guillaume Compain and Clément Monsarrat and Julie Blagojevic and Karl Brillet and Philippe Dumas and Philippe Hammann and Lauriane Kuhn and Isabelle Martiel and Sylvain Engilberge and Vincent Oliéric and Philippe Wolff and Dominique Y Burnouf and Jérôme Wagner and Gilles Guichard},
doi = {10.1021/jacsau.3c00572},
issn = {2691-3704},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {JACS Au},
volume = {4},
number = {2},
pages = {432--440},
abstract = {Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.},
keywords = {ARN-MS, ENNIFAR, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.
Soufi G El, Jorio L Di, Gerber Z, Cluzel N, Assche J Van, Delafoy D, Olaso R, Daviaud C, Loustau T, Schwartz C, Trebouet D, Hernalsteens O, Marechal V, Raffestin S, Rousset D, Lint C Van, Deleuze J F, Boni M, and O Rohr, Villain-Gambier M, Wallet C
Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater Article de journal
Dans: Water Res, vol. 249, p. 120959, 2024, ISSN: 1879-2448.
Résumé | Liens | BibTeX | Étiquettes: ROHR, Unité ARN
@article{pmid38070350,
title = {Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater},
author = {G El Soufi and L Di Jorio and Z Gerber and N Cluzel and J Van Assche and D Delafoy and R Olaso and C Daviaud and T Loustau and C Schwartz and D Trebouet and O Hernalsteens and V Marechal and S Raffestin and D Rousset and C Van Lint and J F Deleuze and M Boni and and O Rohr and M Villain-Gambier and C Wallet},
doi = {10.1016/j.watres.2023.120959},
issn = {1879-2448},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {Water Res},
volume = {249},
pages = {120959},
abstract = {Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.
Brillet Karl, Janczuk-Richter Marta, Poon Amanda, Laukart-Bradley Joanne, Ennifar Eric, Lebars Isabelle
Characterization of SLA RNA promoter from dengue virus and its interaction with the viral non-structural NS5 protein Article de journal
Dans: Biochimie, vol. 222, p. 87–100, 2024, ISSN: 1638-6183.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN
@article{pmid38408720,
title = {Characterization of SLA RNA promoter from dengue virus and its interaction with the viral non-structural NS5 protein},
author = {Karl Brillet and Marta Janczuk-Richter and Amanda Poon and Joanne Laukart-Bradley and Eric Ennifar and Isabelle Lebars},
doi = {10.1016/j.biochi.2024.02.005},
issn = {1638-6183},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {Biochimie},
volume = {222},
pages = {87--100},
abstract = {The Dengue virus (DENV) is the most significant arthropod-borne viral pathogen in humans with 400 million infections annually. DENV comprises four distinct serotypes (DENV-1 to -4) which complicates vaccine development. Any of the four serotypes can cause clinical illness but with distinctive infection dynamics. Variations in sequences identified within the four genomes induce structural differences in crucial RNA motifs that were suggested to be correlated to the degree of pathogenicity among DENV-1 to -4. In particular, the RNA Stem-loop A (SLA) at the 5'-end of the genome, acts as a key regulator of the viral replication cycle by interacting with the viral NS5 polymerase to initiate the minus-strand viral RNA synthesis and later to methylate and cap the synthesized RNA. The molecular details of this interaction remain not fully described. Here, we report the solution secondary structures of SLA from DENV-1 to -4. Our results highlight that the four SLA exhibit structural and dynamic differences. Secondly, to determine whether SLA RNA contains serotype-specific determinants for the recognition by the viral NS5 protein, we investigated interactions between SLA from DENV -1 to -4 and DENV2 NS5 using combined biophysical approaches. Our results show that NS5 from DENV2 is able to bind SLA from other serotypes, but that other viral or host factors may be necessary to stabilize the complex and promote the catalytically active state of the NS5. By contrast, we show that a serotype-specific binding is driven by specific interactions involving conformational changes within the SLA RNA.},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Dengue virus (DENV) is the most significant arthropod-borne viral pathogen in humans with 400 million infections annually. DENV comprises four distinct serotypes (DENV-1 to -4) which complicates vaccine development. Any of the four serotypes can cause clinical illness but with distinctive infection dynamics. Variations in sequences identified within the four genomes induce structural differences in crucial RNA motifs that were suggested to be correlated to the degree of pathogenicity among DENV-1 to -4. In particular, the RNA Stem-loop A (SLA) at the 5'-end of the genome, acts as a key regulator of the viral replication cycle by interacting with the viral NS5 polymerase to initiate the minus-strand viral RNA synthesis and later to methylate and cap the synthesized RNA. The molecular details of this interaction remain not fully described. Here, we report the solution secondary structures of SLA from DENV-1 to -4. Our results highlight that the four SLA exhibit structural and dynamic differences. Secondly, to determine whether SLA RNA contains serotype-specific determinants for the recognition by the viral NS5 protein, we investigated interactions between SLA from DENV -1 to -4 and DENV2 NS5 using combined biophysical approaches. Our results show that NS5 from DENV2 is able to bind SLA from other serotypes, but that other viral or host factors may be necessary to stabilize the complex and promote the catalytically active state of the NS5. By contrast, we show that a serotype-specific binding is driven by specific interactions involving conformational changes within the SLA RNA.
Zhang Jian-Hui, Eriani Gilbert, Zhou Xiao-Long
Pathophysiology of human mitochondrial tRNA metabolism Article de journal
Dans: Trends Endocrinol Metab, 2024, ISSN: 1879-3061.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, Unité ARN
@article{pmid38307811,
title = {Pathophysiology of human mitochondrial tRNA metabolism},
author = {Jian-Hui Zhang and Gilbert Eriani and Xiao-Long Zhou},
doi = {10.1016/j.tem.2024.01.002},
issn = {1879-3061},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {Trends Endocrinol Metab},
abstract = {Mitochondria play multiple critical roles in cellular activity. In particular, mitochondrial translation is pivotal in the regulation of mitochondrial and cellular homeostasis. In this forum article, we discuss human mitochondrial tRNA metabolism and highlight its tight connection with various mitochondrial diseases caused by mutations in aminoacyl-tRNA synthetases, tRNAs, and tRNA-modifying enzymes.},
keywords = {ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mitochondria play multiple critical roles in cellular activity. In particular, mitochondrial translation is pivotal in the regulation of mitochondrial and cellular homeostasis. In this forum article, we discuss human mitochondrial tRNA metabolism and highlight its tight connection with various mitochondrial diseases caused by mutations in aminoacyl-tRNA synthetases, tRNAs, and tRNA-modifying enzymes.
CF Estevez-Castro, MF Rodrigues, A Babarit, FV Ferreira, de Andrade EG, E Marois, R Cogni, ERGR Aguiar, JT Marques, RP Olmo
Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein Article de journal
Dans: BMC Biol , vol. 22, no. 14, 2024.
Résumé | Liens | BibTeX | Étiquettes: Aedes mosquitoes, Double-stranded RNA (dsRNA), dsRNA binding protein (dsRBP), loqs2, M3i, marois, Marques, Olmo, RNA interference (RNAi)
@article{CF2024,
title = {Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein},
author = {Estevez-Castro CF and Rodrigues MF and Babarit A and Ferreira FV and de Andrade EG and Marois E and Cogni R and Aguiar ERGR and Marques JT and Olmo RP},
url = {https://doi.org/10.1186/s12915-024-01821-4},
doi = {10.1186/s12915-024-01821-4},
year = {2024},
date = {2024-01-25},
urldate = {2024-01-25},
journal = {BMC Biol },
volume = {22},
number = {14},
abstract = {Background
Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects.
Results
Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference.
Conclusions
Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.},
keywords = {Aedes mosquitoes, Double-stranded RNA (dsRNA), dsRNA binding protein (dsRBP), loqs2, M3i, marois, Marques, Olmo, RNA interference (RNAi)},
pubstate = {published},
tppubtype = {article}
}
Background
Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects.
Results
Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference.
Conclusions
Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.
Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects.
Results
Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference.
Conclusions
Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.
Green Emily I, Jaouen Etienne, Klug Dennis, Olmo Roenick Proveti, Gautier Amandine, Blandin Stéphanie, Marois Eric
A population modification gene drive targeting both Saglin and Lipophorin disables Plasmodium transmission in Anopheles mosquitoes Article de journal
Dans: Genetics and Genomics, 2024.
