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2020
Incarbone Marco, Scheer Hélene, Hily Jean-Michel, Kuhn Lauriane, Erhardt Mathieu, Dunoyer Patrice, Altenbach Denise, Ritzenthaler Christophe
Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana. Article de journal
Dans: Viruses, vol. 12, no. 10, 2020, ISSN: 1999-4915 1999-4915.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{incarbone_characterization_2020,
title = {Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana.},
author = {Marco Incarbone and Hélene Scheer and Jean-Michel Hily and Lauriane Kuhn and Mathieu Erhardt and Patrice Dunoyer and Denise Altenbach and Christophe Ritzenthaler},
doi = {10.3390/v12101121},
issn = {1999-4915 1999-4915},
year = {2020},
date = {2020-10-01},
journal = {Viruses},
volume = {12},
number = {10},
abstract = {Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to Saccharomyces cerevisiae.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to Saccharomyces cerevisiae.
Normant Vincent, Josts Inokentijs, Kuhn Lauriane, Perraud Quentin, Fritsch Sarah, Hammann Philippe, Mislin Gaëtan L A, Tidow Henning, Schalk Isabelle J
Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter. Article de journal
Dans: ACS chemical biology, vol. 15, no. 10, p. 2741–2751, 2020, ISSN: 1554-8937 1554-8929.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{normant_nocardamine-dependent_2020,
title = {Nocardamine-Dependent Iron Uptake in Pseudomonas aeruginosa: Exclusive Involvement of the FoxA Outer Membrane Transporter.},
author = {Vincent Normant and Inokentijs Josts and Lauriane Kuhn and Quentin Perraud and Sarah Fritsch and Philippe Hammann and Gaëtan L A Mislin and Henning Tidow and Isabelle J Schalk},
doi = {10.1021/acschembio.0c00535},
issn = {1554-8937 1554-8929},
year = {2020},
date = {2020-10-01},
journal = {ACS chemical biology},
volume = {15},
number = {10},
pages = {2741--2751},
abstract = {Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own siderophores or use those produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction of the transcription and expression of the outer membrane transporter FoxA in the presence of NOCA or DFOB in the bacterial environment. Expression of the proteins of the heme- or pyoverdine- and pyochelin-dependent iron uptake pathways was not affected by the presence of these two tris-hydroxamate siderophores. (55)Fe uptake assays using foxA mutants showed ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in light of these results.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own siderophores or use those produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a large panel of exosiderophores. We investigated the ability of P. aeruginosa to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction of the transcription and expression of the outer membrane transporter FoxA in the presence of NOCA or DFOB in the bacterial environment. Expression of the proteins of the heme- or pyoverdine- and pyochelin-dependent iron uptake pathways was not affected by the presence of these two tris-hydroxamate siderophores. (55)Fe uptake assays using foxA mutants showed ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-Fe. We discuss iron uptake by hydroxamate exosiderophores in P. aeruginosa cells in light of these results.
Wolff P, Villette C, Zumsteg J, Heintz D, Antoine L, Chane-Woon-Ming B, Droogmans L, Grosjean H, Westhof E
Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea Article de journal
Dans: RNA, vol. 26, no. 12, p. 1957-1975, 2020.
Résumé | Liens | BibTeX | Étiquettes: (hyper)thermophiles Archaea mass spectrometry modifications tRNA, ARN-MS, ENNIFAR, Unité ARN, WESTHOF
@article{,
title = {Comparative patterns of modified nucleotides in individual tRNA species from a mesophilic and two thermophilic archaea},
author = {P Wolff and C Villette and J Zumsteg and D Heintz and L Antoine and B Chane-Woon-Ming and L Droogmans and H Grosjean and E Westhof},
url = {https://pubmed.ncbi.nlm.nih.gov/32994183/},
doi = {10.1261/rna.077537.120},
year = {2020},
date = {2020-09-29},
journal = {RNA},
volume = {26},
number = {12},
pages = {1957-1975},
abstract = {To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale) and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.},
keywords = {(hyper)thermophiles Archaea mass spectrometry modifications tRNA, ARN-MS, ENNIFAR, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale) and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius. Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.
Kröll-Hermi Ariane, Ebstein Frédéric, Stoetzel Corinne, Geoffroy Véronique, Schaefer Elise, Scheidecker Sophie, Bär Séverine, Takamiya Masanari, Kawakami Koichi, Zieba Barbara A, Studer Fouzia, Pelletier Valerie, Eyermann Carine, Speeg-Schatz Claude, Laugel Vincent, Lipsker Dan, Sandron Florian, McGinn Steven, Boland Anne, Deleuze Jean-François, Kuhn Lauriane, Chicher Johana, Hammann Philippe, Friant Sylvie, Etard Christelle, Krüger Elke, Muller Jean, Strähle Uwe, Dollfus Hélène
Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress. Article de journal
Dans: EMBO molecular medicine, vol. 12, no. 7, p. e11861, 2020, ISSN: 1757-4684 1757-4676 1757-4676.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{kroll-hermi_proteasome_2020,
title = {Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress.},
author = {Ariane Kröll-Hermi and Frédéric Ebstein and Corinne Stoetzel and Véronique Geoffroy and Elise Schaefer and Sophie Scheidecker and Séverine Bär and Masanari Takamiya and Koichi Kawakami and Barbara A Zieba and Fouzia Studer and Valerie Pelletier and Carine Eyermann and Claude Speeg-Schatz and Vincent Laugel and Dan Lipsker and Florian Sandron and Steven McGinn and Anne Boland and Jean-François Deleuze and Lauriane Kuhn and Johana Chicher and Philippe Hammann and Sylvie Friant and Christelle Etard and Elke Krüger and Jean Muller and Uwe Strähle and Hélène Dollfus},
doi = {10.15252/emmm.201911861},
issn = {1757-4684 1757-4676 1757-4676},
year = {2020},
date = {2020-07-01},
journal = {EMBO molecular medicine},
volume = {12},
number = {7},
pages = {e11861},
abstract = {The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development.
Perraud Quentin, Cantero Paola, Roche Béatrice, Gasser Véronique, Normant Vincent P, Kuhn Lauriane, Hammann Philippe, Mislin Gaëtan L A, Ehret-Sabatier Laurence, Schalk Isabelle J
Phenotypic Adaption of Pseudomonas aeruginosa by Hacking Siderophores Produced by Other Microorganisms. Article de journal
Dans: Molecular & cellular proteomics : MCP, vol. 19, no. 4, p. 589–607, 2020, ISSN: 1535-9484 1535-9476 1535-9476.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{perraud_phenotypic_2020,
title = {Phenotypic Adaption of Pseudomonas aeruginosa by Hacking Siderophores Produced by Other Microorganisms.},
author = {Quentin Perraud and Paola Cantero and Béatrice Roche and Véronique Gasser and Vincent P Normant and Lauriane Kuhn and Philippe Hammann and Gaëtan L A Mislin and Laurence Ehret-Sabatier and Isabelle J Schalk},
doi = {10.1074/mcp.RA119.001829},
issn = {1535-9484 1535-9476 1535-9476},
year = {2020},
date = {2020-04-01},
journal = {Molecular & cellular proteomics : MCP},
volume = {19},
number = {4},
pages = {589--607},
abstract = {Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa, an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of P. aeruginosa in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how P. aeruginosa adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa, an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of P. aeruginosa in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how P. aeruginosa adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.
Janel-Bintz Régine, Kuhn Lauriane, Frit Philippe, Chicher Johana, Wagner Jérôme, Haracska Lajos, Hammann Philippe, Cordonnier Agnès M
Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication-Associated Proteins. Article de journal
Dans: Proteomics, vol. 20, no. 3-4, p. e1900184, 2020, ISSN: 1615-9861 1615-9853.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{janel-bintz_proteomic_2020,
title = {Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication-Associated Proteins.},
author = {Régine Janel-Bintz and Lauriane Kuhn and Philippe Frit and Johana Chicher and Jérôme Wagner and Lajos Haracska and Philippe Hammann and Agnès M Cordonnier},
doi = {10.1002/pmic.201900184},
issn = {1615-9861 1615-9853},
year = {2020},
date = {2020-02-01},
journal = {Proteomics},
volume = {20},
number = {3-4},
pages = {e1900184},
abstract = {It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo.
