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2024
Rios-Delgado Gustavo, McReynolds Aubrey K G, Pagella Emma A, Norambuena Javiera, Briaud Paul, Zheng Vincent, Munneke Matthew J, Kim Jisun, Racine Hugo, Carroll Ronan, Zelzion Ehud, Skaar Eric, Bose Jeffrey L, Parker Dane, Lalaouna David, Boyd Jeffrey M
The small non-coding RNA IsrR regulates TCA cycle activity and virulence Journal Article
In: bioRxiv, 2024, ISSN: 2692-8205.
Abstract | Links | BibTeX | Tags: LALAOUNA, ROMBY, Unité ARN
@article{pmid39005296,
title = {The small non-coding RNA IsrR regulates TCA cycle activity and virulence},
author = {Gustavo Rios-Delgado and Aubrey K G McReynolds and Emma A Pagella and Javiera Norambuena and Paul Briaud and Vincent Zheng and Matthew J Munneke and Jisun Kim and Hugo Racine and Ronan Carroll and Ehud Zelzion and Eric Skaar and Jeffrey L Bose and Dane Parker and David Lalaouna and Jeffrey M Boyd},
doi = {10.1101/2024.07.03.601953},
issn = {2692-8205},
year = {2024},
date = {2024-07-01},
urldate = {2024-07-01},
journal = {bioRxiv},
abstract = { has evolved mechanisms to cope with low iron (Fe) availability in host tissues. uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an Δ mutant has decreased expression of , which codes for the Fe-dependent enzyme aconitase. Decreased expression prevented the Δ mutant from growing with amino acids as sole carbon and energy sources. Suppressor analysis determined that a mutation in , which produces a regulatory RNA, permitted growth by decreasing transcription. The decreased AcnA activity of the Δ mutant was partially relieved by an Δ mutation. Directed mutation of bases predicted to facilitate the interaction between the transcript and IsrR, decreased the ability of IsrR to control expression and IsrR bound to the transcript . IsrR also bound to the transcripts coding the alternate TCA cycle proteins , , , and . Whole cell metal analyses suggest that IsrR promotes Fe uptake and increases intracellular Fe not ligated by macromolecules. Lastly, we determined that Fur and IsrR promote infection using murine skin and acute pneumonia models.},
keywords = {LALAOUNA, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
has evolved mechanisms to cope with low iron (Fe) availability in host tissues. uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an Δ mutant has decreased expression of , which codes for the Fe-dependent enzyme aconitase. Decreased expression prevented the Δ mutant from growing with amino acids as sole carbon and energy sources. Suppressor analysis determined that a mutation in , which produces a regulatory RNA, permitted growth by decreasing transcription. The decreased AcnA activity of the Δ mutant was partially relieved by an Δ mutation. Directed mutation of bases predicted to facilitate the interaction between the transcript and IsrR, decreased the ability of IsrR to control expression and IsrR bound to the transcript . IsrR also bound to the transcripts coding the alternate TCA cycle proteins , , , and . Whole cell metal analyses suggest that IsrR promotes Fe uptake and increases intracellular Fe not ligated by macromolecules. Lastly, we determined that Fur and IsrR promote infection using murine skin and acute pneumonia models.
Kohl Maximilian P, Chane-Woon-Ming Béatrice, Bahena-Ceron Roberto, Jaramillo-Ponce Jose, Antoine Laura, Herrgott Lucas, Romby Pascale, Marzi Stefano
Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus Book Chapter
In: Claudio ValverdeVéronique Arluison Véronique Arluison, Claudio Valverde (Ed.): vol. 2741, pp. 73–100, Véronique Arluison, Claudio Valverde, Springer, 2024, ISSN: 1940-6029.
Abstract | Links | BibTeX | Tags: MARZI, ROMBY, Unité ARN
@inbook{pmid38217649,
title = {Ribosome Profiling Methods Adapted to the Study of RNA-Dependent Translation Regulation in Staphylococcus aureus},
author = {Maximilian P Kohl and Béatrice Chane-Woon-Ming and Roberto Bahena-Ceron and Jose Jaramillo-Ponce and Laura Antoine and Lucas Herrgott and Pascale Romby and Stefano Marzi},
editor = {Véronique Arluison, Claudio ValverdeVéronique Arluison, Claudio Valverde},
doi = {10.1007/978-1-0716-3565-0_5},
issn = {1940-6029},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Methods Mol Biol},
volume = {2741},
pages = {73--100},
publisher = {Véronique Arluison, Claudio Valverde},
edition = {Springer},
series = {Bacterial Regulatory RNA, Methods and Protocols},
abstract = {Noncoding RNAs, including regulatory RNAs (sRNAs), are instrumental in regulating gene expression in pathogenic bacteria, allowing them to adapt to various stresses encountered in their host environments. Staphylococcus aureus is a well-studied model for RNA-mediated regulation of virulence and pathogenicity, with sRNAs playing significant roles in shaping S. aureus interactions with human and animal hosts. By modulating the translation and/or stability of target mRNAs, sRNAs regulate the synthesis of virulence factors and regulatory proteins required for pathogenesis. Moreover, perturbation of the levels of RNA modifications in two other classes of noncoding RNAs, rRNAs, and tRNAs, has been proposed to contribute to stress adaptation. However, the study of how these various factors affect translation regulation has often been restricted to specific genes, using in vivo reporters and/or in vitro translation systems. Genome-wide sequencing approaches offer novel perspectives for studying RNA-dependent regulation. In particular, ribosome profiling methods provide a powerful resource for characterizing the overall landscape of translational regulation, contributing to a better understanding of S. aureus physiopathology. Here, we describe protocols that we have adapted to perform ribosome profiling in S. aureus.},
keywords = {MARZI, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Noncoding RNAs, including regulatory RNAs (sRNAs), are instrumental in regulating gene expression in pathogenic bacteria, allowing them to adapt to various stresses encountered in their host environments. Staphylococcus aureus is a well-studied model for RNA-mediated regulation of virulence and pathogenicity, with sRNAs playing significant roles in shaping S. aureus interactions with human and animal hosts. By modulating the translation and/or stability of target mRNAs, sRNAs regulate the synthesis of virulence factors and regulatory proteins required for pathogenesis. Moreover, perturbation of the levels of RNA modifications in two other classes of noncoding RNAs, rRNAs, and tRNAs, has been proposed to contribute to stress adaptation. However, the study of how these various factors affect translation regulation has often been restricted to specific genes, using in vivo reporters and/or in vitro translation systems. Genome-wide sequencing approaches offer novel perspectives for studying RNA-dependent regulation. In particular, ribosome profiling methods provide a powerful resource for characterizing the overall landscape of translational regulation, contributing to a better understanding of S. aureus physiopathology. Here, we describe protocols that we have adapted to perform ribosome profiling in S. aureus.