Résumé | Liens | BibTeX | Étiquettes: Anopheles, blandin, gene drive, Lipophorin, M3i, marois, marque, Marques, Olmo, Plasmodium, Saglin
@article{Green2024,
title = {A population modification gene drive targeting both Saglin and Lipophorin disables Plasmodium transmission in Anopheles mosquitoes},
author = {Emily I Green and Etienne Jaouen and Dennis Klug and Roenick Proveti Olmo and Amandine Gautier and Stéphanie Blandin and Eric Marois},
url = {https://elifesciences.org/articles/93142},
doi = {10.7554/eLife.93142},
year = {2024},
date = {2024-01-12},
journal = {Genetics and Genomics},
abstract = {Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.},
keywords = {Anopheles, blandin, gene drive, Lipophorin, M3i, marois, marque, Marques, Olmo, Plasmodium, Saglin},
pubstate = {published},
tppubtype = {article}
}
Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.
Krishnan A., Ali L. M., Prabhu S. G., Pillai V. N., Chameettachal A., Vivet-Boudou V., Bernacchi S., Mustafa F., Marquet R., Rizvi T. A.
Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging Article de journal
Dans: RNA Journal, vol. 30, p. 68-88, 2024.
Résumé | Liens | BibTeX | Étiquettes: bernacchi, MARQUET, Unité ARN, vivet-boudou
@article{nokey,
title = {Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging},
author = {A. Krishnan and L.M. Ali and S.G. Prabhu and V.N. Pillai and A. Chameettachal and V. Vivet-Boudou and S. Bernacchi and F. Mustafa and R. Marquet and T.A. Rizvi},
editor = {cold spring harbor laboratory press},
url = {https://rnajournal.cshlp.org/content/30/1/68.long},
doi = {10.1261/rna.079840.123},
year = {2024},
date = {2024-01-10},
urldate = {2024-01-10},
journal = {RNA Journal},
volume = {30},
pages = {68-88},
abstract = {The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to
virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous
regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To
better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ
sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed
that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput
selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences,
with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained
nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our
data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays
performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to
mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating
sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion
assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of
FIV-based vectors for human gene therapy.},
keywords = {bernacchi, MARQUET, Unité ARN, vivet-boudou},
pubstate = {published},
tppubtype = {article}
}
The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to
virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous
regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To
better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ
sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed
that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput
selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences,
with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained
nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our
data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays
performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to
mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating
sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion
assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of
FIV-based vectors for human gene therapy.
virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous
regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To
better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ
sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed
that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput
selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences,
with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained
nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our
data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays
performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to
mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating
sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion
assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of
FIV-based vectors for human gene therapy.
Marois Eric
Using the CRISPR / Cas9 system for genome editing in Anopheles mosquitoes En ligne
2024, Visité: 08.01.2024.
Résumé | Liens | BibTeX | Étiquettes: Anopheles, CRISPR/Cas9, Genome editing, M3i, marois, mosquitoes
@online{Marois2024,
title = {Using the CRISPR / Cas9 system for genome editing in Anopheles mosquitoes},
author = { Eric Marois},
url = {https://hal.science/hal-04380430/document},
doi = {HAL Id: hal-04380430},
year = {2024},
date = {2024-01-08},
urldate = {2024-01-08},
journal = {HAL science},
abstract = {The advent of the CRISPR / Cas9 technology permits the targeted editing of mosquito genomes, ranging from site-directed mutagenesis of genes of interest yielding knockout mutations (which arise by insertion / deletion of a few nucleotides) to site-specific insertion of exogenous DNA sequences such as fluorescence markers or even large gene drive cassettes, themselves encoding the components of the CRISPR / Cas9 system. To obtain these heritable targeted changes, genome editing requires the delivery of Cas9 protein and its guide RNA(s) to the developing germ tissue of an embryo. Different species require adaptation of this basic principle to accommodate for their specific biology. Here, we describe a technical pipeline based on delivering the CRISPR/Cas9 components in the form of injected plasmid or as transgenes, resulting in highly efficient gene editing in Anopheles malaria vector mosquitoes. We have reliably employed these methods to mutagenize > 20 different loci of interest in Anopheles coluzzii to date. },
keywords = {Anopheles, CRISPR/Cas9, Genome editing, M3i, marois, mosquitoes},
pubstate = {published},
tppubtype = {online}
}
The advent of the CRISPR / Cas9 technology permits the targeted editing of mosquito genomes, ranging from site-directed mutagenesis of genes of interest yielding knockout mutations (which arise by insertion / deletion of a few nucleotides) to site-specific insertion of exogenous DNA sequences such as fluorescence markers or even large gene drive cassettes, themselves encoding the components of the CRISPR / Cas9 system. To obtain these heritable targeted changes, genome editing requires the delivery of Cas9 protein and its guide RNA(s) to the developing germ tissue of an embryo. Different species require adaptation of this basic principle to accommodate for their specific biology. Here, we describe a technical pipeline based on delivering the CRISPR/Cas9 components in the form of injected plasmid or as transgenes, resulting in highly efficient gene editing in Anopheles malaria vector mosquitoes. We have reliably employed these methods to mutagenize > 20 different loci of interest in Anopheles coluzzii to date.
Khodr Radi, Husser Claire, Ryckelynck Michael
Direct fluoride monitoring using a fluorogenic RNA-based biosensor Chapitre d'ouvrage
Dans: Stockbridge, Randy B. (Ed.): vol. 696, p. 85–107, Randy B. Stockbridge, 2024, ISSN: 1557-7988.
Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN
@inbook{pmid38658090,
title = {Direct fluoride monitoring using a fluorogenic RNA-based biosensor},
author = {Radi Khodr and Claire Husser and Michael Ryckelynck},
editor = {Randy B. Stockbridge},
doi = {10.1016/bs.mie.2023.12.019},
issn = {1557-7988},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Methods Enzymol},
volume = {696},
pages = {85--107},
publisher = {Randy B. Stockbridge},
abstract = {Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.
Janvier Aurélie, Hayek Hassan, Alghoul Fatima, Gross Lauriane, Allmang Christine, Martin Franck, Eriani Gilbert
Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods Chapitre d'ouvrage
Dans: vol. 3234, p. 17–29, M. Cristina Vega, Francisco J. Fernández, Springer, 2024, ISSN: 0065-2598.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, MARTIN, Unité ARN
@inbook{pmid38507197,
title = {Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods},
author = {Aurélie Janvier and Hassan Hayek and Fatima Alghoul and Lauriane Gross and Christine Allmang and Franck Martin and Gilbert Eriani},
doi = {10.1007/978-3-031-52193-5_2},
issn = {0065-2598},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Adv Exp Med Biol},
volume = {3234},
pages = {17--29},
publisher = {M. Cristina Vega, Francisco J. Fernández},
edition = {Springer},
series = {Advanced Technologies for Protein Complex Production and Characterization},
abstract = {Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.},
keywords = {ERIANI, MARTIN, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.
Hayek Hassan, Gross Lauriane, Alghoul Fatima, Martin Franck, Eriani Gilbert, Allmang Christine
Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP) Chapitre d'ouvrage
Dans: vol. 3234, p. 1–15, M. Cristina Vega, Francisco J. Fernández, Springer, 2024, ISSN: 0065-2598.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, MARTIN, PPSE, Unité ARN
@inbook{pmid38507196,
title = {Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP)},
author = {Hassan Hayek and Lauriane Gross and Fatima Alghoul and Franck Martin and Gilbert Eriani and Christine Allmang},
doi = {10.1007/978-3-031-52193-5_1},
issn = {0065-2598},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Adv Exp Med Biol},
volume = {3234},
pages = {1--15},
publisher = {M. Cristina Vega, Francisco J. Fernández},
edition = {Springer},
series = {Advanced Technologies for Protein Complex Production and Characterization},
abstract = {Throughout their life cycle, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Each mRNA is part of multiple successive mRNP complexes that participate in their biogenesis, cellular localization, translation and decay. The dynamic composition of mRNP complexes and their structural remodelling play crucial roles in the control of gene expression. Studying the endogenous composition of different mRNP complexes is a major challenge. In this chapter, we describe the variety of protein-centric immunoprecipitation methods available for the identification of mRNP complexes and the requirements for their experimental settings.},
keywords = {ERIANI, MARTIN, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Throughout their life cycle, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Each mRNA is part of multiple successive mRNP complexes that participate in their biogenesis, cellular localization, translation and decay. The dynamic composition of mRNP complexes and their structural remodelling play crucial roles in the control of gene expression. Studying the endogenous composition of different mRNP complexes is a major challenge. In this chapter, we describe the variety of protein-centric immunoprecipitation methods available for the identification of mRNP complexes and the requirements for their experimental settings.