Simonetti A, Guca E, Bochler A, Kuhn L, Hashem Y
Structural Insights Into the Mammalian Late-Stage Initiation Complexes Article de journal
Dans: Cell Rep, vol. 31, no. 1, p. 107497, 2020, ISBN: 32268096.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, PPSE, Unité ARN
@article{,
title = {Structural Insights Into the Mammalian Late-Stage Initiation Complexes},
author = {A Simonetti and E Guca and A Bochler and L Kuhn and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32268096?dopt=Abstract},
doi = {10.1016/j.celrep.2020.03.061},
isbn = {32268096},
year = {2020},
date = {2020-01-01},
journal = {Cell Rep},
volume = {31},
number = {1},
pages = {107497},
abstract = {In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.},
keywords = {ENNIFAR, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.
Rol-Moreno J, Kuhn L, Marzi S, Simonetti A
Grad-cryo-EM: Tool to Isolate Translation Initiation Complexes From Rabbit Reticulocyte Lysate Suitable for Structural Studies Chapitre d'ouvrage
Dans: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, p. 329-339, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006323.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, PPSE, ROMBY, Unité ARN
@inbook{,
title = {Grad-cryo-EM: Tool to Isolate Translation Initiation Complexes From Rabbit Reticulocyte Lysate Suitable for Structural Studies},
author = {J Rol-Moreno and L Kuhn and S Marzi and A Simonetti},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006323},
doi = {10.1007/978-1-0716-0278-2_21},
isbn = {32006323},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {329-339},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation. The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.},
keywords = {ENNIFAR, PPSE, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation. The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.
Chernorudskiy A, Varone E, Colombo S F, Fumagalli S, Cagnotto A, Cattaneo A, Briens M, Baltzinger M, Kuhn L, Bachi A, Berardi A, Salmona M, Musco G, Borgese N, Lescure A, Zito E
Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity Article de journal
Dans: Proc Natl Acad Sci U S A, vol. 117, no. 35, p. 21288-21298, 2020, ISBN: 32817544.
Résumé | Liens | BibTeX | Étiquettes: LESCURE, PPSE, Unité ARN
@article{,
title = {Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity},
author = {A Chernorudskiy and E Varone and S F Colombo and S Fumagalli and A Cagnotto and A Cattaneo and M Briens and M Baltzinger and L Kuhn and A Bachi and A Berardi and M Salmona and G Musco and N Borgese and A Lescure and E Zito},
url = {https://pubmed.ncbi.nlm.nih.gov/32817544/},
doi = {10.1073/pnas.2003847117},
isbn = {32817544},
year = {2020},
date = {2020-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {117},
number = {35},
pages = {21288-21298},
abstract = {The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.},
keywords = {LESCURE, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.
Pernod K, Schaeffer L, Chicher J, Hok E, Rick C, Geslain R, Eriani G, Westhof E, Ryckelynck M, Martin F
The Nature of the Purine at Position 34 in tRNAs of 4-codon Boxes Is Correlated With Nucleotides at Positions 32 and 38 to Maintain Decoding Fidelity Article de journal
Dans: Nucleic Acids Res, vol. 48, no. 11, p. 6170-6183, 2020, ISBN: 32266934.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, PPSE, RYCKELYNCK, Unité ARN, WESTHOF
@article{,
title = {The Nature of the Purine at Position 34 in tRNAs of 4-codon Boxes Is Correlated With Nucleotides at Positions 32 and 38 to Maintain Decoding Fidelity},
author = {K Pernod and L Schaeffer and J Chicher and E Hok and C Rick and R Geslain and G Eriani and E Westhof and M Ryckelynck and F Martin},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32266934?dopt=Abstract},
doi = {10.1093/nar/gkaa221},
isbn = {32266934},
year = {2020},
date = {2020-01-01},
journal = {Nucleic Acids Res},
volume = {48},
number = {11},
pages = {6170-6183},
abstract = {Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.},
keywords = {ERIANI, PPSE, RYCKELYNCK, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.
Summer Sabrina, Smirnova Anna, Gabriele Alessandro, Toth Ursula, Fasemore Akinyemi Mandela, Förstner Konrad U, Kuhn Lauriane, Chicher Johana, Hammann Philippe, Mitulović Goran, Entelis Nina, Tarassov Ivan, Rossmanith Walter, Smirnov Alexandre
YBEY is an essential biogenesis factor for mitochondrial ribosomes. Article de journal
Dans: Nucleic acids research, vol. 48, no. 17, p. 9762–9786, 2020, ISSN: 1362-4962 0305-1048 0305-1048.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{summer_ybey_2020,
title = {YBEY is an essential biogenesis factor for mitochondrial ribosomes.},
author = {Sabrina Summer and Anna Smirnova and Alessandro Gabriele and Ursula Toth and Akinyemi Mandela Fasemore and Konrad U Förstner and Lauriane Kuhn and Johana Chicher and Philippe Hammann and Goran Mitulović and Nina Entelis and Ivan Tarassov and Walter Rossmanith and Alexandre Smirnov},
doi = {10.1093/nar/gkaa148},
issn = {1362-4962 0305-1048 0305-1048},
year = {2020},
date = {2020-01-01},
journal = {Nucleic acids research},
volume = {48},
number = {17},
pages = {9762--9786},
abstract = {Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.
Bajczyk Mateusz, Lange Heike, Bielewicz Dawid, Szewc Lukasz, Bhat Susheel S, Dolata Jakub, Kuhn Lauriane, Szweykowska-Kulinska Zofia, Gagliardi Dominique, Jarmolowski Artur
SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis. Article de journal
Dans: Nucleic acids research, vol. 48, no. 12, p. 6839–6854, 2020, ISSN: 1362-4962 0305-1048 0305-1048.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{bajczyk_serrate_2020,
title = {SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.},
author = {Mateusz Bajczyk and Heike Lange and Dawid Bielewicz and Lukasz Szewc and Susheel S Bhat and Jakub Dolata and Lauriane Kuhn and Zofia Szweykowska-Kulinska and Dominique Gagliardi and Artur Jarmolowski},
doi = {10.1093/nar/gkaa373},
issn = {1362-4962 0305-1048 0305-1048},
year = {2020},
date = {2020-01-01},
journal = {Nucleic acids research},
volume = {48},
number = {12},
pages = {6839--6854},
abstract = {SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.
Méteignier Louis-Valentin, Ghandour Rabea, Meierhoff Karin, Zimmerman Aude, Chicher Johana, Baumberger Nicolas, Alioua Abdelmalek, Meurer Jörg, Zoschke Reimo, Hammani Kamel
The Arabidopsis mTERF-repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons. Article de journal
Dans: The New phytologist, vol. 227, no. 5, p. 1376–1391, 2020, ISSN: 1469-8137 0028-646X 0028-646X.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{meteignier_arabidopsis_2020,
title = {The Arabidopsis mTERF-repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons.},
author = {Louis-Valentin Méteignier and Rabea Ghandour and Karin Meierhoff and Aude Zimmerman and Johana Chicher and Nicolas Baumberger and Abdelmalek Alioua and Jörg Meurer and Reimo Zoschke and Kamel Hammani},
doi = {10.1111/nph.16625},
issn = {1469-8137 0028-646X 0028-646X},
year = {2020},
date = {2020-01-01},
journal = {The New phytologist},
volume = {227},
number = {5},
pages = {1376--1391},
abstract = {The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5'-ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA-binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5'-ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA-binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.
Chico Jose Manuel, Lechner Esther, Fernandez-Barbero Gemma, Canibano Esther, García-Casado Gloria, Franco-Zorrilla Jose Manuel, Hammann Philippe, Zamarreño Angel M, García-Mina Jose M, Rubio Vicente, Genschik Pascal, Solano Roberto
CUL3(BPM) E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses. Article de journal
Dans: Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 11, p. 6205–6215, 2020, ISSN: 1091-6490 0027-8424 0027-8424.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{chico_cul3bpm_2020,
title = {CUL3(BPM) E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses.},
author = {Jose Manuel Chico and Esther Lechner and Gemma Fernandez-Barbero and Esther Canibano and Gloria García-Casado and Jose Manuel Franco-Zorrilla and Philippe Hammann and Angel M Zamarreño and Jose M García-Mina and Vicente Rubio and Pascal Genschik and Roberto Solano},
doi = {10.1073/pnas.1912199117},
issn = {1091-6490 0027-8424 0027-8424},
year = {2020},
date = {2020-01-01},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {11},
pages = {6205--6215},
abstract = {The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3(BPM) in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3(BPM)-mediated degradation of MYC transcription factors.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3(BPM) in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3(BPM)-mediated degradation of MYC transcription factors.