2023
Bahena-Ceron Roberto, Teixeira Chloe, Ponce Jose R Jaramillo, Wolff Philippe, Couzon Florence, François Pauline, Klaholz Bruno, Vandenesch François, Romby Pascale, Moreau Karen, Marzi Stefano
RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in Journal Article
In: RNA, vol. 30, no. 3, pp. 200-212, 2023, ISSN: 1469-9001.
Abstract | Links | BibTeX | Tags: ARN-MS, ROMBY, Unité ARN
@article{pmid38164596,
title = {RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in },
author = {Roberto Bahena-Ceron and Chloe Teixeira and Jose R Jaramillo Ponce and Philippe Wolff and Florence Couzon and Pauline François and Bruno Klaholz and François Vandenesch and Pascale Romby and Karen Moreau and Stefano Marzi},
doi = {10.1261/rna.079850.123},
issn = {1469-9001},
year = {2023},
date = {2023-12-01},
urldate = {2023-12-01},
journal = {RNA},
volume = {30},
number = {3},
pages = {200-212},
abstract = {rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in responsible for the Gram-positive specific mG2601, which is not modified in (G2574). We also demonstrate the absence of methylation on C1989, equivalent to C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications ( mG2601 and mC1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of Q causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.},
keywords = {ARN-MS, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in responsible for the Gram-positive specific mG2601, which is not modified in (G2574). We also demonstrate the absence of methylation on C1989, equivalent to C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications ( mG2601 and mC1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of Q causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.
Pivard Mariane, Caldelari Isabelle, Brun Virginie, Croisier Delphine, Jaquinod Michel, Anzala Nelson, Gilquin Benoît, Teixeira Chloé, Benito Yvonne, Couzon Florence, Romby Pascale, Moreau Karen, Vandenesch François
Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence Journal Article
In: Microbiol Spectr, pp. e0107323, 2023, ISSN: 2165-0497.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid37347186,
title = {Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence},
author = {Mariane Pivard and Isabelle Caldelari and Virginie Brun and Delphine Croisier and Michel Jaquinod and Nelson Anzala and Benoît Gilquin and Chloé Teixeira and Yvonne Benito and Florence Couzon and Pascale Romby and Karen Moreau and François Vandenesch},
doi = {10.1128/spectrum.01073-23},
issn = {2165-0497},
year = {2023},
date = {2023-06-01},
urldate = {2023-06-01},
journal = {Microbiol Spectr},
pages = {e0107323},
abstract = {Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of mRNA strongly impaired translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in -negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of mRNA induce the differential expression of , drastically impacting mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.
Giuliodori Anna Maria, Belardinelli Riccardo, Duval Melodie, Garofalo Raffaella, Schenckbecher Emma, Hauryliuk Vasili, Ennifar Eric, Marzi Stefano
CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression Journal Article
In: Front Microbiol, vol. 14, pp. 1118329, 2023, ISSN: 1664-302X.
Abstract | Links | BibTeX | Tags: ENNIFAR, ROMBY, Unité ARN
@article{pmid36846801b,
title = { CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression},
author = {Anna Maria Giuliodori and Riccardo Belardinelli and Melodie Duval and Raffaella Garofalo and Emma Schenckbecher and Vasili Hauryliuk and Eric Ennifar and Stefano Marzi},
doi = {10.3389/fmicb.2023.1118329},
issn = {1664-302X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Front Microbiol},
volume = {14},
pages = {1118329},
abstract = { CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.},
keywords = {ENNIFAR, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.
Giuliodori Anna Maria, Londei Paola, Marzi Stefano
2023, ISSN: 1664-302X.
Links | BibTeX | Tags: MARZI, ROMBY, Unité ARN
@misc{pmid37383636,
title = {Editorial: Interview with the translational apparatus: stories of intriguing circuits and mechanisms to regulate translation in bacteria, volume II},
author = {Anna Maria Giuliodori and Paola Londei and Stefano Marzi},
doi = {10.3389/fmicb.2023.1195257},
issn = {1664-302X},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Front Microbiol},
volume = {14},
pages = {1195257},
keywords = {MARZI, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {misc}
}
Bahena-Ceron R., Ponce J. R. Jaramillo, Kanazawa H., Antoine L., Wolff P., Marchand V., Klaholz B., Motorin Y, Romby P., Marzi S.
Methods to Analyze Post-transcriptional Modifications Applied to Stable RNAs in Staphylococcus aureus Book Chapter
In: Springer, (Ed.): RNA Structure and Function, vol. 14, pp. 233-258, 2023.
Abstract | Links | BibTeX | Tags: ROMBY, ROMBY MARZI, Unité ARN
@inbook{nokey,
title = {Methods to Analyze Post-transcriptional Modifications Applied to Stable RNAs in Staphylococcus aureus},
author = {R. Bahena-Ceron and J. R. Jaramillo Ponce and H. Kanazawa and L. Antoine and P. Wolff and V. Marchand and B. Klaholz and Y Motorin and P. Romby and S. Marzi},
editor = {Springer},
url = {https://link.springer.com/chapter/10.1007/978-3-031-36390-0_11},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
booktitle = {RNA Structure and Function},
volume = {14},
pages = {233-258},
abstract = {RNA modifications contribute to the various functions of RNAs in all living organisms. Some of these modifications are dynamic and contribute to the regulation of gene expression. In bacteria, their roles in stress, environmental adaptation, and in infections caused by pathogens have been recently fully recognized. In this review, we describe several methodologies including mass spectrometry, next-generation RNA sequencing methods, biochemical approaches, and cryo-EM structural analysis that are used to detect and localize the modifications in tRNAs and rRNAs. We illustrate how the combination of methods was necessary to avoid technical biases for a successful mapping of the modifications in tRNAs and rRNAs in Staphylococcus aureus.},
keywords = {ROMBY, ROMBY MARZI, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
RNA modifications contribute to the various functions of RNAs in all living organisms. Some of these modifications are dynamic and contribute to the regulation of gene expression. In bacteria, their roles in stress, environmental adaptation, and in infections caused by pathogens have been recently fully recognized. In this review, we describe several methodologies including mass spectrometry, next-generation RNA sequencing methods, biochemical approaches, and cryo-EM structural analysis that are used to detect and localize the modifications in tRNAs and rRNAs. We illustrate how the combination of methods was necessary to avoid technical biases for a successful mapping of the modifications in tRNAs and rRNAs in Staphylococcus aureus.