Quignon E., Ferhadian D., Hache A., Vivet-Boudou V., Isel C., Printz-Schweigert A., Donchet A., Crépin T., Marquet R.
Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments Article de journal
Dans: Viruses, vol. 16, no. 3, p. 421, 2024, ISBN: 10.3390/v16030421.
Résumé | Liens | BibTeX | Étiquettes: MARQUET, MARQUET influenza A virus NP nucleoprotein vRNA RNA structure RNA chaperon chemical probing, Unité ARN
@article{nokey,
title = {Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments},
author = {E. Quignon and D. Ferhadian and A. Hache and V. Vivet-Boudou and C. Isel and A. Printz-Schweigert and A. Donchet and T. Crépin and R. Marquet},
url = {https://www.mdpi.com/1999-4915/16/3/421},
isbn = {10.3390/v16030421},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Viruses},
volume = {16},
number = {3},
pages = {421},
abstract = {Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.},
keywords = {MARQUET, MARQUET influenza A virus NP nucleoprotein vRNA RNA structure RNA chaperon chemical probing, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.
Nerantzaki Maria, Husser Claire, Ryckelynck Michael, Lutz Jean-François
Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy Article de journal
Dans: J Am Chem Soc, 2024, ISSN: 1520-5126.
Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN
@article{pmid38286022,
title = {Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy},
author = {Maria Nerantzaki and Claire Husser and Michael Ryckelynck and Jean-François Lutz},
doi = {10.1021/jacs.3c13953},
issn = {1520-5126},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {J Am Chem Soc},
abstract = {Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.
Baldaccini Morgane, Gaucherand Léa, Chane-Woon-Ming Béatrice, Messmer Mélanie, Gucciardi Floriane, Pfeffer Sébastien
The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways Article de journal
Dans: EMBO J, 2024, ISSN: 1460-2075.
Résumé | Liens | BibTeX | Étiquettes: PFEFFER, Unité ARN
@article{pmid38287188,
title = {The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways},
author = {Morgane Baldaccini and Léa Gaucherand and Béatrice Chane-Woon-Ming and Mélanie Messmer and Floriane Gucciardi and Sébastien Pfeffer},
doi = {10.1038/s44318-024-00035-2},
issn = {1460-2075},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {EMBO J},
abstract = {In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.},
keywords = {PFEFFER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
Kohl Maximilian P, Chane-Woon-Ming Béatrice, Bahena-Ceron Roberto, Jaramillo-Ponce Jose, Antoine Laura, Herrgott Lucas, Romby Pascale, Marzi Stefano
Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus Chapitre d'ouvrage
Dans: Claudio ValverdeVéronique Arluison Véronique Arluison, Claudio Valverde (Ed.): vol. 2741, p. 73–100, Véronique Arluison, Claudio Valverde, Springer, 2024, ISSN: 1940-6029.
Résumé | Liens | BibTeX | Étiquettes: MARZI, ROMBY, Unité ARN
@inbook{pmid38217649,
title = {Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus},
author = {Maximilian P Kohl and Béatrice Chane-Woon-Ming and Roberto Bahena-Ceron and Jose Jaramillo-Ponce and Laura Antoine and Lucas Herrgott and Pascale Romby and Stefano Marzi},
editor = {Véronique Arluison, Claudio ValverdeVéronique Arluison, Claudio Valverde},
doi = {10.1007/978-1-0716-3565-0_5},
issn = {1940-6029},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Methods Mol Biol},
volume = {2741},
pages = {73--100},
publisher = {Véronique Arluison, Claudio Valverde},
edition = {Springer},
series = {Bacterial Regulatory RNA, Methods and Protocols},
abstract = {Noncoding RNAs, including regulatory RNAs (sRNAs), are instrumental in regulating gene expression in pathogenic bacteria, allowing them to adapt to various stresses encountered in their host environments. Staphylococcus aureus is a well-studied model for RNA-mediated regulation of virulence and pathogenicity, with sRNAs playing significant roles in shaping S. aureus interactions with human and animal hosts. By modulating the translation and/or stability of target mRNAs, sRNAs regulate the synthesis of virulence factors and regulatory proteins required for pathogenesis. Moreover, perturbation of the levels of RNA modifications in two other classes of noncoding RNAs, rRNAs, and tRNAs, has been proposed to contribute to stress adaptation. However, the study of how these various factors affect translation regulation has often been restricted to specific genes, using in vivo reporters and/or in vitro translation systems. Genome-wide sequencing approaches offer novel perspectives for studying RNA-dependent regulation. In particular, ribosome profiling methods provide a powerful resource for characterizing the overall landscape of translational regulation, contributing to a better understanding of S. aureus physiopathology. Here, we describe protocols that we have adapted to perform ribosome profiling in S. aureus.},
keywords = {MARZI, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Noncoding RNAs, including regulatory RNAs (sRNAs), are instrumental in regulating gene expression in pathogenic bacteria, allowing them to adapt to various stresses encountered in their host environments. Staphylococcus aureus is a well-studied model for RNA-mediated regulation of virulence and pathogenicity, with sRNAs playing significant roles in shaping S. aureus interactions with human and animal hosts. By modulating the translation and/or stability of target mRNAs, sRNAs regulate the synthesis of virulence factors and regulatory proteins required for pathogenesis. Moreover, perturbation of the levels of RNA modifications in two other classes of noncoding RNAs, rRNAs, and tRNAs, has been proposed to contribute to stress adaptation. However, the study of how these various factors affect translation regulation has often been restricted to specific genes, using in vivo reporters and/or in vitro translation systems. Genome-wide sequencing approaches offer novel perspectives for studying RNA-dependent regulation. In particular, ribosome profiling methods provide a powerful resource for characterizing the overall landscape of translational regulation, contributing to a better understanding of S. aureus physiopathology. Here, we describe protocols that we have adapted to perform ribosome profiling in S. aureus.
Lechner Antony, Wolff Philippe
In-Gel Cyanoethylation for Pseudouridines Mass Spectrometry Detection of Bacterial Regulatory RNA Chapitre d'ouvrage
Dans: Véronique Arluison, Claudio Valverde (Ed.): vol. 2741, p. 273–287, Véronique Arluison, Claudio Valverde, 2024, ISSN: 1940-6029.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, Unité ARN
@inbook{pmid38217659,
title = {In-Gel Cyanoethylation for Pseudouridines Mass Spectrometry Detection of Bacterial Regulatory RNA},
author = {Antony Lechner and Philippe Wolff},
editor = {Véronique Arluison, Claudio Valverde},
doi = {10.1007/978-1-0716-3565-0_15},
issn = {1940-6029},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Methods Mol Biol},
volume = {2741},
pages = {273--287},
publisher = {Véronique Arluison, Claudio Valverde},
series = {Bacterial Regulatory RNA, Methods and Protocols},
abstract = {Regulatory RNAs, as well as many RNA families, contain chemically modified nucleotides, including pseudouridines (ψ). To map nucleotide modifications, approaches based on enzymatic digestion of RNA followed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis were implemented several years ago. However, detection of ψ by mass spectrometry (MS) is challenging as ψ exhibits the same mass as uridine. Thus, a chemical labeling strategy using acrylonitrile was developed to detect this mass-silent modification. Acrylonitrile reacts specifically to ψ to form 1-cyanoethylpseudouridine (Ceψ), resulting in a mass shift of ψ detectable by MS. Here, a protocol detailing the steps from the purification of RNA by polyacrylamide gel electrophoresis, including in-gel labeling of ψ, to MS data interpretation to map ψ and other modifications is proposed. To demonstrate its efficiency, the protocol was applied to bacterial regulatory RNAs from E. coli: 6S RNA and transfer-messenger RNA (tmRNA, also known as 10Sa RNA). Moreover, ribonuclease P (RNase P) was also mapped using this approach. This method enabled the detection of several ψ at single nucleotide resolution.},
keywords = {ARN-MS, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Regulatory RNAs, as well as many RNA families, contain chemically modified nucleotides, including pseudouridines (ψ). To map nucleotide modifications, approaches based on enzymatic digestion of RNA followed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis were implemented several years ago. However, detection of ψ by mass spectrometry (MS) is challenging as ψ exhibits the same mass as uridine. Thus, a chemical labeling strategy using acrylonitrile was developed to detect this mass-silent modification. Acrylonitrile reacts specifically to ψ to form 1-cyanoethylpseudouridine (Ceψ), resulting in a mass shift of ψ detectable by MS. Here, a protocol detailing the steps from the purification of RNA by polyacrylamide gel electrophoresis, including in-gel labeling of ψ, to MS data interpretation to map ψ and other modifications is proposed. To demonstrate its efficiency, the protocol was applied to bacterial regulatory RNAs from E. coli: 6S RNA and transfer-messenger RNA (tmRNA, also known as 10Sa RNA). Moreover, ribonuclease P (RNase P) was also mapped using this approach. This method enabled the detection of several ψ at single nucleotide resolution.