Bochler A, Querido J B, Prilepskaja T, Soufari H, Simonetti A, Cistia M L Del, Kuhn L, Ribeiro A R, Valášek L S, Hashem Y
Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts Article de journal
Dans: Cell Rep, vol. 33, no. 12, p. 108534, 2020, ISBN: 33357443.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ES6(S) ES7(S) ES9(S) Trypanosoma cruzi cryo-EM eIF1 eIF2 eIF3 eIF5-CTD k-DDX60 the 43S pre-initiation complex translation initiation, HASCHEM, PPSE, Unité ARN
@article{,
title = {Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts},
author = {A Bochler and J B Querido and T Prilepskaja and H Soufari and A Simonetti and M L Del Cistia and L Kuhn and A R Ribeiro and L S Valášek and Y Hashem},
url = {https://pubmed.ncbi.nlm.nih.gov/33357443/},
doi = {10.1016/j.celrep.2020.108534},
isbn = {33357443},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Cell Rep},
volume = {33},
number = {12},
pages = {108534},
abstract = {Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.},
keywords = {ENNIFAR, ES6(S) ES7(S) ES9(S) Trypanosoma cruzi cryo-EM eIF1 eIF2 eIF3 eIF5-CTD k-DDX60 the 43S pre-initiation complex translation initiation, HASCHEM, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.
Wolff P, Ennifar E
Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes Chapitre d'ouvrage
Dans: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, p. 111-118, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006311.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ENNIFAR, Native mass spectrometry RNA, Unité ARN
@inbook{,
title = {Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes},
author = {P Wolff and E Ennifar},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006311},
doi = {10.1007/978-1-0716-0278-2_9},
isbn = {32006311},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {111-118},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.},
keywords = {ARN-MS, ENNIFAR, Native mass spectrometry RNA, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.
Lechner A, Wolff P, Leize-Wagner E, François Y N
Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry Article de journal
Dans: Anal Chem, vol. 92, no. 10, p. 7363-7370, 2020, ISBN: 32343557.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ENNIFAR, Unité ARN
@article{,
title = {Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis-High Resolution Mass Spectrometry},
author = {A Lechner and P Wolff and E Leize-Wagner and Y N François},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32343557?dopt=Abstract},
doi = {10.1021/acs.analchem.0c01345},
isbn = {32343557},
year = {2020},
date = {2020-01-01},
journal = {Anal Chem},
volume = {92},
number = {10},
pages = {7363-7370},
abstract = {Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.},
keywords = {ARN-MS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE-MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.
Antoine L, Wolff P
Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry Chapitre d'ouvrage
Dans: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, p. 101-110, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006310.
Résumé | Liens | BibTeX | Étiquettes: 2D Gel isolation Nano-LC-MS/MS Posttranscriptional tRNA modifications, ARN-MS, ENNIFAR, ROMBY, Unité ARN
@inbook{,
title = {Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry},
author = {L Antoine and P Wolff},
editor = {V Arluison and F Wien},
url = {https://pubmed.ncbi.nlm.nih.gov/32006310},
doi = {10.1007/978-1-0716-0278-2_8},
isbn = {32006310},
year = {2020},
date = {2020-01-01},
booktitle = {RNA Spectroscopy: Methods and Protocols},
volume = {2113},
pages = {101-110},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
series = {Methods in Molecular Biology},
abstract = {RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.},
keywords = {2D Gel isolation Nano-LC-MS/MS Posttranscriptional tRNA modifications, ARN-MS, ENNIFAR, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
RNA modification mapping by mass spectrometry (MS) is based on the use of specific ribonucleases (RNases) that generate short oligonucleotide digestion products which are further separated by nano-liquid chromatography and analyzed by MS and MS/MS. Recent developments in MS instrumentation allow the possibility to deeply explore posttranscriptional modifications. Notably, development of nano-liquid chromatography and nano-electrospray drastically increases the detection sensitivity and allows the identification and sequencing of RNA digested fragments separated and extracted from two-dimensional polyacrylamide gels, as long as the mapping and characterization of ribonucleotide modifications.
2019
Bouchoucha Ayoub, Waltz Florent, Bonnard Géraldine, Arrivé Mathilde, Hammann Philippe, Kuhn Lauriane, Schelcher Cédric, Zuber Hélène, Gobert Anthony, Giegé Philippe
Determination of protein-only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease. Article de journal
Dans: The Plant journal : for cell and molecular biology, vol. 100, no. 3, p. 549–561, 2019, ISSN: 1365-313X 0960-7412.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{bouchoucha_determination_2019,
title = {Determination of protein-only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease.},
author = {Ayoub Bouchoucha and Florent Waltz and Géraldine Bonnard and Mathilde Arrivé and Philippe Hammann and Lauriane Kuhn and Cédric Schelcher and Hélène Zuber and Anthony Gobert and Philippe Giegé},
doi = {10.1111/tpj.14458},
issn = {1365-313X 0960-7412},
year = {2019},
date = {2019-11-01},
journal = {The Plant journal : for cell and molecular biology},
volume = {100},
number = {3},
pages = {549--561},
abstract = {The essential type of endonuclease that removes 5' leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called 'PRORP', for 'PROtein-only RNase P', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The essential type of endonuclease that removes 5' leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called 'PRORP', for 'PROtein-only RNase P', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.
Lange Heike, Ndecky Simon Y A, Gomez-Diaz Carlos, Pflieger David, Butel Nicolas, Zumsteg Julie, Kuhn Lauriane, Piermaria Christina, Chicher Johana, Christie Michael, Karaaslan Ezgi S, Lang Patricia L M, Weigel Detlef, Vaucheret Hervé, Hammann Philippe, Gagliardi Dominique
RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis. Article de journal
Dans: Nature communications, vol. 10, no. 1, p. 3871, 2019, ISSN: 2041-1723 2041-1723.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{lange_rst1_2019,
title = {RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis.},
author = {Heike Lange and Simon Y A Ndecky and Carlos Gomez-Diaz and David Pflieger and Nicolas Butel and Julie Zumsteg and Lauriane Kuhn and Christina Piermaria and Johana Chicher and Michael Christie and Ezgi S Karaaslan and Patricia L M Lang and Detlef Weigel and Hervé Vaucheret and Philippe Hammann and Dominique Gagliardi},
doi = {10.1038/s41467-019-11807-4},
issn = {2041-1723 2041-1723},
year = {2019},
date = {2019-08-01},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3871},
abstract = {The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.
Lalaouna D, Baude J, Wu Z, Tomasini A, Chicher J, Marzi S, Vandenesch F, Romby P, Caldelari I, Moreau K
RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation Article de journal
Dans: Nucleic Acids Res, vol. 47, no. 18, p. 9871-9887, 2019, ISBN: 31504767.
Résumé | Liens | BibTeX | Étiquettes: PPSE, ROMBY, Unité ARN
@article{Lalaouna2019,
title = {RsaC sRNA modulates the oxidative stress response of \textit{Staphylococcus aureus} during manganese starvation},
author = {D Lalaouna and J Baude and Z Wu and A Tomasini and J Chicher and S Marzi and F Vandenesch and P Romby and I Caldelari and K Moreau},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31504767?dopt=Abstract},
doi = {10.1093/nar/gkz728},
isbn = {31504767},
year = {2019},
date = {2019-01-01},
journal = {Nucleic Acids Res},
volume = {47},
number = {18},
pages = {9871-9887},
abstract = {The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5′ end while its 3′ part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3′UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.},
keywords = {PPSE, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5′ end while its 3′ part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3′UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.