2022
Belinite Margarita, Khusainov Iskander, Marzi Stefano
30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis Journal Article
In: Bio Protoc, vol. 12, no. 20, 2022, ISSN: 2331-8325.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid36353712b,
title = { 30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis},
author = {Margarita Belinite and Iskander Khusainov and Stefano Marzi},
doi = {10.21769/BioProtoc.4532},
issn = {2331-8325},
year = {2022},
date = {2022-10-01},
urldate = {2022-10-01},
journal = {Bio Protoc},
volume = {12},
number = {20},
abstract = {The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K and Mg , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K and Mg , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.
Diallo Idrissa, Ho Jeffrey, Lambert Marine, Benmoussa Abderrahim, Husseini Zeinab, Lalaouna David, Massé Eric, Provost Patrick
A tRNA-derived fragment present in E. coli OMVs regulates host cell gene expression and proliferation Journal Article
In: PLoS Pathog, vol. 18, no. 9, pp. e1010827, 2022, ISSN: 1553-7374.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid36108089,
title = {A tRNA-derived fragment present in E. coli OMVs regulates host cell gene expression and proliferation},
author = {Idrissa Diallo and Jeffrey Ho and Marine Lambert and Abderrahim Benmoussa and Zeinab Husseini and David Lalaouna and Eric Massé and Patrick Provost},
doi = {10.1371/journal.ppat.1010827},
issn = {1553-7374},
year = {2022},
date = {2022-09-01},
urldate = {2022-09-01},
journal = {PLoS Pathog},
volume = {18},
number = {9},
pages = {e1010827},
abstract = {RNA-sequencing has led to a spectacular increase in the repertoire of bacterial sRNAs and improved our understanding of their biological functions. Bacterial sRNAs have also been found in outer membrane vesicles (OMVs), raising questions about their potential involvement in bacteria-host relationship, but few studies have documented this issue. Recent RNA-Sequencing analyses of bacterial RNA unveiled the existence of abundant very small RNAs (vsRNAs) shorter than 16 nt. These especially include tRNA fragments (tRFs) that are selectively loaded in OMVs and are predicted to target host mRNAs. Here, in Escherichia coli (E. coli), we report the existence of an abundant vsRNA, Ile-tRF-5X, which is selectively modulated by environmental stress, while remaining unaffected by inhibition of transcription or translation. Ile-tRF-5X is released through OMVs and can be transferred to human HCT116 cells, where it promoted MAP3K4 expression. Our findings provide a novel perspective and paradigm on the existing symbiosis between bacteria and human cells.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA-sequencing has led to a spectacular increase in the repertoire of bacterial sRNAs and improved our understanding of their biological functions. Bacterial sRNAs have also been found in outer membrane vesicles (OMVs), raising questions about their potential involvement in bacteria-host relationship, but few studies have documented this issue. Recent RNA-Sequencing analyses of bacterial RNA unveiled the existence of abundant very small RNAs (vsRNAs) shorter than 16 nt. These especially include tRNA fragments (tRFs) that are selectively loaded in OMVs and are predicted to target host mRNAs. Here, in Escherichia coli (E. coli), we report the existence of an abundant vsRNA, Ile-tRF-5X, which is selectively modulated by environmental stress, while remaining unaffected by inhibition of transcription or translation. Ile-tRF-5X is released through OMVs and can be transferred to human HCT116 cells, where it promoted MAP3K4 expression. Our findings provide a novel perspective and paradigm on the existing symbiosis between bacteria and human cells.
McKellar Stuart W, Ivanova Ivayla, Arede Pedro, Zapf Rachel L, Mercier Noémie, Chu Liang-Cui, Mediati Daniel G, Pickering Amy C, Briaud Paul, Foster Robert G, Kudla Grzegorz, Fitzgerald J Ross, Caldelari Isabelle, Carroll Ronan K, Tree Jai J, Granneman Sander
RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression Journal Article
In: Nat Commun, vol. 13, no. 1, pp. 3560, 2022, ISSN: 2041-1723.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid35732654,
title = {RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression},
author = {Stuart W McKellar and Ivayla Ivanova and Pedro Arede and Rachel L Zapf and Noémie Mercier and Liang-Cui Chu and Daniel G Mediati and Amy C Pickering and Paul Briaud and Robert G Foster and Grzegorz Kudla and J Ross Fitzgerald and Isabelle Caldelari and Ronan K Carroll and Jai J Tree and Sander Granneman},
doi = {10.1038/s41467-022-31173-y},
issn = {2041-1723},
year = {2022},
date = {2022-06-01},
urldate = {2022-06-01},
journal = {Nat Commun},
volume = {13},
number = {1},
pages = {3560},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.
Desgranges E., Barrientos L., Herrgott L., Marzi S., Toledo-Arana A., Moreau K., Vandenesch F., Romby P., Caldelari I.
In: Mol Microbiol, 2022, ISBN: 34783400, (1365-2958 (Electronic) 0950-382X (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{nokey,
title = {The 3'UTR-derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose-6-phosphate uptake in Staphylococcus aureus},
author = {E. Desgranges and L. Barrientos and L. Herrgott and S. Marzi and A. Toledo-Arana and K. Moreau and F. Vandenesch and P. Romby and I. Caldelari},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34783400},
doi = {10.1111/mmi.14845},
isbn = {34783400},
year = {2022},
date = {2022-01-01},
urldate = {2021-01-01},
journal = {Mol Microbiol},
abstract = {Staphylococcus aureus RsaG is a 3' untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5' to 3' end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5' end of RsaG, leading to its accumulation. RsaG together with uhpT are induced when bacteria are internalized into host cells or in presence of mucus-secreting cells. Using MS2 affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, to degrade, or to repress translation of its mRNA targets. While RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors a sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.},
note = {1365-2958 (Electronic)
0950-382X (Linking)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus RsaG is a 3' untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5' to 3' end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5' end of RsaG, leading to its accumulation. RsaG together with uhpT are induced when bacteria are internalized into host cells or in presence of mucus-secreting cells. Using MS2 affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, to degrade, or to repress translation of its mRNA targets. While RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors a sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.