2023
Ponce José R Jaramillo, Frugier Magali
Plasmodium, the Apicomplexa Outlier When It Comes to Protein Synthesis Article de journal
Dans: Biomolecules, vol. 14, no. 1, p. 46, 2023, ISSN: 2218-273X.
Résumé | Liens | BibTeX | Étiquettes: FRUGIER, Unité ARN
@article{pmid38254646,
title = {Plasmodium, the Apicomplexa Outlier When It Comes to Protein Synthesis},
author = {José R Jaramillo Ponce and Magali Frugier},
doi = {10.3390/biom14010046},
issn = {2218-273X},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Biomolecules},
volume = {14},
number = {1},
pages = {46},
abstract = {Plasmodium is an obligate intracellular parasite that has numerous interactions with different hosts during its elaborate life cycle. This is also the case for the other parasites belonging to the same phylum Apicomplexa. In this study, we bioinformatically identified the components of the multi-synthetase complexes (MSCs) of several Apicomplexa parasites and modelled their assembly using AlphaFold2. It appears that none of these MSCs resemble the two MSCs that we have identified and characterized in Plasmodium. Indeed, tRip, the central protein involved in the association of the two Plasmodium MSCs is different from its homologues, suggesting also that the tRip-dependent import of exogenous tRNAs is not conserved in other apicomplexan parasites. Based on this observation, we searched for obvious differences that could explain the singularity of Plasmodium protein synthesis by comparing tRNA genes and amino acid usage in the different genomes. We noted a contradiction between the large number of asparagine residues used in Plasmodium proteomes and the single gene encoding the tRNA that inserts them into proteins. This observation remains true for all the Plasmodia strains studied, even those that do not contain long asparagine homorepeats. },
keywords = {FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Plasmodium is an obligate intracellular parasite that has numerous interactions with different hosts during its elaborate life cycle. This is also the case for the other parasites belonging to the same phylum Apicomplexa. In this study, we bioinformatically identified the components of the multi-synthetase complexes (MSCs) of several Apicomplexa parasites and modelled their assembly using AlphaFold2. It appears that none of these MSCs resemble the two MSCs that we have identified and characterized in Plasmodium. Indeed, tRip, the central protein involved in the association of the two Plasmodium MSCs is different from its homologues, suggesting also that the tRip-dependent import of exogenous tRNAs is not conserved in other apicomplexan parasites. Based on this observation, we searched for obvious differences that could explain the singularity of Plasmodium protein synthesis by comparing tRNA genes and amino acid usage in the different genomes. We noted a contradiction between the large number of asparagine residues used in Plasmodium proteomes and the single gene encoding the tRNA that inserts them into proteins. This observation remains true for all the Plasmodia strains studied, even those that do not contain long asparagine homorepeats.
Bahena-Ceron Roberto, Teixeira Chloe, Ponce Jose R Jaramillo, Wolff Philippe, Couzon Florence, François Pauline, Klaholz Bruno, Vandenesch François, Romby Pascale, Moreau Karen, Marzi Stefano
RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in Article de journal
Dans: RNA, vol. 30, no. 3, p. 200-212, 2023, ISSN: 1469-9001.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ROMBY, Unité ARN
@article{pmid38164596,
title = {RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in },
author = {Roberto Bahena-Ceron and Chloe Teixeira and Jose R Jaramillo Ponce and Philippe Wolff and Florence Couzon and Pauline François and Bruno Klaholz and François Vandenesch and Pascale Romby and Karen Moreau and Stefano Marzi},
doi = {10.1261/rna.079850.123},
issn = {1469-9001},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {RNA},
volume = {30},
number = {3},
pages = {200-212},
abstract = {rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in responsible for the Gram-positive specific mG2601, which is not modified in (G2574). We also demonstrate the absence of methylation on C1989, equivalent to C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications ( mG2601 and mC1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of Q causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.},
keywords = {ARN-MS, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in responsible for the Gram-positive specific mG2601, which is not modified in (G2574). We also demonstrate the absence of methylation on C1989, equivalent to C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications ( mG2601 and mC1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of Q causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.
Kompatscher Maria, Bartosik Karolina, Erharter Kevin, Plangger Raphael, Juen Fabian Sebastian, Kreutz Christoph, Micura Ronald, Westhof Eric, Erlacher Matthias D
Contribution of tRNA sequence and modifications to the decoding preferences of E. coli and M. mycoides tRNAGlyUCC for synonymous glycine codons Article de journal
Dans: Nucleic Acids Res, 2023, ISSN: 1362-4962.
Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF
@article{pmid38050960,
title = {Contribution of tRNA sequence and modifications to the decoding preferences of E. coli and M. mycoides tRNAGlyUCC for synonymous glycine codons},
author = {Maria Kompatscher and Karolina Bartosik and Kevin Erharter and Raphael Plangger and Fabian Sebastian Juen and Christoph Kreutz and Ronald Micura and Eric Westhof and Matthias D Erlacher},
doi = {10.1093/nar/gkad1136},
issn = {1362-4962},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Nucleic Acids Res},
abstract = {tRNA superwobbling, used by certain bacteria and organelles, is an intriguing decoding concept in which a single tRNA isoacceptor is used to decode all synonymous codons of a four-fold degenerate codon box. While Escherichia coli relies on three tRNAGly isoacceptors to decode the four glycine codons (GGN), Mycoplasma mycoides requires only a single tRNAGly. Both organisms express tRNAGly with the anticodon UCC, which are remarkably similar in sequence but different in their decoding ability. By systematically introducing mutations and altering the number and type of tRNA modifications using chemically synthesized tRNAs, we elucidated the contribution of individual nucleotides and chemical groups to decoding by the E. coli and M. mycoides tRNAGly. The tRNA sequence was identified as the key factor for superwobbling, revealing the T-arm sequence as a novel pivotal element. In addition, the presence of tRNA modifications, although not essential for providing superwobbling, was shown to delicately fine-tune and balance the decoding of synonymous codons. This emphasizes that the tRNA sequence and its modifications together form an intricate system of high complexity that is indispensable for accurate and efficient decoding.},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
tRNA superwobbling, used by certain bacteria and organelles, is an intriguing decoding concept in which a single tRNA isoacceptor is used to decode all synonymous codons of a four-fold degenerate codon box. While Escherichia coli relies on three tRNAGly isoacceptors to decode the four glycine codons (GGN), Mycoplasma mycoides requires only a single tRNAGly. Both organisms express tRNAGly with the anticodon UCC, which are remarkably similar in sequence but different in their decoding ability. By systematically introducing mutations and altering the number and type of tRNA modifications using chemically synthesized tRNAs, we elucidated the contribution of individual nucleotides and chemical groups to decoding by the E. coli and M. mycoides tRNAGly. The tRNA sequence was identified as the key factor for superwobbling, revealing the T-arm sequence as a novel pivotal element. In addition, the presence of tRNA modifications, although not essential for providing superwobbling, was shown to delicately fine-tune and balance the decoding of synonymous codons. This emphasizes that the tRNA sequence and its modifications together form an intricate system of high complexity that is indispensable for accurate and efficient decoding.
Skerniskyte Jurate, Mulet Céline, André Antonin C, Anderson Mark C, Injarabian Louise, Buck Achim, Prade Verena M, Sansonetti Philippe J, Reibel-Foisset Sophie, Walch Axel K, Lebel Michel, Lykkesfeldt Jens, Marteyn Benoit S
Ascorbate deficiency increases progression of shigellosis in guinea pigs and mice infection models Article de journal
Dans: Gut Microbes, vol. 15, no. 2, p. 2271597, 2023, ISSN: 1949-0984.