Arbogast Florent, Arnold Johan, Hammann Philippe, Kuhn Lauriane, Chicher Johana, Murera Diane, Weishaar Justine, Muller Sylviane, Fauny Jean-Daniel, Gros Frédéric
ATG5 is required for B cell polarization and presentation of particulate antigens. Article de journal
Dans: Autophagy, vol. 15, no. 2, p. 280–294, 2019, ISSN: 1554-8635 1554-8627 1554-8627.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{arbogast_atg5_2019,
title = {ATG5 is required for B cell polarization and presentation of particulate antigens.},
author = {Florent Arbogast and Johan Arnold and Philippe Hammann and Lauriane Kuhn and Johana Chicher and Diane Murera and Justine Weishaar and Sylviane Muller and Jean-Daniel Fauny and Frédéric Gros},
doi = {10.1080/15548627.2018.1516327},
issn = {1554-8635 1554-8627 1554-8627},
year = {2019},
date = {2019-01-01},
journal = {Autophagy},
volume = {15},
number = {2},
pages = {280--294},
abstract = {The involvement of macroautophagy/autophagy proteins in B-cell receptor (BCR) trafficking, although suspected, is not well understood. We show that ATG5 (autophagy related 5) contributes to BCR polarization after stimulation and internalization into LAMP1 (lysosomal-associated membrane protein 1)(+) and major histocompatibility complex class II (MHC-II)(+) compartments. BCR polarization is crucial in the context of immobilized antigen processing. Moreover, antigen presentation to cognate T cells is decreased in the absence of ATG5 when the model antigen OVAL/ovalbumin is provided in an immobilized form in contrast to the normal presentation of soluble OVAL. We further show that ATG5 is required for centrosome polarization and actin nucleation in the immune synapse area. This event is accompanied by an increased interaction between ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and the microtubule-organizing center-associated protein PCM1 (pericentriolar material 1). In the human B cell line BJAB, PCM1 is required for BCR polarization after stimulation. We thus propose that the ATG12 (autophagy related 12)-ATG5-ATG16L1 complex under BCR stimulation allows its interaction with PCM1 and consequently facilitates centrosome relocalization to the immune synapse, optimizing the presentation of particulate antigens. Abbreviations: ACTB: actin beta; ACTR2/3: ARP2/3 actin-related protein 2/3; APC: antigen-presenting cells; ATG: autophagy-related; BCR: B cell receptor; BECN1/Beclin 1: beclin 1, autophagy related; CDC42: cell division cycle 42; Cr2: complement receptor 2; CSFE: carboxyfluorescein succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EEA1: early endosome antigen 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothyocyanate; GC: germinal center; GJA1/CX3: gap junction protein, alpha 1; Ig: immunoglobulin; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; LM: littermate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen activated protein kinase; MHC-II: major histocompatibility complex class II; MIIC: MHC class II compartment; OVAL: ovalbumin; PBS: phosphate-buffered saline; PCM1: pericentriolar material 1; PtdIns3K: phosphatidylinositol 3-kinase; PTPRC/CD45RB/B220; Protein tyrosine phosphatase, receptor type, C; SYK: spleen tyrosine kinase; TBS: Tris-buffered saline; TCR: T cell receptor; ULK1: unc-51 like kinase 1.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The involvement of macroautophagy/autophagy proteins in B-cell receptor (BCR) trafficking, although suspected, is not well understood. We show that ATG5 (autophagy related 5) contributes to BCR polarization after stimulation and internalization into LAMP1 (lysosomal-associated membrane protein 1)(+) and major histocompatibility complex class II (MHC-II)(+) compartments. BCR polarization is crucial in the context of immobilized antigen processing. Moreover, antigen presentation to cognate T cells is decreased in the absence of ATG5 when the model antigen OVAL/ovalbumin is provided in an immobilized form in contrast to the normal presentation of soluble OVAL. We further show that ATG5 is required for centrosome polarization and actin nucleation in the immune synapse area. This event is accompanied by an increased interaction between ATG16L1 (autophagy related 16-like 1 [S. cerevisiae]) and the microtubule-organizing center-associated protein PCM1 (pericentriolar material 1). In the human B cell line BJAB, PCM1 is required for BCR polarization after stimulation. We thus propose that the ATG12 (autophagy related 12)-ATG5-ATG16L1 complex under BCR stimulation allows its interaction with PCM1 and consequently facilitates centrosome relocalization to the immune synapse, optimizing the presentation of particulate antigens. Abbreviations: ACTB: actin beta; ACTR2/3: ARP2/3 actin-related protein 2/3; APC: antigen-presenting cells; ATG: autophagy-related; BCR: B cell receptor; BECN1/Beclin 1: beclin 1, autophagy related; CDC42: cell division cycle 42; Cr2: complement receptor 2; CSFE: carboxyfluorescein succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EEA1: early endosome antigen 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothyocyanate; GC: germinal center; GJA1/CX3: gap junction protein, alpha 1; Ig: immunoglobulin; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; LM: littermate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen activated protein kinase; MHC-II: major histocompatibility complex class II; MIIC: MHC class II compartment; OVAL: ovalbumin; PBS: phosphate-buffered saline; PCM1: pericentriolar material 1; PtdIns3K: phosphatidylinositol 3-kinase; PTPRC/CD45RB/B220; Protein tyrosine phosphatase, receptor type, C; SYK: spleen tyrosine kinase; TBS: Tris-buffered saline; TCR: T cell receptor; ULK1: unc-51 like kinase 1.
Waltz Florent, Nguyen Tan-Trung, Arrivé Mathilde, Bochler Anthony, Chicher Johana, Hammann Philippe, Kuhn Lauriane, Quadrado Martine, Mireau Hakim, Hashem Yaser, Giegé Philippe
Small is big in Arabidopsis mitochondrial ribosome. Article de journal
Dans: Nature plants, vol. 5, no. 1, p. 106–117, 2019, ISSN: 2055-0278 2055-0278.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{waltz_small_2019,
title = {Small is big in Arabidopsis mitochondrial ribosome.},
author = {Florent Waltz and Tan-Trung Nguyen and Mathilde Arrivé and Anthony Bochler and Johana Chicher and Philippe Hammann and Lauriane Kuhn and Martine Quadrado and Hakim Mireau and Yaser Hashem and Philippe Giegé},
doi = {10.1038/s41477-018-0339-y},
issn = {2055-0278 2055-0278},
year = {2019},
date = {2019-01-01},
journal = {Nature plants},
volume = {5},
number = {1},
pages = {106--117},
abstract = {Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.
Trinquier Aude, Ulmer Jonathan E, Gilet Laetitia, Figaro Sabine, Hammann Philippe, Kuhn Lauriane, Braun Frédérique, Condon Ciarán
tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp. Article de journal
Dans: Molecular cell, vol. 74, no. 6, p. 1227–1238.e3, 2019, ISSN: 1097-4164 1097-2765.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{trinquier_trna_2019,
title = {tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.},
author = {Aude Trinquier and Jonathan E Ulmer and Laetitia Gilet and Sabine Figaro and Philippe Hammann and Lauriane Kuhn and Frédérique Braun and Ciarán Condon},
doi = {10.1016/j.molcel.2019.03.030},
issn = {1097-4164 1097-2765},
year = {2019},
date = {2019-01-01},
journal = {Molecular cell},
volume = {74},
number = {6},
pages = {1227--1238.e3},
abstract = {rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.
Antoine L, Wolff P, Westhof E, Romby P, Marzi S
Mapping post-transcriptional modifications in Staphylococcus aureus tRNAs by nanoLC/MSMS Article de journal
Dans: Biochimie, vol. 164, p. 60-69, 2019, ISBN: 31295507.
Résumé | Liens | BibTeX | Étiquettes: 2D gel isolation Staphylococcus aureus nanoLC/MSMS post-transcriptional tRNA modifications, ARN-MS, ENNIFAR, ROMBY, Unité ARN, WESTHOF
@article{,
title = {Mapping post-transcriptional modifications in Staphylococcus aureus tRNAs by nanoLC/MSMS},
author = {L Antoine and P Wolff and E Westhof and P Romby and S Marzi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31295507?dopt=Abstract},
doi = {10.1016/j.biochi.2019.07.003},
isbn = {31295507},
year = {2019},
date = {2019-01-01},
journal = {Biochimie},
volume = {164},
pages = {60-69},
abstract = {RNA modifications are involved in numerous biological processes. These modifications are constitutive or modulated in response to adaptive processes and can impact RNA base pairing formation, protein recognition, RNA structure and stability. tRNAs are the most abundantly modified RNA molecules. Analysis of the roles of their modifications in response to stress, environmental changes, and infections caused by pathogens, has fueled new research areas. Nevertheless, the detection of modified nucleotides in RNAs is still a challenging task. We present here a reliable method to identify and localize tRNA modifications, which was applied to the human pathogenic bacteria, Staphyloccocus aureus. The method is based on a separation of tRNA species on a two-dimensional polyacrylamide gel electrophoresis followed by nano liquid chromatography-mass spectrometry. We provided a list of modifications mapped on 25 out of the 40 tRNA species (one isoacceptor for each amino acid). This method can be easily used to monitor the dynamics of tRNA modifications in S. aureus in response to stress adaptation and during infection of the host, a relatively unexplored field.},
keywords = {2D gel isolation Staphylococcus aureus nanoLC/MSMS post-transcriptional tRNA modifications, ARN-MS, ENNIFAR, ROMBY, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
RNA modifications are involved in numerous biological processes. These modifications are constitutive or modulated in response to adaptive processes and can impact RNA base pairing formation, protein recognition, RNA structure and stability. tRNAs are the most abundantly modified RNA molecules. Analysis of the roles of their modifications in response to stress, environmental changes, and infections caused by pathogens, has fueled new research areas. Nevertheless, the detection of modified nucleotides in RNAs is still a challenging task. We present here a reliable method to identify and localize tRNA modifications, which was applied to the human pathogenic bacteria, Staphyloccocus aureus. The method is based on a separation of tRNA species on a two-dimensional polyacrylamide gel electrophoresis followed by nano liquid chromatography-mass spectrometry. We provided a list of modifications mapped on 25 out of the 40 tRNA species (one isoacceptor for each amino acid). This method can be easily used to monitor the dynamics of tRNA modifications in S. aureus in response to stress adaptation and during infection of the host, a relatively unexplored field.