Laveilhe A., Fochesato S., Lalaouna D., Heulin T., Achouak W.
Phytobeneficial traits of rhizobacteria under the control of multiple molecular dialogues Journal Article
In: Microb Biotechnol, vol. 15, iss. 7, pp. 2083-2096, 2022, ISBN: 35502577, (1751-7915 (Electronic) 1751-7915 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{nokey,
title = {Phytobeneficial traits of rhizobacteria under the control of multiple molecular dialogues},
author = {A. Laveilhe and S. Fochesato and D. Lalaouna and T. Heulin and W. Achouak},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35502577},
doi = {10.1111/1751-7915.14023},
isbn = {35502577},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Microb Biotechnol},
volume = {15},
issue = {7},
pages = {2083-2096},
abstract = {Pseudomonads play crucial roles in plant growth promotion and control of plant diseases. However, under natural conditions, other microorganisms competing for the same nutrient resources in the rhizosphere may exert negative control over their phytobeneficial characteristics. We assessed the expression of phytobeneficial genes involved in biocontrol, biostimulation and iron regulation such as, phlD, hcnA, acdS, and iron-small regulatory RNAs prrF1 and prrF2 in Pseudomonas brassicacearum co-cultivated with three phytopathogenic fungi, and two rhizobacteria in the presence or absence of Brassica napus, and in relation to iron availability. We found that the antifungal activity of P. brassicacearum depends mostly on the production of DAPG and not on HCN whose production is suppressed by fungi. We have also shown that the two-competing bacterial strains modulate the plant growth promotion activity of P. brassicacearum by modifying the expression of phlD, hcnA and acdS according to iron availability. Overall, it allows us to better understand the complexity of the multiple molecular dialogues that take place underground between microorganisms and between plants and its rhizosphere microbiota and to show that synergy in favour of phytobeneficial gene expression may exist between different bacterial species.},
note = {1751-7915 (Electronic)
1751-7915 (Linking)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Pseudomonads play crucial roles in plant growth promotion and control of plant diseases. However, under natural conditions, other microorganisms competing for the same nutrient resources in the rhizosphere may exert negative control over their phytobeneficial characteristics. We assessed the expression of phytobeneficial genes involved in biocontrol, biostimulation and iron regulation such as, phlD, hcnA, acdS, and iron-small regulatory RNAs prrF1 and prrF2 in Pseudomonas brassicacearum co-cultivated with three phytopathogenic fungi, and two rhizobacteria in the presence or absence of Brassica napus, and in relation to iron availability. We found that the antifungal activity of P. brassicacearum depends mostly on the production of DAPG and not on HCN whose production is suppressed by fungi. We have also shown that the two-competing bacterial strains modulate the plant growth promotion activity of P. brassicacearum by modifying the expression of phlD, hcnA and acdS according to iron availability. Overall, it allows us to better understand the complexity of the multiple molecular dialogues that take place underground between microorganisms and between plants and its rhizosphere microbiota and to show that synergy in favour of phytobeneficial gene expression may exist between different bacterial species.
Charbonnier Mathilde, González-Espinoza Gabriela, Kehl-Fie Thomas E, Lalaouna David
Battle for Metals: Regulatory RNAs at the Front Line Journal Article
In: Front Cell Infect Microbiol, vol. 12, pp. 952948, 2022, ISSN: 2235-2988.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{pmid35865816,
title = {Battle for Metals: Regulatory RNAs at the Front Line},
author = {Mathilde Charbonnier and Gabriela González-Espinoza and Thomas E Kehl-Fie and David Lalaouna},
doi = {10.3389/fcimb.2022.952948},
issn = {2235-2988},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Front Cell Infect Microbiol},
volume = {12},
pages = {952948},
abstract = {Metal such as iron, zinc, manganese, and nickel are essential elements for bacteria. These nutrients are required in crucial structural and catalytic roles in biological processes, including precursor biosynthesis, DNA replication, transcription, respiration, and oxidative stress responses. While essential, in excess these nutrients can also be toxic. The immune system leverages both of these facets, to limit bacterial proliferation and combat invaders. Metal binding immune proteins reduce the bioavailability of metals at the infection sites starving intruders, while immune cells intoxicate pathogens by providing metals in excess leading to enzyme mismetallation and/or reactive oxygen species generation. In this dynamic metal environment, maintaining metal homeostasis is a critical process that must be precisely coordinated. To achieve this, bacteria utilize diverse metal uptake and efflux systems controlled by metalloregulatory proteins. Recently, small regulatory RNAs (sRNAs) have been revealed to be critical post-transcriptional regulators, working in conjunction with transcription factors to promote rapid adaptation and to fine-tune bacterial adaptation to metal abundance. In this mini review, we discuss the expanding role for sRNAs in iron homeostasis, but also in orchestrating adaptation to the availability of other metals like manganese and nickel. Furthermore, we describe the sRNA-mediated interdependency between metal homeostasis and oxidative stress responses, and how regulatory networks controlled by sRNAs contribute to survival and virulence.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Metal such as iron, zinc, manganese, and nickel are essential elements for bacteria. These nutrients are required in crucial structural and catalytic roles in biological processes, including precursor biosynthesis, DNA replication, transcription, respiration, and oxidative stress responses. While essential, in excess these nutrients can also be toxic. The immune system leverages both of these facets, to limit bacterial proliferation and combat invaders. Metal binding immune proteins reduce the bioavailability of metals at the infection sites starving intruders, while immune cells intoxicate pathogens by providing metals in excess leading to enzyme mismetallation and/or reactive oxygen species generation. In this dynamic metal environment, maintaining metal homeostasis is a critical process that must be precisely coordinated. To achieve this, bacteria utilize diverse metal uptake and efflux systems controlled by metalloregulatory proteins. Recently, small regulatory RNAs (sRNAs) have been revealed to be critical post-transcriptional regulators, working in conjunction with transcription factors to promote rapid adaptation and to fine-tune bacterial adaptation to metal abundance. In this mini review, we discuss the expanding role for sRNAs in iron homeostasis, but also in orchestrating adaptation to the availability of other metals like manganese and nickel. Furthermore, we describe the sRNA-mediated interdependency between metal homeostasis and oxidative stress responses, and how regulatory networks controlled by sRNAs contribute to survival and virulence.
2021
Lalaouna D, Fochesato S, Harir M, Ortet P, Schmitt-Kopplin P, Heulin T, Achouak W
In: Microorganisms, vol. 9, no. 2, pp. 250, 2021.