Résumé | Liens | BibTeX | Étiquettes: MARTEYN, Unité ARN
@article{pmid37876025,
title = {Ascorbate deficiency increases progression of shigellosis in guinea pigs and mice infection models},
author = {Jurate Skerniskyte and Céline Mulet and Antonin C André and Mark C Anderson and Louise Injarabian and Achim Buck and Verena M Prade and Philippe J Sansonetti and Sophie Reibel-Foisset and Axel K Walch and Michel Lebel and Jens Lykkesfeldt and Benoit S Marteyn},
doi = {10.1080/19490976.2023.2271597},
issn = {1949-0984},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {Gut Microbes},
volume = {15},
number = {2},
pages = {2271597},
abstract = { spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of entire life cycle and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (, 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased penetration into the colon epithelium layer in a Gulo mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both infection strategies and immune responses to infection.},
keywords = {MARTEYN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of entire life cycle and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (, 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased penetration into the colon epithelium layer in a Gulo mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both infection strategies and immune responses to infection.
A Hinze, J Pelletier, M Ghaninia, E Marois, Hill, R S Ignell
Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii Article de journal
Dans: Proc Biol Sci 290(2011):20232092, vol. 290, no. 2011, 2023.
Résumé | Liens | BibTeX | Étiquettes: CRISPR–Cas9, human odour, M3i, marois, mosquito flight, odour valence, SSR
@article{Hinze2023,
title = {Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii},
author = {Hinze A and Pelletier J and Ghaninia M and Marois E and Hill and S Ignell R},
url = {https://doi.org/10.1098/rspb.2023.2092},
doi = {10.1098/rspb.2023.2092},
year = {2023},
date = {2023-11-29},
urldate = {2023-11-29},
booktitle = {Proc Biol Sci},
journal = {Proc Biol Sci 290(2011):20232092},
volume = {290},
number = {2011},
abstract = {The attraction of anthropophilic mosquitoes to human host cues, such as body odour and carbon dioxide, gradually increases during adult maturation. This acquisition of host-seeking behaviour correlates with age-dependent changes in odorant receptor (OR) transcript abundance and sensitivity of olfactory sensory neurons (OSNs). One OR gene of the human malaria vector, Anopheles coluzzii, AcolOR39, is significantly downregulated in mature females, and a cognate ligand of AcolOR39, sulcatone, a major component of human emanations, mediates the observed behavioural inhibition of newly emerged (teneral) females to human body odour. Knockout of AcolOR39, using CRISPR–Cas9 mutagenesis, selectively abolished sulcatone detection in OSNs, housed in trichoid sensilla. However, knockout of AcolOR39 altered neither the response rate nor the flight behaviour of teneral females in a wind tunnel, indicating the involvement of other genes, and thus a redundancy, in regulating the acquisition of host seeking in mosquitoes.},
keywords = {CRISPR–Cas9, human odour, M3i, marois, mosquito flight, odour valence, SSR},
pubstate = {published},
tppubtype = {article}
}
The attraction of anthropophilic mosquitoes to human host cues, such as body odour and carbon dioxide, gradually increases during adult maturation. This acquisition of host-seeking behaviour correlates with age-dependent changes in odorant receptor (OR) transcript abundance and sensitivity of olfactory sensory neurons (OSNs). One OR gene of the human malaria vector, Anopheles coluzzii, AcolOR39, is significantly downregulated in mature females, and a cognate ligand of AcolOR39, sulcatone, a major component of human emanations, mediates the observed behavioural inhibition of newly emerged (teneral) females to human body odour. Knockout of AcolOR39, using CRISPR–Cas9 mutagenesis, selectively abolished sulcatone detection in OSNs, housed in trichoid sensilla. However, knockout of AcolOR39 altered neither the response rate nor the flight behaviour of teneral females in a wind tunnel, indicating the involvement of other genes, and thus a redundancy, in regulating the acquisition of host seeking in mosquitoes.
SC Weng, I Antoshechkin, E Marois, O Akbari
Efficient Sex Separation by Exploiting Differential Alternative Splicing of a Dominant Marker in Aedes aegypti Article de journal
Dans: PLoS Genetics, 2023.
Résumé | Liens | BibTeX | Étiquettes: Aedes aegypti, Introns, larvae, M3i, marois, mosquitoes, pupae, rna sequencing, transcriptome analyses
@article{nokey,
title = {Efficient Sex Separation by Exploiting Differential Alternative Splicing of a Dominant Marker in Aedes aegypti},
author = {Weng SC and Antoshechkin I and Marois E and Akbari O},
url = {https://doi.org/10.1371/journal.pgen.1011065},
doi = {10.1371/journal.pgen.1011065},
year = {2023},
date = {2023-11-27},
urldate = {2023-11-27},
journal = {PLoS Genetics},
abstract = {Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.},
keywords = {Aedes aegypti, Introns, larvae, M3i, marois, mosquitoes, pupae, rna sequencing, transcriptome analyses},
pubstate = {published},
tppubtype = {article}
}
Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.
Arrivé Mathilde, Bruggeman Mathieu, Skaltsogiannis Vasileios, Coudray Léna, Quan Yi-Fat, Schelcher Cédric, Cognat Valérie, Hammann Philippe, Chicher Johana, Wolff Philippe, Gobert Anthony, Giegé Philippe
A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei Article de journal
Dans: Nat Plants, vol. 9, iss. 12, p. 2031-2041, 2023, ISSN: 2055-0278.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, PPSE, Unité ARN
@article{pmid37945696,
title = {A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei},
author = {Mathilde Arrivé and Mathieu Bruggeman and Vasileios Skaltsogiannis and Léna Coudray and Yi-Fat Quan and Cédric Schelcher and Valérie Cognat and Philippe Hammann and Johana Chicher and Philippe Wolff and Anthony Gobert and Philippe Giegé},
doi = {10.1038/s41477-023-01564-0},
issn = {2055-0278},
year = {2023},
date = {2023-11-01},
urldate = {2023-11-01},
journal = {Nat Plants},
volume = {9},
issue = {12},
pages = {2031-2041},
abstract = {RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the mG26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a mG modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the mG-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.},
keywords = {ARN-MS, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the mG26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a mG modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the mG-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.
Lu Kunpeng, Pan Yifei, Shen Jianghong, Yang Lin, Zhan Chengyu, Liang Shubo, Tai Shuaishuai, Wan Linrong, Li Tian, Cheng Tingcai, Ma Bi, Pan Guoqing, He Ningjia, Lu Cheng, Westhof Eric, Xiang Zhonghuai, Han Min-Jin, Tong Xiaoling, Dai Fangyin
SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data Article de journal
Dans: Nucleic Acids Res, vol. 52, iss. D1, p. D1024-D1032, 2023, ISSN: 1362-4962.
Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF
@article{pmid37941143,
title = {SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data},
author = {Kunpeng Lu and Yifei Pan and Jianghong Shen and Lin Yang and Chengyu Zhan and Shubo Liang and Shuaishuai Tai and Linrong Wan and Tian Li and Tingcai Cheng and Bi Ma and Guoqing Pan and Ningjia He and Cheng Lu and Eric Westhof and Zhonghuai Xiang and Min-Jin Han and Xiaoling Tong and Fangyin Dai},
doi = {10.1093/nar/gkad956},
issn = {1362-4962},
year = {2023},
date = {2023-11-01},
urldate = {2023-11-01},
journal = {Nucleic Acids Res},
volume = {52},
issue = {D1},
pages = {D1024-D1032},
abstract = {The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.
Das Rhiju, Kretsch Rachael C, Simpkin Adam J, Mulvaney Thomas, Pham Phillip, Rangan Ramya, Bu Fan, Keegan Ronan M, Topf Maya, Rigden Daniel J, Miao Zhichao, Westhof Eric
Assessment of three-dimensional RNA structure prediction in CASP15 Article de journal
Dans: Proteins, vol. 91, iss. 12, p. 1747-1770, 2023, ISSN: 1097-0134.