André C, Martiel I, Wolff P, Landolfo M, Lorber B, da Veiga C Silva, Dejaegere A, Dumas P, Guichard G, Olieric V, Wagner J G, Burnouf D Y
Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps Article de journal
Dans: ACS Infect Dis, vol. 5, no. 6, p. 1022-1034, 2019, ISBN: 30912430.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ENNIFAR, FRUGIER, ITC ligand−target interaction new antibacterials development sliding clamp, Unité ARN
@article{,
title = {Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps},
author = {C André and I Martiel and P Wolff and M Landolfo and B Lorber and C Silva da Veiga and A Dejaegere and P Dumas and G Guichard and V Olieric and J G Wagner and D Y Burnouf},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30912430?dopt=Abstract},
doi = {10.1021/acsinfecdis.9b00089},
isbn = {30912430},
year = {2019},
date = {2019-01-01},
urldate = {2019-01-01},
journal = {ACS Infect Dis},
volume = {5},
number = {6},
pages = {1022-1034},
abstract = {Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drugs development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets but the complex stability varies according to a pocket specific network of interactions.},
keywords = {ARN-MS, ENNIFAR, FRUGIER, ITC ligand−target interaction new antibacterials development sliding clamp, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drugs development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets but the complex stability varies according to a pocket specific network of interactions.
2018
Olmo Roenick P, Ferreira Alvaro G A, Izidoro-Toledo Tatiane C, Aguiar Eric R G R, de Faria Isaque J S, de Souza Kátia P R, Osório Kátia P, Kuhn Lauriane, Hammann Philippe, de Andrade Elisa G, Todjro Yaovi Mathias, Rocha Marcele N, Leite Thiago H J F, Amadou Siad C G, Armache Juliana N, Paro Simona, de Oliveira Caroline D, Carvalho Fabiano D, Moreira Luciano A, Marois Eric, Imler Jean-Luc, Marques João T
Control of dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-binding protein Loqs2. Article de journal
Dans: Nature microbiology, vol. 3, no. 12, p. 1385–1393, 2018, ISSN: 2058-5276 2058-5276.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{olmo_control_2018,
title = {Control of dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-binding protein Loqs2.},
author = {Roenick P Olmo and Alvaro G A Ferreira and Tatiane C Izidoro-Toledo and Eric R G R Aguiar and Isaque J S de Faria and Kátia P R de Souza and Kátia P Osório and Lauriane Kuhn and Philippe Hammann and Elisa G de Andrade and Yaovi Mathias Todjro and Marcele N Rocha and Thiago H J F Leite and Siad C G Amadou and Juliana N Armache and Simona Paro and Caroline D de Oliveira and Fabiano D Carvalho and Luciano A Moreira and Eric Marois and Jean-Luc Imler and João T Marques},
doi = {10.1038/s41564-018-0268-6},
issn = {2058-5276 2058-5276},
year = {2018},
date = {2018-12-01},
journal = {Nature microbiology},
volume = {3},
number = {12},
pages = {1385--1393},
abstract = {Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes(1). In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV(2-5). However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes(1). In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV(2-5). However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.
Chicois Clara, Scheer Hélène, Garcia Shahïnez, Zuber Hélène, Mutterer Jérôme, Chicher Johana, Hammann Philippe, Gagliardi Dominique, Garcia Damien
The UPF1 interactome reveals interaction networks between RNA degradation and translation repression factors in Arabidopsis. Article de journal
Dans: The Plant journal : for cell and molecular biology, vol. 96, no. 1, p. 119–132, 2018, ISSN: 1365-313X 0960-7412, (Place: England).
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{chicois_upf1_2018,
title = {The UPF1 interactome reveals interaction networks between RNA degradation and translation repression factors in Arabidopsis.},
author = {Clara Chicois and Hélène Scheer and Shahïnez Garcia and Hélène Zuber and Jérôme Mutterer and Johana Chicher and Philippe Hammann and Dominique Gagliardi and Damien Garcia},
doi = {10.1111/tpj.14022},
issn = {1365-313X 0960-7412},
year = {2018},
date = {2018-10-01},
journal = {The Plant journal : for cell and molecular biology},
volume = {96},
number = {1},
pages = {119--132},
abstract = {The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.},
note = {Place: England},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.
Goto Akira, Okado Kiyoshi, Martins Nelson, Cai Hua, Barbier Vincent, Lamiable Olivier, Troxler Laurent, Santiago Estelle, Kuhn Lauriane, Paik Donggi, Silverman Neal, Holleufer Andreas, Hartmann Rune, Liu Jiyong, Peng Tao, Hoffmann Jules A, Meignin Carine, Daeffler Laurent, Imler Jean-Luc
The Kinase IKKβ Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila Article de journal
Dans: Immunity, no. 49, p. 225-234, 2018.
Résumé | Liens | BibTeX | Étiquettes: hoffmann, imler, M3i, meignin, PPSE
@article{Goto2018,
title = {The Kinase IKKβ Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila},
author = {Akira Goto and Kiyoshi Okado and Nelson Martins and Hua Cai and Vincent Barbier and Olivier Lamiable and Laurent Troxler and Estelle Santiago and Lauriane Kuhn and Donggi Paik and Neal Silverman and Andreas Holleufer and Rune Hartmann and Jiyong Liu and Tao Peng and Jules A Hoffmann and Carine Meignin and Laurent Daeffler and Jean-Luc Imler},
editor = {Elsevier Inc.},
url = {https://doi.org/10.1016/j.immuni.2018.07.013},
doi = {j.immuni.2018.07.013},
year = {2018},
date = {2018-08-21},
journal = {Immunity},
number = {49},
pages = {225-234},
abstract = {Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKβ and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.},
keywords = {hoffmann, imler, M3i, meignin, PPSE},
pubstate = {published},
tppubtype = {article}
}
Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKβ and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.
Wang Fengjuan, Bonam Srinivasa Reddy, Schall Nicolas, Kuhn Lauriane, Hammann Philippe, Chaloin Olivier, Madinier Jean-Baptiste, Briand Jean-Paul, Page Nicolas, Muller Sylviane
Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival. Article de journal
Dans: Scientific reports, vol. 8, no. 1, p. 16820, 2018, ISSN: 2045-2322 2045-2322.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{wang_blocking_2018,
title = {Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival.},
author = {Fengjuan Wang and Srinivasa Reddy Bonam and Nicolas Schall and Lauriane Kuhn and Philippe Hammann and Olivier Chaloin and Jean-Baptiste Madinier and Jean-Paul Briand and Nicolas Page and Sylviane Muller},
doi = {10.1038/s41598-018-34887-6},
issn = {2045-2322 2045-2322},
year = {2018},
date = {2018-01-01},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16820},
abstract = {The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.