Abstract | Links | BibTeX | Tags: GacA-dependent, nutritional stress, Pseudomonas brassicacearum, ROMBY, Rsm sRNAs, sRNAs stability, Unité ARN
@article{,
title = {Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments},
author = {D Lalaouna and S Fochesato and M Harir and P Ortet and P Schmitt-Kopplin and T Heulin and W Achouak},
url = {https://www.mdpi.com/2076-2607/9/2/250},
doi = {10.3390/microorganisms9020250},
year = {2021},
date = {2021-01-01},
journal = {Microorganisms},
volume = {9},
number = {2},
pages = {250},
abstract = {In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.},
keywords = {GacA-dependent, nutritional stress, Pseudomonas brassicacearum, ROMBY, Rsm sRNAs, sRNAs stability, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.
Mercier N, Prévost K, Massé E, Romby P, Caldelari I, Lalaouna D
MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria Journal Article
In: J Vis Exp, pp. e61731, 2021.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{Mercier2021,
title = {MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria},
author = {N Mercier and K Prévost and E Massé and P Romby and I Caldelari and D Lalaouna},
url = {https://www.jove.com/t/61731/ms2-affinity-purification-coupled-with-rna-sequencing-gram-positive},
doi = {10.3791/61731},
year = {2021},
date = {2021-01-01},
journal = {J Vis Exp},
pages = {e61731},
abstract = {Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5’ extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5’ extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.
Catalan-Moreno A, Cela M, Menendez-Gil P, Irurzun N, Caballero C J, Caldelari I, Toledo-Arana A
RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures Journal Article
In: Nucleic Acids Res, pp. on press, 2021, ISBN: 33660769, (1362-4962 (Electronic) 0305-1048 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{Catalan-Moreno2021,
title = {RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures},
author = {A Catalan-Moreno and M Cela and P Menendez-Gil and N Irurzun and C J Caballero and I Caldelari and A Toledo-Arana},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33660769},
doi = {0.1093/nar/gkab117},
isbn = {33660769},
year = {2021},
date = {2021-01-01},
journal = {Nucleic Acids Res},
pages = {on press},
abstract = {Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5'UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22 degrees C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37 degrees C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37 degrees C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.},
note = {1362-4962 (Electronic)
0305-1048 (Linking)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5'UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22 degrees C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37 degrees C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37 degrees C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.
Ortet P, Fochesato S, Bitbol A F, Whitworth D E, Lalaouna D, Santaella C, Heulin T, Achouak W, Barakat M
Evolutionary history expands the range of signaling interactions in hybrid multikinase networks Journal Article
In: Sci Rep, vol. 11, no. 1, pp. 11763, 2021, ISBN: 34083699.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Evolutionary history expands the range of signaling interactions in hybrid multikinase networks},
author = {P Ortet and S Fochesato and A F Bitbol and D E Whitworth and D Lalaouna and C Santaella and T Heulin and W Achouak and M Barakat},
url = {https://pubmed.ncbi.nlm.nih.gov/34083699/},
doi = {10.1038/s41598-021-91260-w},
isbn = {34083699},
year = {2021},
date = {2021-01-01},
journal = {Sci Rep},
volume = {11},
number = {1},
pages = {11763},
abstract = {Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS-GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS-GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.
Giuliodori A M, Marzi S
Editorial: Interview With the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria Journal Article
In: Front Microbiol, vol. 12, pp. 707354, 2021, ISBN: 34220790, (1664-302X (Print) 1664-302X (Linking) Editorial).
Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Editorial: Interview With the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria},
author = {A M Giuliodori and S Marzi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34220790},
doi = {10.3389/fmicb.2021.707354},
isbn = {34220790},
year = {2021},
date = {2021-01-01},
journal = {Front Microbiol},
volume = {12},
pages = {707354},
note = {1664-302X (Print)
1664-302X (Linking)
Editorial},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Barrientos L, Mercier N, Lalaouna D, Caldelari I
Assembling the Current Pieces: The Puzzle of RNA-Mediated Regulation in Staphylococcus aureus Journal Article
In: Front Microbiol, vol. 12, pp. 706690, 2021, ISBN: 34367109, (1664-302X (Print) 1664-302X (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Assembling the Current Pieces: The Puzzle of RNA-Mediated Regulation in Staphylococcus aureus},
author = {L Barrientos and N Mercier and D Lalaouna and I Caldelari},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34367109},
doi = {10.3389/fmicb.2021.706690},
isbn = {34367109},
year = {2021},
date = {2021-01-01},
journal = {Front Microbiol},
volume = {12},
pages = {706690},
abstract = {The success of the major opportunistic human Staphylococcus aureus relies on the production of numerous virulence factors, which allow rapid colonization and dissemination in any tissues. Indeed, regulation of its virulence is multifactorial, and based on the production of transcriptional factors, two-component systems (TCS) and small regulatory RNAs (sRNAs). Advances in high-throughput sequencing technologies have unveiled the existence of hundreds of potential RNAs with regulatory functions, but only a fraction of which have been validated in vivo. These discoveries have modified our thinking and understanding of bacterial physiology and virulence fitness by placing sRNAs, alongside transcriptional regulators, at the center of complex and intertwined regulatory networks that allow S. aureus to rapidly adapt to the environmental cues present at infection sites. In this review, we describe the recently acquired knowledge of characterized regulatory RNAs in S. aureus that are associated with metal starvation, nutrient availability, stress responses and virulence. These findings highlight the importance of sRNAs for the comprehension of S. aureus infection processes while raising questions about the interplay between these key regulators and the pathways they control.},
note = {1664-302X (Print)
1664-302X (Linking)
Journal Article
Review},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The success of the major opportunistic human Staphylococcus aureus relies on the production of numerous virulence factors, which allow rapid colonization and dissemination in any tissues. Indeed, regulation of its virulence is multifactorial, and based on the production of transcriptional factors, two-component systems (TCS) and small regulatory RNAs (sRNAs). Advances in high-throughput sequencing technologies have unveiled the existence of hundreds of potential RNAs with regulatory functions, but only a fraction of which have been validated in vivo. These discoveries have modified our thinking and understanding of bacterial physiology and virulence fitness by placing sRNAs, alongside transcriptional regulators, at the center of complex and intertwined regulatory networks that allow S. aureus to rapidly adapt to the environmental cues present at infection sites. In this review, we describe the recently acquired knowledge of characterized regulatory RNAs in S. aureus that are associated with metal starvation, nutrient availability, stress responses and virulence. These findings highlight the importance of sRNAs for the comprehension of S. aureus infection processes while raising questions about the interplay between these key regulators and the pathways they control.