Résumé | Liens | BibTeX | Étiquettes: Unité ARN, WESTHOF
@article{pmid37876231,
title = {Assessment of three-dimensional RNA structure prediction in CASP15},
author = {Rhiju Das and Rachael C Kretsch and Adam J Simpkin and Thomas Mulvaney and Phillip Pham and Ramya Rangan and Fan Bu and Ronan M Keegan and Maya Topf and Daniel J Rigden and Zhichao Miao and Eric Westhof},
doi = {10.1002/prot.26602},
issn = {1097-0134},
year = {2023},
date = {2023-10-01},
urldate = {2023-10-01},
journal = {Proteins},
volume = {91},
issue = {12},
pages = {1747-1770},
abstract = {The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report assessments of RNA structure predictions in CASP15, the first CASP exercise that involved RNA structure modeling. Forty-two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, which did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and x-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global fold of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as noncanonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.},
keywords = {Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
The prediction of RNA three-dimensional structures remains an unsolved problem. Here, we report assessments of RNA structure predictions in CASP15, the first CASP exercise that involved RNA structure modeling. Forty-two predictor groups submitted models for at least one of twelve RNA-containing targets. These models were evaluated by the RNA-Puzzles organizers and, separately, by a CASP-recruited team using metrics (GDT, lDDT) and approaches (Z-score rankings) initially developed for assessment of proteins and generalized here for RNA assessment. The two assessments independently ranked the same predictor groups as first (AIchemy_RNA2), second (Chen), and third (RNAPolis and GeneSilico, tied); predictions from deep learning approaches were significantly worse than these top ranked groups, which did not use deep learning. Further analyses based on direct comparison of predicted models to cryogenic electron microscopy (cryo-EM) maps and x-ray diffraction data support these rankings. With the exception of two RNA-protein complexes, models submitted by CASP15 groups correctly predicted the global fold of the RNA targets. Comparisons of CASP15 submissions to designed RNA nanostructures as well as molecular replacement trials highlight the potential utility of current RNA modeling approaches for RNA nanotechnology and structural biology, respectively. Nevertheless, challenges remain in modeling fine details such as noncanonical pairs, in ranking among submitted models, and in prediction of multiple structures resolved by cryo-EM or crystallography.
Silva P Malaka De, Bennett Rebecca J, Kuhn Lauriane, Ngondo Patryk, Debande Lorine, Njamkepo Elisabeth, Ho Brian, Weill François-Xavier, Marteyn Benoît S, Jenkins Claire, Baker Kate S
Escherichia coli killing by epidemiologically successful sublineages of Shigella sonnei is mediated by colicins Article de journal
Dans: EBioMedicine, vol. 97, p. 104822, 2023, ISSN: 2352-3964.
Résumé | Liens | BibTeX | Étiquettes: MARTEYN, PPSE, Unité ARN
@article{pmid37806286,
title = {Escherichia coli killing by epidemiologically successful sublineages of Shigella sonnei is mediated by colicins},
author = {P Malaka De Silva and Rebecca J Bennett and Lauriane Kuhn and Patryk Ngondo and Lorine Debande and Elisabeth Njamkepo and Brian Ho and François-Xavier Weill and Benoît S Marteyn and Claire Jenkins and Kate S Baker},
doi = {10.1016/j.ebiom.2023.104822},
issn = {2352-3964},
year = {2023},
date = {2023-10-01},
urldate = {2023-10-01},
journal = {EBioMedicine},
volume = {97},
pages = {104822},
abstract = {BACKGROUND: Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S. sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S. sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E. coli killing in S. sonnei.nnMETHODS: We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S. sonnei surveillance isolates to identify the genetic features associated with E. coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics.nnFINDINGS: Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E. coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent.nnINTERPRETATION: Colicins are responsible for E. coli killing by S. sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S. sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings.nnFUNDING: Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.},
keywords = {MARTEYN, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S. sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S. sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E. coli killing in S. sonnei.nnMETHODS: We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S. sonnei surveillance isolates to identify the genetic features associated with E. coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics.nnFINDINGS: Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E. coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent.nnINTERPRETATION: Colicins are responsible for E. coli killing by S. sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S. sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings.nnFUNDING: Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.
AI Kalita, E Marois, M Kozielska, FJ Weissing, E Jaouen, Möckel MM and Rühle F, F Butter, MF Basilicata, CI Keller Valsecchi
The sex-specific factor SOA controls dosage compensation in Anopheles mosquitoes Article de journal
Dans: Nature, vol. 623, p. 175–182, 2023.
Résumé | Liens | BibTeX | Étiquettes: Anopheles, M3i, marois, SOA
@article{AI2023,
title = {The sex-specific factor SOA controls dosage compensation in Anopheles mosquitoes},
author = {Kalita AI and Marois E and Kozielska M and Weissing FJ and Jaouen E and Möckel MM and, Rühle F and Butter F and Basilicata MF and Keller Valsecchi CI},
url = {https://www.nature.com/articles/s41586-023-06641-0},
doi = {10.1038/s41586-023-06641-0.},
year = {2023},
date = {2023-09-28},
journal = {Nature},
volume = {623},
pages = {175–182},
abstract = {The Anopheles mosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved1. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation (SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.},
keywords = {Anopheles, M3i, marois, SOA},
pubstate = {published},
tppubtype = {article}
}
The Anopheles mosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved1. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation (SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.
SR Abbo, de Almeida JPP, RP Olmo, C Balvers, JS Griep, C Linthout, CJM Koenraadt, BM Silva, JJ Fros, ERGR Aguiar, E Marois, GP Pijlman, JT Marques
The virome of the invasive Asian bush mosquito Aedes japonicus in Europe Article de journal
Dans: Virus Evol., vol. 9, iss. 2, 2023.
Résumé | Liens | BibTeX | Étiquettes: Aedes japonicus, anphevirus, bunyavirus, M3i, marois, Marques, metagenomics, mosquito, Olmo, rhabdovirus, RNA Interference, totivirus, virome
@article{Abbo2023,
title = {The virome of the invasive Asian bush mosquito Aedes japonicus in Europe},
author = {Abbo SR and de Almeida JPP and Olmo RP and Balvers C and Griep JS and Linthout C and Koenraadt CJM and Silva BM and Fros JJ and Aguiar ERGR and Marois E and Pijlman GP and Marques JT},
doi = {10.1093/ve/vead041},
year = {2023},
date = {2023-09-22},
urldate = {2023-09-22},
journal = {Virus Evol.},
volume = {9},
issue = {2},
abstract = {The Asian bush mosquito Aedes japonicus is rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus, and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that are receiving increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating in Ae. japonicus populations in the Netherlands and France. Applying a small RNA-based metagenomic approach to Ae. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus, Ae. japonicus narnavirus 1 (AejapNV1), and three new virus species that we named Ae. japonicus totivirus 1 (AejapTV1), Ae. japonicus anphevirus 1 (AejapAV1) and Ae. japonicus bunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses: Ae. japonicus bunyavirus 2 (AejapBV2) and Ae. japonicus rhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of twenty-one nucleotide-sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families; however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified an ∼1-kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1, and AejapBV1 by reverse transcriptase polymerase chain reaction (PCR) in wild-caught Ae. japonicus mosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100 per cent) in adult females, adult males, and larvae. Using a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome among Ae. japonicus mosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated with Ae. japonicus.},
keywords = {Aedes japonicus, anphevirus, bunyavirus, M3i, marois, Marques, metagenomics, mosquito, Olmo, rhabdovirus, RNA Interference, totivirus, virome},
pubstate = {published},
tppubtype = {article}
}
The Asian bush mosquito Aedes japonicus is rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus, and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that are receiving increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating in Ae. japonicus populations in the Netherlands and France. Applying a small RNA-based metagenomic approach to Ae. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus, Ae. japonicus narnavirus 1 (AejapNV1), and three new virus species that we named Ae. japonicus totivirus 1 (AejapTV1), Ae. japonicus anphevirus 1 (AejapAV1) and Ae. japonicus bunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses: Ae. japonicus bunyavirus 2 (AejapBV2) and Ae. japonicus rhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of twenty-one nucleotide-sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families; however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified an ∼1-kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1, and AejapBV1 by reverse transcriptase polymerase chain reaction (PCR) in wild-caught Ae. japonicus mosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100 per cent) in adult females, adult males, and larvae. Using a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome among Ae. japonicus mosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated with Ae. japonicus.
Labaronne E., Decimo D., Bertrand L., Guiguettaz L., Sohier T. J. M., Cluet D., Vivet-Boudou V., Dahoui C., François P., Hatin I., Lambotte O., Samri A., Autran B., Etienne L., Goujon C., Paillart J. -C., Namy O., Ramirez B. C., Ohlmann T., Morris A., Ricci E. P.
Dans: (bioRxiv), 2023.