Poirier Isabelle, Pallud Marie, Kuhn Lauriane, Hammann Philippe, Demortière Arnaud, Jamali Arash, Chicher Johana, Caplat Christelle, Gallon Régis Kevin, Bertrand Martine
Toxicological effects of CdSe nanocrystals on the marine diatom Phaeodactylum tricornutum: The first mass spectrometry-based proteomic approach. Article de journal
Dans: Ecotoxicology and environmental safety, vol. 152, p. 78–90, 2018, ISSN: 1090-2414 0147-6513.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{poirier_toxicological_2018,
title = {Toxicological effects of CdSe nanocrystals on the marine diatom Phaeodactylum tricornutum: The first mass spectrometry-based proteomic approach.},
author = {Isabelle Poirier and Marie Pallud and Lauriane Kuhn and Philippe Hammann and Arnaud Demortière and Arash Jamali and Johana Chicher and Christelle Caplat and Régis Kevin Gallon and Martine Bertrand},
doi = {10.1016/j.ecoenv.2018.01.043},
issn = {1090-2414 0147-6513},
year = {2018},
date = {2018-01-01},
journal = {Ecotoxicology and environmental safety},
volume = {152},
pages = {78--90},
abstract = {In the marine environment, benthic diatoms from estuarine and coastal sediments are among the first targets of nanoparticle pollution whose potential toxicity on marine organisms is still largely unknown. It is therefore relevant to improve our knowledge of interactions between these new pollutants and microalgae, the key players in the control of marine resources. In this study, the response of P. tricornutum to CdSe nanocrystals (CdSe NPs) of 5 nm (NP5) and 12 nm (NP12) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. NP5 and NP12 affected cell growth but oxygen production was only slightly decreased by NP5 after 1-d incubation time. In our experimental conditions, a high CdSe NP dissolution was observed during the first day of culture, leading to Cd bioaccumulation and oxidative stress, particularly with NP12. However, after a 7-day incubation time, proteomic analysis highlighted that P. tricornutum responded to CdSe NP toxicity by regulating numerous proteins involved in protection against oxidative stress, cellular redox homeostasis, Ca(2+) regulation and signalling, S-nitrosylation and S-glutathionylation processes and cell damage repair. These proteome changes allowed algae cells to regulate their intracellular ROS level in contaminated cultures. P. tricornutum was also capable to control its intracellular Cd concentration at a sufficiently low level to preserve its growth. To our knowledge, this is the first work allowing the identification of proteins differentially expressed by P. tricornutum subjected to NPs and thus the understanding of some molecular pathways involved in its cellular response to nanoparticles. SIGNIFICANCE: The microalgae play a key role in the control of marine resources. Moreover, they produce 50% of the atmospheric oxygen. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Since estuarine and coastal sediments concentrate pollutants, benthic microalgae which live in superficial sediments will be among the first targets of nanoparticle pollution. Thus, it is relevant to improve our knowledge of interactions between diatoms and nanoparticles. Proteomics is a powerful tool for understanding the molecular mechanisms triggered by nanoparticle exposure, and our study is the first one to use this tool to identify proteins differentially expressed by P. tricornutum subjected to CdSe nanocrystals. This work is fundamental to improve our knowledge about the defence mechanisms developed by algae cells to counteract damage caused by CdSe NPs.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
In the marine environment, benthic diatoms from estuarine and coastal sediments are among the first targets of nanoparticle pollution whose potential toxicity on marine organisms is still largely unknown. It is therefore relevant to improve our knowledge of interactions between these new pollutants and microalgae, the key players in the control of marine resources. In this study, the response of P. tricornutum to CdSe nanocrystals (CdSe NPs) of 5 nm (NP5) and 12 nm (NP12) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. NP5 and NP12 affected cell growth but oxygen production was only slightly decreased by NP5 after 1-d incubation time. In our experimental conditions, a high CdSe NP dissolution was observed during the first day of culture, leading to Cd bioaccumulation and oxidative stress, particularly with NP12. However, after a 7-day incubation time, proteomic analysis highlighted that P. tricornutum responded to CdSe NP toxicity by regulating numerous proteins involved in protection against oxidative stress, cellular redox homeostasis, Ca(2+) regulation and signalling, S-nitrosylation and S-glutathionylation processes and cell damage repair. These proteome changes allowed algae cells to regulate their intracellular ROS level in contaminated cultures. P. tricornutum was also capable to control its intracellular Cd concentration at a sufficiently low level to preserve its growth. To our knowledge, this is the first work allowing the identification of proteins differentially expressed by P. tricornutum subjected to NPs and thus the understanding of some molecular pathways involved in its cellular response to nanoparticles. SIGNIFICANCE: The microalgae play a key role in the control of marine resources. Moreover, they produce 50% of the atmospheric oxygen. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Since estuarine and coastal sediments concentrate pollutants, benthic microalgae which live in superficial sediments will be among the first targets of nanoparticle pollution. Thus, it is relevant to improve our knowledge of interactions between diatoms and nanoparticles. Proteomics is a powerful tool for understanding the molecular mechanisms triggered by nanoparticle exposure, and our study is the first one to use this tool to identify proteins differentially expressed by P. tricornutum subjected to CdSe nanocrystals. This work is fundamental to improve our knowledge about the defence mechanisms developed by algae cells to counteract damage caused by CdSe NPs.
Montavon Thomas, Kwon Yerim, Zimmermann Aude, Hammann Philippe, Vincent Timothée, Cognat Valérie, Bergdoll Marc, Michel Fabrice, Dunoyer Patrice
Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates. Article de journal
Dans: The Plant journal : for cell and molecular biology, vol. 95, no. 2, p. 204–218, 2018, ISSN: 1365-313X 0960-7412, (Place: England).
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{montavon_characterization_2018,
title = {Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates.},
author = {Thomas Montavon and Yerim Kwon and Aude Zimmermann and Philippe Hammann and Timothée Vincent and Valérie Cognat and Marc Bergdoll and Fabrice Michel and Patrice Dunoyer},
doi = {10.1111/tpj.13941},
issn = {1365-313X 0960-7412},
year = {2018},
date = {2018-01-01},
journal = {The Plant journal : for cell and molecular biology},
volume = {95},
number = {2},
pages = {204--218},
abstract = {In the model plant Arabidopsis thaliana, four Dicer-like proteins (DCL1-4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII-like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4-dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4-dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold-back double-stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR-dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.},
note = {Place: England},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
In the model plant Arabidopsis thaliana, four Dicer-like proteins (DCL1-4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII-like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4-dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4-dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold-back double-stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR-dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.
Rozov A, Wolff P, Grosjean H, Yusupov M, Yusupova G, Westhof E
Tautomeric G*U pairs within the molecular ribosomal grip and fidelity of decoding in bacteria Article de journal
Dans: Nucleic Acids Res, vol. 46, no. 14, p. 7425-7435, 2018, ISBN: 29931292.
Résumé | Liens | BibTeX | Étiquettes: ARN-MS, ENNIFAR, Unité ARN, WESTHOF
@article{,
title = {Tautomeric G*U pairs within the molecular ribosomal grip and fidelity of decoding in bacteria},
author = {A Rozov and P Wolff and H Grosjean and M Yusupov and G Yusupova and E Westhof},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29931292?dopt=Abstract},
doi = {10.1093/nar/gky547},
isbn = {29931292},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Nucleic Acids Res},
volume = {46},
number = {14},
pages = {7425-7435},
abstract = {We report new crystallographic structures of Thermus thermophilus ribosomes complexed with long mRNAs and native Escherichia coli tRNAs. They complete the full set of combinations of Watson-Crick GC and miscoding GU pairs at the first two positions of the codon-anticodon duplex in ribosome functional complexes. Within the tight decoding center, miscoding GU pairs occur, in all combinations, with a non-wobble geometry structurally indistinguishable from classical coding Watson-Crick pairs at the same first two positions. The contacts with the ribosomal grip surrounding the decoding center are all quasi-identical, except in the crowded environment of the amino group of a guanosine at the second position; in which case a G in the codons may be preferred. In vivo experimental data show that the translational errors due to miscoding by GU pairs at the first two positions are the most frequently encountered ones, especially at the second position and with a G on the codon. Such preferred miscodings involve a switch from an A-U to a GU pair in the tRNA/mRNA complex and very rarely from a G = C to a GU pair. It is concluded that the frequencies of such occurrences are only weakly affected by the codon/anticodon structures but depend mainly on the stability and lifetime of the complex, the modifications present in the anticodon loop, especially those at positions 34 and 37, in addition to the relative concentration of cognate/near-cognate tRNA species present in the cellular tRNA pool.},
keywords = {ARN-MS, ENNIFAR, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
We report new crystallographic structures of Thermus thermophilus ribosomes complexed with long mRNAs and native Escherichia coli tRNAs. They complete the full set of combinations of Watson-Crick GC and miscoding GU pairs at the first two positions of the codon-anticodon duplex in ribosome functional complexes. Within the tight decoding center, miscoding GU pairs occur, in all combinations, with a non-wobble geometry structurally indistinguishable from classical coding Watson-Crick pairs at the same first two positions. The contacts with the ribosomal grip surrounding the decoding center are all quasi-identical, except in the crowded environment of the amino group of a guanosine at the second position; in which case a G in the codons may be preferred. In vivo experimental data show that the translational errors due to miscoding by GU pairs at the first two positions are the most frequently encountered ones, especially at the second position and with a G on the codon. Such preferred miscodings involve a switch from an A-U to a GU pair in the tRNA/mRNA complex and very rarely from a G = C to a GU pair. It is concluded that the frequencies of such occurrences are only weakly affected by the codon/anticodon structures but depend mainly on the stability and lifetime of the complex, the modifications present in the anticodon loop, especially those at positions 34 and 37, in addition to the relative concentration of cognate/near-cognate tRNA species present in the cellular tRNA pool.