Mediati D G, Lalaouna D, Tree J J
Burning the Candle at Both Ends: Have Exoribonucleases Driven Divergence of Regulatory RNA Mechanisms in Bacteria? Journal Article
In: mBio, pp. e0104121, 2021, ISBN: 34372700, (2150-7511 (Electronic) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Burning the Candle at Both Ends: Have Exoribonucleases Driven Divergence of Regulatory RNA Mechanisms in Bacteria?},
author = {D G Mediati and D Lalaouna and J J Tree},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34372700},
doi = {10.1128/mBio.01041-21},
isbn = {34372700},
year = {2021},
date = {2021-01-01},
journal = {mBio},
pages = {e0104121},
abstract = {Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model Gammaproteobacteria Escherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling approximately 20 to 25% of the entire transcriptome. While the model Firmicutes Bacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3'-->5' exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3' UTR cleavage fragments that contain stabilizing 3' structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5' and 3' ends, efficiently degrading mRNA 3' UTR fragments. Here, we propose that the lack of 5'-->3' exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3' UTR ends as the "default" setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3' UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.},
note = {2150-7511 (Electronic)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Regulatory RNAs have emerged as ubiquitous gene regulators in all bacterial species studied to date. The combination of sequence-specific RNA interactions and malleable RNA structure has allowed regulatory RNA to adopt different mechanisms of gene regulation in a diversity of genetic backgrounds. In the model Gammaproteobacteria Escherichia coli and Salmonella, the regulatory RNA chaperone Hfq appears to play a global role in gene regulation, directly controlling approximately 20 to 25% of the entire transcriptome. While the model Firmicutes Bacillus subtilis and Staphylococcus aureus encode a Hfq homologue, its role has been significantly depreciated. These bacteria also have marked differences in RNA turnover. E. coli and Salmonella degrade RNA through internal endonucleolytic and 3'-->5' exonucleolytic cleavage that appears to allow transient accumulation of mRNA 3' UTR cleavage fragments that contain stabilizing 3' structures. In contrast, B. subtilis and S. aureus are able to exonucleolytically attack internally cleaved RNA from both the 5' and 3' ends, efficiently degrading mRNA 3' UTR fragments. Here, we propose that the lack of 5'-->3' exoribonuclease activity in Gammaproteobacteria has allowed the accumulation of mRNA 3' UTR ends as the "default" setting. This in turn may have provided a larger pool of unconstrained RNA sequences that has fueled the expansion of Hfq function and small RNA (sRNA) regulation in E. coli and Salmonella. Conversely, the exoribonuclease RNase J may be a significant barrier to the evolution of 3' UTR sRNAs in B. subtilis and S. aureus that has limited the pool of RNA ligands available to Hfq and other sRNA chaperones, depreciating their function in these model Firmicutes.
Antoine L, Bahena-Ceron R, Bunwaree H Devi, Gobry M, Loegler V, Romby P, Marzi S
RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence Journal Article
In: Genes (Basel), vol. 12, no. 8, 2021, ISBN: 34440299, (2073-4425 (Electronic) 2073-4425 (Linking) Journal Article Review).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence},
author = {L Antoine and R Bahena-Ceron and H Devi Bunwaree and M Gobry and V Loegler and P Romby and S Marzi},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34440299},
doi = {10.3390/genes12081125},
isbn = {34440299},
year = {2021},
date = {2021-01-01},
journal = {Genes (Basel)},
volume = {12},
number = {8},
abstract = {RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.},
note = {2073-4425 (Electronic)
2073-4425 (Linking)
Journal Article
Review},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.
Lalaouna D., Prévost K., Park S., Chénard T., Bouchard M - P., Caron M - P., Vanderpool C. K., Fei J., Massé E.
Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli Journal Article
In: Non-Coding RNA, vol. 7, no. 4, pp. 64, 2021.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{nokey,
title = {Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli},
author = {D. Lalaouna and K. Prévost and S. Park and T. Chénard and M - P. Bouchard and M - P. Caron and C. K. Vanderpool and J. Fei and E. Massé},
url = {https://www.mdpi.com/2311-553X/7/4/64},
doi = {10.3390/ncrna7040064},
year = {2021},
date = {2021-01-01},
journal = {Non-Coding RNA},
volume = {7},
number = {4},
pages = {64},
abstract = {Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.
Belinite M., Khusainov I., Soufari H., Marzi S., Romby P., Yusupov M., Hashem Y.
Stabilization of Ribosomal RNA of the Small Subunit by Spermidine in Staphylococcus aureus Journal Article
In: Front Mol Biosci, vol. 8, pp. 738752, 2021, ISBN: 34869582, (2296-889X (Print) 2296-889X (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{nokey,
title = {Stabilization of Ribosomal RNA of the Small Subunit by Spermidine in Staphylococcus aureus},
author = {M. Belinite and I. Khusainov and H. Soufari and S. Marzi and P. Romby and M. Yusupov and Y. Hashem},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34869582},
doi = {10.3389/fmolb.2021.738752},
isbn = {34869582},
year = {2021},
date = {2021-01-01},
journal = {Front Mol Biosci},
volume = {8},
pages = {738752},
abstract = {Cryo-electron microscopy is now used as a method of choice in structural biology for studying protein synthesis, a process mediated by the ribosome machinery. In order to achieve high-resolution structures using this approach, one needs to obtain homogeneous and stable samples, which requires optimization of ribosome purification in a species-dependent manner. This is especially critical for the bacterial small ribosomal subunit that tends to be unstable in the absence of ligands. Here, we report a protocol for purification of stable 30 S from the Gram-positive bacterium Staphylococcus aureus and its cryo-EM structures: in presence of spermidine at a resolution ranging between 3.4 and 3.6 A and in its absence at 5.3 A. Using biochemical characterization and cryo-EM, we demonstrate the importance of spermidine for stabilization of the 30 S via preserving favorable conformation of the helix 44.},
note = {2296-889X (Print)
2296-889X (Linking)
Journal Article},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Cryo-electron microscopy is now used as a method of choice in structural biology for studying protein synthesis, a process mediated by the ribosome machinery. In order to achieve high-resolution structures using this approach, one needs to obtain homogeneous and stable samples, which requires optimization of ribosome purification in a species-dependent manner. This is especially critical for the bacterial small ribosomal subunit that tends to be unstable in the absence of ligands. Here, we report a protocol for purification of stable 30 S from the Gram-positive bacterium Staphylococcus aureus and its cryo-EM structures: in presence of spermidine at a resolution ranging between 3.4 and 3.6 A and in its absence at 5.3 A. Using biochemical characterization and cryo-EM, we demonstrate the importance of spermidine for stabilization of the 30 S via preserving favorable conformation of the helix 44.