Liens | BibTeX | Étiquettes: PAILLART, Unité ARN
@article{nokey,
title = {Extensive uORF translation from HIV-1 transcripts elicits specific T cell immune responses in infected individuals and conditions DDX3 dependency for expression of main ORFs},
author = {E. Labaronne and D. Decimo and L. Bertrand and L. Guiguettaz and T.J.M. Sohier and D. Cluet and V. Vivet-Boudou and C. Dahoui and P. François and I. Hatin and O. Lambotte and A. Samri and B. Autran and L. Etienne and C. Goujon and J.-C. Paillart and O. Namy and B.C. Ramirez and T. Ohlmann and A. Morris and E.P. Ricci},
doi = {10.1101/2022.04.29.489990},
year = {2023},
date = {2023-09-20},
urldate = {2023-09-20},
journal = {(bioRxiv)},
keywords = {PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
H Cai, L Li, KM Slavik, J Huang, T Yin, X Ai, L Hédelin, G Haas, Z Xiang, Y Yang, X Li, Y Chen, Z Wei, H Deng, D Chen, R Jiao, N Martins, C Meignin, PJ Kranzusch, JL Imler
The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila Article de journal
Dans: Immunity, vol. 56, iss. 9, p. 1991-2005, 2023.
Résumé | Liens | BibTeX | Étiquettes: c-di-GMP, cGAMP, cGAS, cGLR, cyclic dinucleotide, Drosophila, Evolution, Hua, imler, M3i, meignin, pattern recognition receptor, STING, virus
@article{nokey,
title = {The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila},
author = {Cai H and Li L and Slavik KM and Huang J and Yin T and Ai X and Hédelin L and Haas G and Xiang Z and Yang Y and Li X and Chen Y and Wei Z and Deng H and Chen D and Jiao R and Martins N and Meignin C and Kranzusch PJ and Imler JL},
editor = {Elsevier Inc. },
url = {https://pubmed.ncbi.nlm.nih.gov/37659413/},
doi = {10.1016/j.immuni.2023.08.006 },
year = {2023},
date = {2023-09-12},
urldate = {2023-09-12},
journal = {Immunity},
volume = {56},
issue = {9},
pages = {1991-2005},
abstract = {In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity. },
keywords = {c-di-GMP, cGAMP, cGAS, cGLR, cyclic dinucleotide, Drosophila, Evolution, Hua, imler, M3i, meignin, pattern recognition receptor, STING, virus},
pubstate = {published},
tppubtype = {article}
}
In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.
Marois Eric
Screening Mosquito Larvae Under a Fluorescence Binocular Microscope Article de journal
Dans: Cold Spring Harb Protoc, 2023.
Liens | BibTeX | Étiquettes: fluorescent protein, M3i, marois
@article{Marois2023,
title = {Screening Mosquito Larvae Under a Fluorescence Binocular Microscope},
author = {Eric Marois},
doi = {10.1101/pdb.prot108306 },
year = {2023},
date = {2023-09-11},
urldate = {2023-09-11},
journal = {Cold Spring Harb Protoc},
keywords = {fluorescent protein, M3i, marois},
pubstate = {published},
tppubtype = {article}
}
Tidu Antonin, Martin Franck
The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes Article de journal
Dans: Biochimie, vol. 217, p. 20-30, 2023, ISSN: 1638-6183.
Résumé | Liens | BibTeX | Étiquettes: MARTIN, Unité ARN
@article{pmid37741547,
title = {The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes},
author = {Antonin Tidu and Franck Martin},
doi = {10.1016/j.biochi.2023.09.017},
issn = {1638-6183},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {Biochimie},
volume = {217},
pages = {20-30},
abstract = {Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.},
keywords = {MARTIN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.
Dufossez Robin, Krafft Marie-Pierre, Ursuegui Sylvain, Mosser Michel, Mouftakhir Safae, Pernod Ketty, Chaubet Guilhem, Ryckelynck Michael, Wagner Alain
Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface Article de journal
Dans: ACS Appl Mater Interfaces, vol. 15, iss. 38, p. 45498-45505, 2023, ISSN: 1944-8252.
Résumé | Liens | BibTeX | Étiquettes: RYCKELYNCK, Unité ARN
@article{pmid37704020,
title = {Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface},
author = {Robin Dufossez and Marie-Pierre Krafft and Sylvain Ursuegui and Michel Mosser and Safae Mouftakhir and Ketty Pernod and Guilhem Chaubet and Michael Ryckelynck and Alain Wagner},
doi = {10.1021/acsami.3c10655},
issn = {1944-8252},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {ACS Appl Mater Interfaces},
volume = {15},
issue = {38},
pages = {45498-45505},
abstract = {Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.
Zheng Wen-Qiang, Zhang Jian-Hui, Li Zi-Han, Liu Xiuxiu, Zhang Yong, Huang Shuo, Li Jinsong, Zhou Bin, Eriani Gilbert, Wang En-Duo, Zhou Xiao-Long
Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy Article de journal
Dans: Proc Natl Acad Sci U S A, vol. 120, no. 37, p. e2309714120, 2023, ISSN: 1091-6490.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, Unité ARN
@article{pmid37669377,
title = {Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy},
author = {Wen-Qiang Zheng and Jian-Hui Zhang and Zi-Han Li and Xiuxiu Liu and Yong Zhang and Shuo Huang and Jinsong Li and Bin Zhou and Gilbert Eriani and En-Duo Wang and Xiao-Long Zhou},
doi = {10.1073/pnas.2309714120},
issn = {1091-6490},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {Proc Natl Acad Sci U S A},
volume = {120},
number = {37},
pages = {e2309714120},
abstract = {Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by ), disruption of which accumulated Ser-tRNA and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.},
keywords = {ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by ), disruption of which accumulated Ser-tRNA and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.
Saeb Sepideh, Wallet Clémentine, Rohr Olivier, Schwartz Christian, Loustau Thomas
Targeting and eradicating latent CNS reservoirs of HIV-1: Original strategies and new models Article de journal
Dans: Biochem Pharmacol, vol. 214, p. 115679, 2023, ISSN: 1873-2968.
Résumé | Liens | BibTeX | Étiquettes: ROHR, Unité ARN
@article{pmid37399950,
title = {Targeting and eradicating latent CNS reservoirs of HIV-1: Original strategies and new models},
author = {Sepideh Saeb and Clémentine Wallet and Olivier Rohr and Christian Schwartz and Thomas Loustau},
doi = {10.1016/j.bcp.2023.115679},
issn = {1873-2968},
year = {2023},
date = {2023-08-01},
urldate = {2023-08-01},
journal = {Biochem Pharmacol},
volume = {214},
pages = {115679},
abstract = {Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.
PELLETIER Julien, DAWIT Mengistu, GHANINIA Majid, MAROIS Eric, IGNELL Rickard
A mosquito-specific antennal protein is critical for the attraction to human odor in the malaria vector Anopheles gambiae Article de journal
Dans: Insect Biochemistry and Molecular Biology, vol. 159, iss. August 2023, 2023.
Résumé | Liens | BibTeX | Étiquettes: antenna, chemoreceptor, M3i, marois, mosquitoes, olfaction
@article{IGNELL2023,
title = {A mosquito-specific antennal protein is critical for the attraction to human odor in the malaria vector Anopheles gambiae},
author = {Julien PELLETIER AND Mengistu DAWIT AND Majid GHANINIA AND Eric MAROIS AND Rickard IGNELL},
editor = { },
url = {https://doi.org/10.1016/j.ibmb.2023.103988},
doi = {j.ibmb.2023.103988},
year = {2023},
date = {2023-07-11},
urldate = {2023-07-11},
journal = {Insect Biochemistry and Molecular Biology},
volume = {159},
issue = {August 2023},
abstract = {Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.},
keywords = {antenna, chemoreceptor, M3i, marois, mosquitoes, olfaction},
pubstate = {published},
tppubtype = {article}
}
Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.