2017
Kuhn Lauriane, Majzoub Karim, Einhorn Evelyne, Chicher Johana, Pompon Julien, Imler Jean-Luc, Hammann Philippe, Meignin Carine
Definition of a RACK1 Interaction Network in Drosophila melanogaster Using SWATH-MS Article de journal
Dans: G3 (Bethesda), 2017, ISSN: 2160-1836.
Résumé | Liens | BibTeX | Étiquettes: imler, M3i, meignin, PPSE
@article{kuhn_definition_2017,
title = {Definition of a RACK1 Interaction Network in Drosophila melanogaster Using SWATH-MS},
author = {Lauriane Kuhn and Karim Majzoub and Evelyne Einhorn and Johana Chicher and Julien Pompon and Jean-Luc Imler and Philippe Hammann and Carine Meignin},
doi = {10.1534/g3.117.042564},
issn = {2160-1836},
year = {2017},
date = {2017-12-31},
journal = {G3 (Bethesda)},
abstract = {Receptor for Activated C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster, we recently showed that RACK1 is required at the ribosome for IRES-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus, an IRES-containing virus.},
keywords = {imler, M3i, meignin, PPSE},
pubstate = {published},
tppubtype = {article}
}
Receptor for Activated C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster, we recently showed that RACK1 is required at the ribosome for IRES-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus, an IRES-containing virus.
Gross L, Vicens Q, Einhorn E, Noireterre A, Schaeffer L, Kuhn L, Imler JL, Eriani G, Meignin C, Martin F
The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure Article de journal
Dans: Nucleic Acids Res, vol. 45, no. 15, p. 8993-9004, 2017, ISBN: 28911115.
Résumé | Liens | BibTeX | Étiquettes: ERIANI, meignin, PPSE, Unité ARN
@article{,
title = {The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure},
author = {L Gross and Q Vicens and E Einhorn and A Noireterre and L Schaeffer and L Kuhn and JL Imler and G Eriani and C Meignin and F Martin},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28911115?dopt=Abstract},
doi = {10.1093/nar/gkx622},
isbn = {28911115},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Nucleic Acids Res},
volume = {45},
number = {15},
pages = {8993-9004},
abstract = {Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.},
keywords = {ERIANI, meignin, PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.
Carapito C, Kuhn L, Karim L, Rompais M, Rabilloud T, Schwenzer H, Sissler M
Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases. Article de journal
Dans: Methods, vol. 113, p. 111-119, 2017, ISBN: 27793688.
Résumé | Liens | BibTeX | Étiquettes: PPSE, SISSLER, Unité ARN
@article{,
title = {Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.},
author = {C Carapito and L Kuhn and L Karim and M Rompais and T Rabilloud and H Schwenzer and M Sissler},
url = {https://www.ncbi.nlm.nih.gov/pubmed/27793688?dopt=Abstract},
doi = {10.1016/j.ymeth.2016.10.012},
isbn = {27793688},
year = {2017},
date = {2017-01-01},
journal = {Methods},
volume = {113},
pages = {111-119},
abstract = {Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.},
keywords = {PPSE, SISSLER, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.
Tomasini A, Moreau K, Chicher J, Geissmann T, Vandenesch F, Romby P, Marzi S, Caldelari I
The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms. Article de journal
Dans: Nucleic Acids Res, vol. 45, no. 11, p. 6746-6760, 2017, ISBN: 28379505.
Résumé | Liens | BibTeX | Étiquettes: PPSE, ROMBY, Unité ARN
@article{,
title = {The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms.},
author = {A Tomasini and K Moreau and J Chicher and T Geissmann and F Vandenesch and P Romby and S Marzi and I Caldelari},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28379505},
doi = {10.1093/nar/gkx219},
isbn = {28379505},
year = {2017},
date = {2017-01-01},
journal = {Nucleic Acids Res},
volume = {45},
number = {11},
pages = {6746-6760},
abstract = {The virulon of Staphyloccocus aureus is controlled by intricate connections between transcriptional and post-transcriptional regulators including proteins and small non-coding RNAs (sRNAs). Many of the sRNAs regulate gene expression through base-pairings with mRNAs. However, characterization of the direct sRNA targets in Gram-positive bacteria remained a difficult challenge. Here, we have applied and adapted the MS2-affinity purification approach coupled to RNA sequencing (MAPS) to determine the targetome of RsaA sRNA of S. aureus, known to repress the synthesis of the transcriptional regulator MgrA. Several mRNAs were enriched with RsaA expanding its regulatory network. Besides mgrA, several of these mRNAs encode a family of SsaA-like enzymes involved in peptidoglycan metabolism and the secreted anti-inflammatory FLIPr protein. Using a combination of in vivo and in vitro approaches, these mRNAs were validated as direct RsaA targets. Quantitative differential proteomics of wild-type and mutant strains corroborated the MAPS results. Additionally, it revealed that RsaA indirectly activated the synthesis of surface proteins supporting previous data that RsaA stimulated biofilm formation and favoured chronic infections. All together, this study shows that MAPS could also be easily applied in Gram-positive bacteria for identification of sRNA targetome.},
keywords = {PPSE, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The virulon of Staphyloccocus aureus is controlled by intricate connections between transcriptional and post-transcriptional regulators including proteins and small non-coding RNAs (sRNAs). Many of the sRNAs regulate gene expression through base-pairings with mRNAs. However, characterization of the direct sRNA targets in Gram-positive bacteria remained a difficult challenge. Here, we have applied and adapted the MS2-affinity purification approach coupled to RNA sequencing (MAPS) to determine the targetome of RsaA sRNA of S. aureus, known to repress the synthesis of the transcriptional regulator MgrA. Several mRNAs were enriched with RsaA expanding its regulatory network. Besides mgrA, several of these mRNAs encode a family of SsaA-like enzymes involved in peptidoglycan metabolism and the secreted anti-inflammatory FLIPr protein. Using a combination of in vivo and in vitro approaches, these mRNAs were validated as direct RsaA targets. Quantitative differential proteomics of wild-type and mutant strains corroborated the MAPS results. Additionally, it revealed that RsaA indirectly activated the synthesis of surface proteins supporting previous data that RsaA stimulated biofilm formation and favoured chronic infections. All together, this study shows that MAPS could also be easily applied in Gram-positive bacteria for identification of sRNA targetome.
Khusainov I, Vicens Q, Bochler A, Grosse F, Myasnikov A, Ménétret J F, Chicher J, Marzi S, Romby P, Yusupova G, Yusupov M, Hashem Y
Structure of the 70S ribosome from human pathogen Staphylococcus aureus. Article de journal
Dans: Nucleic Acids Res, vol. 45, no. 2, p. 1026, 2017, ISBN: 28123039.
Résumé | Liens | BibTeX | Étiquettes: PPSE, ROMBY, Unité ARN
@article{,
title = {Structure of the 70S ribosome from human pathogen Staphylococcus aureus.},
author = {I Khusainov and Q Vicens and A Bochler and F Grosse and A Myasnikov and J F Ménétret and J Chicher and S Marzi and P Romby and G Yusupova and M Yusupov and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28123039?dopt=Abstract},
doi = {10.1093/nar/gkw1126},
isbn = {28123039},
year = {2017},
date = {2017-01-01},
journal = {Nucleic Acids Res},
volume = {45},
number = {2},
pages = {1026},
abstract = {Erratum for Structure of the 70S ribosome from human pathogen Staphylococcus aureus.},
keywords = {PPSE, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Erratum for Structure of the 70S ribosome from human pathogen Staphylococcus aureus.
Querido J Brito, Mancera-Martinez E, Vicens Q, Bochler A, Chicher J, Simonetti A, Hashem Y
The cryo-EM Structure of a Novel 40S Kinetoplastid-Specific Ribosomal Protein Article de journal
Dans: Structure, vol. 25, no. 12, p. 1785-1794, 2017, ISBN: 29107485.