2020
Lalaouna D, Romby P
Manganese: the battle of the two armies Journal Article
In: Project Repository Journal, 2020.
Links | BibTeX | Tags: ROMBY, Unité ARN
@article{Lalouna2020,
title = {Manganese: the battle of the two armies},
author = {D Lalaouna and P Romby},
url = {https://univoak.eu/islandora/object/islandora:105630},
year = {2020},
date = {2020-07-01},
journal = {Project Repository Journal},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Menendez-Gil P, Caballero C J, Catalan-Moreno A, Irurzun N, Barrio-Hernandez I, Caldelari I, Toledo-Arana A
Differential evolution in 3'UTRs leads to specific gene expression in Staphylococcus Journal Article
In: Nucleic Acids Res, vol. 48, no. 5, pp. 2544-2563, 2020, ISBN: 32016395.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Differential evolution in 3'UTRs leads to specific gene expression in Staphylococcus},
author = {P Menendez-Gil and C J Caballero and A Catalan-Moreno and N Irurzun and I Barrio-Hernandez and I Caldelari and A Toledo-Arana},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32016395},
doi = {10.1093/nar/gkaa047},
isbn = {32016395},
year = {2020},
date = {2020-01-01},
journal = {Nucleic Acids Res},
volume = {48},
number = {5},
pages = {2544-2563},
abstract = {The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity.
Georg J, Lalaouna D, Hou S, Lott S C, Caldelari I, Marzi S, Hess W R, Romby P
The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs Journal Article
In: Mol Microbiol, vol. 113, no. 3, pp. 603-612, 2020, ISBN: 31705780.
Abstract | Links | BibTeX | Tags: ROMBY, Staphylococcus aureus CopraRNA MAPS post-transcriptional regulation sRNAs, Unité ARN
@article{,
title = {The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs},
author = {J Georg and D Lalaouna and S Hou and S C Lott and I Caldelari and S Marzi and W R Hess and P Romby},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31705780},
doi = {10.1111/mmi.14420},
isbn = {31705780},
year = {2020},
date = {2020-01-01},
journal = {Mol Microbiol},
volume = {113},
number = {3},
pages = {603-612},
abstract = {Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic miRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNA's targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more detail. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable.},
keywords = {ROMBY, Staphylococcus aureus CopraRNA MAPS post-transcriptional regulation sRNAs, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic miRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNA's targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more detail. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable.
Antoine L, Wolff P
Mapping of Posttranscriptional tRNA Modifications by Two-Dimensional Gel Electrophoresis Mass Spectrometry Book Chapter
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 101-110, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006310.
Abstract | Links | BibTeX | Tags: 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.
Desgranges E, Caldelari I, Marzi S, Lalaouna D
Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs Journal Article
In: Biochim Biophys Acta Gene Regul Mech, vol. 1863, no. 3, pp. 194506, 2020, ISBN: 32068131.
Abstract | Links | BibTeX | Tags: Bacteria Post-transcriptional regulation RNA sequencing Regulatory network Small regulatory RNA Targetome, ROMBY, Unité ARN
@article{,
title = {Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs},
author = {E Desgranges and I Caldelari and S Marzi and D Lalaouna},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32068131},
doi = {10.1016/j.bbagrm.2020.194506},
isbn = {32068131},
year = {2020},
date = {2020-01-01},
journal = {Biochim Biophys Acta Gene Regul Mech},
volume = {1863},
number = {3},
pages = {194506},
abstract = {Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.},
keywords = {Bacteria Post-transcriptional regulation RNA sequencing Regulatory network Small regulatory RNA Targetome, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.
Bouhedda F, Fam K T, Collot M, Autour A, Marzi S, Klymchenko A, Ryckelynck M
A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells Journal Article
In: Nat Chem Biol, vol. 16, no. 1, pp. 69-76, 2020, ISBN: 31636432.
Abstract | Links | BibTeX | Tags: ROMBY, RYCKELYNCK, Unité ARN
@article{,
title = {A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells},
author = {F Bouhedda and K T Fam and M Collot and A Autour and S Marzi and A Klymchenko and M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31636432},
doi = {https://doi.org/10.1038/s41589-019-0381-8},
isbn = {31636432},
year = {2020},
date = {2020-01-01},
journal = {Nat Chem Biol},
volume = {16},
number = {1},
pages = {69-76},
abstract = {Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.},
keywords = {ROMBY, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.
Rol-Moreno J, Kuhn L, Marzi S, Simonetti A
In: Arluison, V; Wien, F (Ed.): RNA Spectroscopy: Methods and Protocols, vol. 2113, pp. 329-339, Springer Protocols, Humana Press, New York, NY, 2020, ISBN: 32006323.
Abstract | Links | BibTeX | Tags: 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.
2019
P Brown RELISH Consortium, Zhou Y
Large expert-curated database for benchmarking document similarity detection in biomedical literature search Journal Article
In: Database, vol. 2019, no. 2019, pp. baz085, 2019.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{Brown2019,
title = {Large expert-curated database for benchmarking document similarity detection in biomedical literature search},
author = {P Brown, RELISH Consortium and Y Zhou},
doi = {https://doi.org/10.1093/database/baz085},
year = {2019},
date = {2019-10-29},
journal = {Database},
volume = {2019},
number = {2019},
pages = {baz085},
abstract = {Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.
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 Journal Article
In: Nucleic Acids Res, vol. 47, no. 18, pp. 9871-9887, 2019, ISBN: 31504767.
Abstract | Links | BibTeX | Tags: 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.
Desgranges E, Marzi S, Moreau K, Romby P, Caldelari I
Noncoding RNA Journal Article
In: Microbiol Spectr, vol. 7, no. 2, pp. 1-2, 2019, ISBN: 31004423.
Abstract | Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Noncoding RNA},
author = {E Desgranges and S Marzi and K Moreau and P Romby and I Caldelari},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31004423?dopt=Abstract},
doi = {10.1128/microbiolspec.GPP3-0038-2018},
isbn = {31004423},
year = {2019},
date = {2019-01-01},
journal = {Microbiol Spectr},
volume = {7},
number = {2},
pages = {1-2},
abstract = {Regulatory RNAs, present in many bacterial genomes and particularly in pathogenic bacteria such as Staphylococcus aureus, control the expression of genes encoding virulence factors or metabolic proteins. They are extremely diverse and include noncoding RNAs (sRNA), antisense RNAs, and some 5' or 3' untranslated regions of messenger RNAs that act as sensors for metabolites, tRNAs, or environmental conditions (e.g., temperature, pH). In this review we focus on specific examples of sRNAs of S. aureus that illustrate how numerous sRNAs and associated proteins are embedded in complex networks of regulation. In addition, we discuss the CRISPR-Cas systems defined as an RNA-interference-like mechanism, which also exist in staphylococcal strains.},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Regulatory RNAs, present in many bacterial genomes and particularly in pathogenic bacteria such as Staphylococcus aureus, control the expression of genes encoding virulence factors or metabolic proteins. They are extremely diverse and include noncoding RNAs (sRNA), antisense RNAs, and some 5' or 3' untranslated regions of messenger RNAs that act as sensors for metabolites, tRNAs, or environmental conditions (e.g., temperature, pH). In this review we focus on specific examples of sRNAs of S. aureus that illustrate how numerous sRNAs and associated proteins are embedded in complex networks of regulation. In addition, we discuss the CRISPR-Cas systems defined as an RNA-interference-like mechanism, which also exist in staphylococcal strains.
Desgranges E, Bronesky D, Corvaglia A, François P, Caballero C, Prado L, Toledo-Arana A, Lasa I, Moreau K, Vandenesch F, Marzi S, Romby P, Caldelari I
[RsaI, a multifaceted regulatory RNA, modulates the metabolism of the opportunistic pathogen Staphylococcus aureus] Journal Article
In: Med Sci (Paris), vol. 35, no. 12, pp. 1221-1223, 2019, ISBN: 31903946.
Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {[RsaI, a multifaceted regulatory RNA, modulates the metabolism of the opportunistic pathogen Staphylococcus aureus]},
author = {E Desgranges and D Bronesky and A Corvaglia and P François and C Caballero and L Prado and A Toledo-Arana and I Lasa and K Moreau and F Vandenesch and S Marzi and P Romby and I Caldelari},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31903946?dopt=Abstract},
doi = {10.1051/medsci/2019235},
isbn = {31903946},
year = {2019},
date = {2019-01-01},
journal = {Med Sci (Paris)},
volume = {35},
number = {12},
pages = {1221-1223},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Bronesky D, Desgranges E, Corvaglia A, François P, Caballero C J, Prado L, Toledo-Arana A, Lasa I, Moreau K, Vandenesch F, Marzi S, Romby P, Caldelari I
A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus Journal Article
In: EMBO J, vol. 38, no. 6, pp. e99363, 2019, ISBN: 30760492.
Abstract | Links | BibTeX | Tags: ROMBY, sRNA carbon metabolism catabolite control protein A pathogenic bacteria regulatory RNAs translational regulation, Unité ARN
@article{,
title = {A multifaceted small RNA modulates gene expression upon glucose limitation in \textit{Staphylococcus aureus}},
author = {D Bronesky and E Desgranges and A Corvaglia and P François and C J Caballero and L Prado and A Toledo-Arana and I Lasa and K Moreau and F Vandenesch and S Marzi and P Romby and I Caldelari},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30760492},
doi = {10.15252/embj.201899363},
isbn = {30760492},
year = {2019},
date = {2019-01-01},
journal = {EMBO J},
volume = {38},
number = {6},
pages = {e99363},
abstract = {Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.},
keywords = {ROMBY, sRNA carbon metabolism catabolite control protein A pathogenic bacteria regulatory RNAs translational regulation, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.
Antoine L, Wolff P, Westhof E, Romby P, Marzi S
Mapping post-transcriptional modifications in Staphylococcus aureus tRNAs by nanoLC/MSMS Journal Article
In: Biochimie, vol. 164, pp. 60-69, 2019, ISBN: 31295507.
Abstract | Links | BibTeX | Tags: 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.
2018
Lalaouna D, Desgranges E, Caldelari I, Marzi S
MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus Journal Article
In: Methods Enzymol, vol. 612, pp. 393-411, 2018, ISBN: 30502950.
Abstract | Links | BibTeX | Tags: Galaxy workflow Interactome MS2-affinity purification coupled with RNA sequencing RNA:RNA interaction Small regulatory RNA Staphylococcus aureus Targetome, ROMBY, Unité ARN
@article{,
title = {MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus},
author = {D Lalaouna and E Desgranges and I Caldelari and S Marzi},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30502950?dopt=Abstract},
doi = {10.1016/bs.mie.2018.08.022},
isbn = {30502950},
year = {2018},
date = {2018-01-01},
journal = {Methods Enzymol},
volume = {612},
pages = {393-411},
abstract = {Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.},
keywords = {Galaxy workflow Interactome MS2-affinity purification coupled with RNA sequencing RNA:RNA interaction Small regulatory RNA Staphylococcus aureus Targetome, ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.
2017
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. Journal Article
In: Nucleic Acids Res, vol. 45, no. 11, pp. 6746-6760, 2017, ISBN: 28379505.
Abstract | Links | BibTeX | Tags: 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.
Lioliou E, Fechter P, Caldelari I, Jester B C, Dubrac S, Helfer A C, Boisset S, Vandenesch F, Romby P, Geissmann T
Erratum Journal Article
In: RNA Biol, vol. 14, no. 12, pp. 1827, 2017, ISBN: 29243975.
Links | BibTeX | Tags: ROMBY, Unité ARN
@article{,
title = {Erratum},
author = {E Lioliou and P Fechter and I Caldelari and B C Jester and S Dubrac and A C Helfer and S Boisset and F Vandenesch and P Romby and T Geissmann},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29243975?dopt=Abstract},
doi = {10.1080/15476286.2016.1184436},
isbn = {29243975},
year = {2017},
date = {2017-01-01},
journal = {RNA Biol},
volume = {14},
number = {12},
pages = {1827},
keywords = {ROMBY, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
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. Journal Article
In: Nucleic Acids Res, vol. 45, no. 2, pp. 1026, 2017, ISBN: 28123039.
Abstract | Links | BibTeX | Tags: 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.