Tardivat Yann, Sosnowski Piotr, Tidu Antonin, Westhof Eric, Eriani Gilbert, Martin Franck
SARS-CoV-2 NSP1 induces mRNA cleavages on the ribosome Article de journal
Dans: Nucleic Acids Res, vol. 51, iss. 16, p. 8677-8690, 2023, ISSN: 1362-4962.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, MARTIN, Unité ARN
@article{pmid37503833b,
title = {SARS-CoV-2 NSP1 induces mRNA cleavages on the ribosome},
author = {Yann Tardivat and Piotr Sosnowski and Antonin Tidu and Eric Westhof and Gilbert Eriani and Franck Martin},
doi = {10.1093/nar/gkad627},
issn = {1362-4962},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {Nucleic Acids Res},
volume = {51},
issue = {16},
pages = {8677-8690},
abstract = {In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the non-structural protein NSP1 inhibits translation of host mRNAs by binding to the mRNA entry channel of the ribosome and, together with the 5'-untranslated region (UTR) of the viral mRNAs, allows the evasion of that inhibition. Here, we show that NSP1 mediates endonucleolytic cleavages of both host and viral mRNAs in the 5'UTR, but with different cleavage patterns. The first pattern is observed in host mRNAs with cleavages interspersed regularly and close to the 5' cap (6-11 nt downstream of the cap). Those cleavage positions depend more on the position relative to the 5' cap than on the sequence itself. The second cleavage pattern occurs at high NSP1 concentrations and only in SARS-CoV-2 RNAs, with the cleavages clustered at positions 45, 46 and 49. Both patterns of cleavage occur with the mRNA and NSP1 bound to the ribosome, with the SL1 hairpin at the 5' end sufficient to protect from NSP1-mediated degradation at low NSP1 concentrations. We show further that the N-terminal domain of NSP1 is necessary and sufficient for efficient cleavage. We suggest that in the ribosome-bound NSP1 protein the catalytic residues of the N-terminal domain are unmasked by the remodelling of the α1- and α2-helices of the C-terminal domain.},
keywords = {ERIANI, MARTIN, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the non-structural protein NSP1 inhibits translation of host mRNAs by binding to the mRNA entry channel of the ribosome and, together with the 5'-untranslated region (UTR) of the viral mRNAs, allows the evasion of that inhibition. Here, we show that NSP1 mediates endonucleolytic cleavages of both host and viral mRNAs in the 5'UTR, but with different cleavage patterns. The first pattern is observed in host mRNAs with cleavages interspersed regularly and close to the 5' cap (6-11 nt downstream of the cap). Those cleavage positions depend more on the position relative to the 5' cap than on the sequence itself. The second cleavage pattern occurs at high NSP1 concentrations and only in SARS-CoV-2 RNAs, with the cleavages clustered at positions 45, 46 and 49. Both patterns of cleavage occur with the mRNA and NSP1 bound to the ribosome, with the SL1 hairpin at the 5' end sufficient to protect from NSP1-mediated degradation at low NSP1 concentrations. We show further that the N-terminal domain of NSP1 is necessary and sufficient for efficient cleavage. We suggest that in the ribosome-bound NSP1 protein the catalytic residues of the N-terminal domain are unmasked by the remodelling of the α1- and α2-helices of the C-terminal domain.
Pitolli Martina, Cela Marta, Paulus Caroline, Rudinger-Thirion Joëlle, Frugier Magali
RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium Article de journal
Dans: Biochimie, vol. 217, p. 106-115, 2023, ISSN: 1638-6183.
Résumé | Liens | BibTeX | Étiquettes: FRUGIER, Unité ARN
@article{pmid37414209,
title = {RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium},
author = {Martina Pitolli and Marta Cela and Caroline Paulus and Joëlle Rudinger-Thirion and Magali Frugier},
doi = {10.1016/j.biochi.2023.06.011},
issn = {1638-6183},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {Biochimie},
volume = {217},
pages = {106-115},
abstract = {Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.},
keywords = {FRUGIER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.
Wright Duncan E, Krol Alain
FEBS fellowships: supporting excellent science for over four decades Divers
2023, ISSN: 2211-5463.
Résumé | Liens | BibTeX | Étiquettes: KROL, Unité ARN
@misc{pmid37394995,
title = {FEBS fellowships: supporting excellent science for over four decades},
author = {Duncan E Wright and Alain Krol},
doi = {10.1002/2211-5463.13659},
issn = {2211-5463},
year = {2023},
date = {2023-07-01},
urldate = {2023-07-01},
journal = {FEBS Open Bio},
volume = {13},
number = {7},
pages = {1138--1139},
abstract = {The Federation of European Biochemical Societies (FEBS) awarded FEBS Long-Term Fellowships from 1979 until 2020, at which time the scheme was replaced with the FEBS Excellence Award. Over four decades, FEBS awarded a huge number of Long-Term Fellowships, helping to support and promote the careers of excellent young researchers across Europe. To celebrate the exciting work performed by the FEBS Long-Term Fellows, we present here a special 'In the Limelight' issue of FEBS Open Bio, containing four Mini-reviews and four Research Protocols authored by the fellows themselves. The four Review articles provide timely updates on the respective research fields, while the Research Protocols describe how to perform challenging experimental methods in detail. We hope this issue will be a valuable resource for the community, and a celebration of the high-quality work done by young scientists.},
keywords = {KROL, Unité ARN},
pubstate = {published},
tppubtype = {misc}
}
The Federation of European Biochemical Societies (FEBS) awarded FEBS Long-Term Fellowships from 1979 until 2020, at which time the scheme was replaced with the FEBS Excellence Award. Over four decades, FEBS awarded a huge number of Long-Term Fellowships, helping to support and promote the careers of excellent young researchers across Europe. To celebrate the exciting work performed by the FEBS Long-Term Fellows, we present here a special 'In the Limelight' issue of FEBS Open Bio, containing four Mini-reviews and four Research Protocols authored by the fellows themselves. The four Review articles provide timely updates on the respective research fields, while the Research Protocols describe how to perform challenging experimental methods in detail. We hope this issue will be a valuable resource for the community, and a celebration of the high-quality work done by young scientists.
C Lutrat, M Burckbuchler, RP Olmo, R Beugnon, A Fontaine, OS Akbari, R Argiles-Herrero, T Baldet, J Bouyer, E Marois
Combining two Genetic Sexing Strains allows sorting of non-transgenic males for Aedes genetic control Article de journal
Dans: Commun Biol., vol. 6, iss. 1, p. 646, 2023.
Résumé | Liens | BibTeX | Étiquettes: Aedes, M3i, marois, mosquitoes, Olmo, vectoring
@article{Lutrat2023,
title = {Combining two Genetic Sexing Strains allows sorting of non-transgenic males for Aedes genetic control},
author = {Lutrat C and Burckbuchler M and Olmo RP and Beugnon R and Fontaine A and Akbari OS and Argiles-Herrero R and Baldet T and Bouyer J and Marois E},
url = {https://www.nature.com/articles/s42003-023-05030-7},
doi = {10.1038/s42003-023-05030-7},
year = {2023},
date = {2023-06-16},
urldate = {2023-06-16},
journal = {Commun Biol.},
volume = {6},
issue = {1},
pages = {646},
abstract = {Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01–0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.},
keywords = {Aedes, M3i, marois, mosquitoes, Olmo, vectoring},
pubstate = {published},
tppubtype = {article}
}
Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01–0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.
Pivard Mariane, Caldelari Isabelle, Brun Virginie, Croisier Delphine, Jaquinod Michel, Anzala Nelson, Gilquin Benoît, Teixeira Chloé, Benito Yvonne, Couzon Florence, Romby Pascale, Moreau Karen, Vandenesch François
Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence Article de journal
Dans: Microbiol Spectr, p. e0107323, 2023, ISSN: 2165-0497.
Résumé | Liens | BibTeX | Étiquettes: ROMBY, Unité ARN
@article{pmid37347186,
title = {Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence},
author = {Mariane Pivard and Isabelle Caldelari and Virginie Brun and Delphine Croisier and Michel Jaquinod and Nelson Anzala and Benoît Gilquin and Chloé Teixeira and Yvonne Benito and Florence Couzon and Pascale Romby and Karen Moreau and François Vandenesch},
doi = {10.1128/spectrum.01073-23},
issn = {2165-0497},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Microbiol Spectr},
pages = {e0107323},
abstract = {Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.
Gaucherand Lea, Iyer Amrita, Gilabert Isabel, Rycroft Chris H, Gaglia Marta M
Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs Article de journal
Dans: Nat Microbiol, 2023, ISSN: 2058-5276.
Résumé | Liens | BibTeX | Étiquettes: PFEFFER, Unité ARN
@article{pmid37349586,
title = {Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs},
author = {Lea Gaucherand and Amrita Iyer and Isabel Gilabert and Chris H Rycroft and Marta M Gaglia},
doi = {10.1038/s41564-023-01409-8},
issn = {2058-5276},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Nat Microbiol},
abstract = {Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.},
keywords = {PFEFFER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5' rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.