Résumé | Liens | BibTeX | Étiquettes: PPSE, Unité ARN
@article{,
title = {The cryo-EM Structure of a Novel 40S Kinetoplastid-Specific Ribosomal Protein},
author = {J Brito Querido and E Mancera-Martinez and Q Vicens and A Bochler and J Chicher and A Simonetti and Y Hashem},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29107485?dopt=Abstract},
doi = {10.1016/j.str.2017.09.014},
isbn = {29107485},
year = {2017},
date = {2017-01-01},
journal = {Structure},
volume = {25},
number = {12},
pages = {1785-1794},
abstract = {Kinetoplastids are potentially lethal protozoan pathogens affecting more than 20 million people worldwide. There is a critical need for more specific targets for the development of safer anti-kinetoplastid therapeutic molecules that can replace the scarce and highly cytotoxic current drugs. The kinetoplastid ribosome represents a potential therapeutic target due to its relative structural divergence when compared with its human counterpart. However, several kinetoplastid-specific ribosomal features remain uncharacterized. Here, we present the near-atomic cryoelectron microscopy structure of a novel bona fide kinetoplastid-specific ribosomal (r-) protein (KSRP) bound to the ribosome. KSRP is an essential protein located at the solvent face of the 40S subunit, where it binds and stabilizes kinetoplastid-specific domains of rRNA, suggesting its role in ribosome integrity. KSRP also interacts with the r-protein eS6 at a region that is only conserved in kinetoplastids. The kinetoplastid-specific ribosomal environment of KSRP provides a promising target for the design of safer anti-kinetoplastidian drugs.},
keywords = {PPSE, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Kinetoplastids are potentially lethal protozoan pathogens affecting more than 20 million people worldwide. There is a critical need for more specific targets for the development of safer anti-kinetoplastid therapeutic molecules that can replace the scarce and highly cytotoxic current drugs. The kinetoplastid ribosome represents a potential therapeutic target due to its relative structural divergence when compared with its human counterpart. However, several kinetoplastid-specific ribosomal features remain uncharacterized. Here, we present the near-atomic cryoelectron microscopy structure of a novel bona fide kinetoplastid-specific ribosomal (r-) protein (KSRP) bound to the ribosome. KSRP is an essential protein located at the solvent face of the 40S subunit, where it binds and stabilizes kinetoplastid-specific domains of rRNA, suggesting its role in ribosome integrity. KSRP also interacts with the r-protein eS6 at a region that is only conserved in kinetoplastids. The kinetoplastid-specific ribosomal environment of KSRP provides a promising target for the design of safer anti-kinetoplastidian drugs.
Kuhn Lauriane, Majzoub Karim, Einhorn Evelyne, Chicher Johana, Pompon Julien, Imler Jean-Luc, Hammann Philippe, Meignin Carine
Definition of a RACK1 Interaction Network in Drosophila melanogaster Using SWATH-MS Article de journal
Dans: G3 (Bethesda), 2017, ISSN: 2160-1836.
Résumé | Liens | BibTeX | Étiquettes: meignin, PPSE
@article{kuhn_definition_2017b,
title = {Definition of a RACK1 Interaction Network in Drosophila melanogaster Using SWATH-MS},
author = {Lauriane Kuhn and Karim Majzoub and Evelyne Einhorn and Johana Chicher and Julien Pompon and Jean-Luc Imler and Philippe Hammann and Carine Meignin},
doi = {10.1534/g3.117.042564},
issn = {2160-1836},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {G3 (Bethesda)},
abstract = {Receptor for Activated C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster, we recently showed that RACK1 is required at the ribosome for IRES-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus, an IRES-containing virus.},
keywords = {meignin, PPSE},
pubstate = {published},
tppubtype = {article}
}
Receptor for Activated C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster, we recently showed that RACK1 is required at the ribosome for IRES-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus, an IRES-containing virus.
Gross Lauriane, Vicens Quentin, Einhorn Evelyne, Noireterre Audrey, Schaeffer Laure, Kuhn Lauriane, Imler Jean-Luc, Eriani Gilbert, Meignin Carine, Martin Franck
The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure Article de journal
Dans: Nucleic Acids Research, vol. 45, no. 15, p. 8993–9004, 2017, ISSN: 1362-4962.
Résumé | Liens | BibTeX | Étiquettes: meignin, PPSE
@article{gross_ires5utr_2017,
title = {The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure},
author = {Lauriane Gross and Quentin Vicens and Evelyne Einhorn and Audrey Noireterre and Laure Schaeffer and Lauriane Kuhn and Jean-Luc Imler and Gilbert Eriani and Carine Meignin and Franck Martin},
doi = {10.1093/nar/gkx622},
issn = {1362-4962},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Nucleic Acids Research},
volume = {45},
number = {15},
pages = {8993--9004},
abstract = {Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.},
keywords = {meignin, PPSE},
pubstate = {published},
tppubtype = {article}
}
Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.
Carapito Christine, Kuhn Lauriane, Karim Loukmane, Rompais Magali, Rabilloud Thierry, Schwenzer Hagen, Sissler Marie
Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases. Article de journal
Dans: Methods (San Diego, Calif.), vol. 113, p. 111–119, 2017, ISSN: 1095-9130 1046-2023.
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{carapito_two_2017,
title = {Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.},
author = {Christine Carapito and Lauriane Kuhn and Loukmane Karim and Magali Rompais and Thierry Rabilloud and Hagen Schwenzer and Marie Sissler},
doi = {10.1016/j.ymeth.2016.10.012},
issn = {1095-9130 1046-2023},
year = {2017},
date = {2017-01-01},
journal = {Methods (San Diego, Calif.)},
volume = {113},
pages = {111--119},
abstract = {Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.
Debard Sylvain, Bader Gaétan, Craene Johan-Owen De, Enkler Ludovic, Bär Séverine, Laporte Daphné, Hammann Philippe, Myslinski Evelyne, Senger Bruno, Friant Sylvie, Becker Hubert Dominique
Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells. Article de journal
Dans: Methods (San Diego, Calif.), vol. 113, p. 91–104, 2017, ISSN: 1095-9130 1046-2023, (Place: United States).
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{debard_nonconventional_2017,
title = {Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.},
author = {Sylvain Debard and Gaétan Bader and Johan-Owen De Craene and Ludovic Enkler and Séverine Bär and Daphné Laporte and Philippe Hammann and Evelyne Myslinski and Bruno Senger and Sylvie Friant and Hubert Dominique Becker},
doi = {10.1016/j.ymeth.2016.09.017},
issn = {1095-9130 1046-2023},
year = {2017},
date = {2017-01-01},
journal = {Methods (San Diego, Calif.)},
volume = {113},
pages = {91--104},
abstract = {By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.},
note = {Place: United States},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.
Incarbone M, Zimmermann A, Hammann P, Erhardt M, Michel F, Dunoyer P
Neutralization of mobile antiviral small RNA through peroxisomal import. Article de journal
Dans: Nature plants, vol. 3, p. 17094, 2017, ISSN: 2055-0278 2055-0278, (Place: England).
Résumé | Liens | BibTeX | Étiquettes: PPSE
@article{incarbone_neutralization_2017,
title = {Neutralization of mobile antiviral small RNA through peroxisomal import.},
author = {M Incarbone and A Zimmermann and P Hammann and M Erhardt and F Michel and P Dunoyer},
doi = {10.1038/nplants.2017.94},
issn = {2055-0278 2055-0278},
year = {2017},
date = {2017-01-01},
journal = {Nature plants},
volume = {3},
pages = {17094},
abstract = {In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles(1-3). In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)(4), whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.},
note = {Place: England},
keywords = {PPSE},
pubstate = {published},
tppubtype = {article}
}
In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles(1-3). In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)(4), whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.
Montavon Thomas, Kwon Yerim, Zimmermann Aude, Hammann Philippe, Vincent Timothée, Cognat Valérie, Michel Fabrice, Dunoyer Patrice
A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing. Article de journal
Dans: Nucleic acids research, vol. 45, no. 3, p. 1330–1344, 2017, ISSN: 1362-4962 0305-1048 0305-1048.
Résumé | Liens | BibTeX | Étiquettes: Inverted Repeat Sequences, PPSE
@article{montavon_specific_2017,
title = {A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.},
author = {Thomas Montavon and Yerim Kwon and Aude Zimmermann and Philippe Hammann and Timothée Vincent and Valérie Cognat and Fabrice Michel and Patrice Dunoyer},
doi = {10.1093/nar/gkw1264},
issn = {1362-4962 0305-1048 0305-1048},
year = {2017},
date = {2017-01-01},
journal = {Nucleic acids research},
volume = {45},
number = {3},
pages = {1330--1344},
abstract = {In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.},
keywords = {Inverted Repeat Sequences, PPSE},
pubstate = {published},
tppubtype = {article}
}
In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (