Papers by Eliane Hajnsdorf
Microorganisms, 2021
The ribosome is the universal catalyst for protein synthesis. Despite extensive studies, the dive... more The ribosome is the universal catalyst for protein synthesis. Despite extensive studies, the diversity of structures and functions of this ribonucleoprotein is yet to be fully understood. Deciphering the biogenesis of the ribosome in a step-by-step manner revealed that this complexity is achieved through a plethora of effectors involved in the maturation and assembly of ribosomal RNAs and proteins. Conserved from bacteria to eukaryotes, double-stranded specific RNase III enzymes play a large role in the regulation of gene expression and the processing of ribosomal RNAs. In this review, we describe the canonical role of RNase III in the biogenesis of the ribosome comparing conserved and unique features from bacteria to eukaryotes. Furthermore, we report additional roles in ribosome biogenesis re-enforcing the importance of RNase III.
kan (IBPC 903 and CA244-PAP, respectively) (lanes 2 and 4) and (IBPC 690) (lane 6) derivatives. C... more kan (IBPC 903 and CA244-PAP, respectively) (lanes 2 and 4) and (IBPC 690) (lane 6) derivatives. Cells were grown in LB medium until mid-log phase and soluble proteins analyzed by SDS-PAGE electrophoresis and revealed by Coomassie Blue staining.
<b>Copyright information:</b>Taken from "Hfq variant with altered RNA binding fu... more <b>Copyright information:</b>Taken from "Hfq variant with altered RNA binding functions"Nucleic Acids Research 2006;34(2):709-720.Published online 30 Jan 2006PMCID:PMC1356530.© The Author 2006. Published by Oxford University Press. All rights reserved Sequence alignment of Hfqs of and . Secondary structural elements are highlighted in grey. B indicate beta sheet. The amino acid replacements are indicated (by bars).
Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was init... more Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5 0 monophosphate and a 3 0 stable hairpin is highly susceptible to poly(A)-dependent degradation.
In order to respond to ever-changing environmental cues, bacteria have evolved resilient regulato... more In order to respond to ever-changing environmental cues, bacteria have evolved resilient regulatory mechanisms controlling gene expression. At the post-transcriptional level, this is achieved by a combination of RNA-binding proteins, such as ribonucleases (RNases) and RNA chaperones, and regulatory RNAs including antisense RNAs (asRNAs). AsRNAs bound to their complementary mRNA are primary targets for the double-strand-specific endoribonuclease, RNase III. By comparing primary and processed transcripts in an rnc strain, mutated for RNase III, and its isogenic wild type strain, we detected several asRNAs. We confirmed the existence of RNase III-sensitive asRNA for crp, ompR, phoP and flhD genes, encoding master regulators of gene expression. AsflhD, the asRNA to the master regulator of motility flhDC, is slightly induced under heat-shock conditions in a sigma24 (RpoE)-dependent manner. We demonstrate that expression of AsflhD asRNA is involved in the transcriptional attenuation of fl...
Communications Biology, 2020
Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosom... more Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2020
Bacteria exhibit an amazing diversity of mechanisms controlling gene expression to both maintain ... more Bacteria exhibit an amazing diversity of mechanisms controlling gene expression to both maintain essential functions and modulate accessory functions in response to environmental cues. Over the years, it has become clear that bacterial regulation of gene expression is still far from fully understood. This review focuses on antisense RNAs (asRNAs), a class of RNA regulators defined by their location in cis and their perfect complementarity with their targets, as opposed to small RNAs (sRNAs) which act in trans with only short regions of complementarity. For a long time, only few functional asRNAs in bacteria were known and were almost exclusively found on mobile genetic elements (MGEs), thus, their importance among the other regulators was underestimated. However, the extensive application of transcriptomic approaches has revealed the ubiquity of asRNAs in bacteria. This review aims to present the landscape of studied asRNAs in bacteria by comparing 67 characterized asRNAs from both Gram-positive and Gram-negative bacteria. First we describe the inherent ambiguity in the existence of asRNAs in bacteria, second, we highlight their diversity and their involvement in all aspects of bacterial life. Finally we compare their location and potential mode of action toward their target between Gram-negative and Gram-positive bacteria and present tendencies and exceptions that could lead to a better understanding of asRNA functions.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2018
Post-transcriptional addition of poly(A) tails to the 3′ end of RNA is one of the fundamental eve... more Post-transcriptional addition of poly(A) tails to the 3′ end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including ant...
Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNA... more Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNAs but relatively few examples have been investigated. We used a combination of 5′-tagRACE and RNA-seq to analyze the total RNA content from a wild-type strain and from a poly(A)polymerase deleted mutant. A total of 178 transcripts were either upor down-regulated in the mutant when compared to the wild-type strain. Poly(A)polymerase up-regulates the expression of all genes related to the FliA regulon and several previously unknown transcripts, including numerous transporters. Notable down-regulation of genes in the expression of antigen 43 and components of the type 1 fimbriae was detected. The major consequence of the absence of poly(A)polymerase was the accumulation of numerous sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the processing of entire transcripts. A new algorithm to analyze the position and composition of post-transcriptional modifications bas...
BMC Molecular Biology, 2007
Background The bacterial Sm-like protein Hfq is known as an important regulator involved in many ... more Background The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A) polymerase I (PAP). As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme). Therefore, it was assumed that Hfq might not only influence the poly(A) polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme. Results Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially ...
Journal of Bacteriology, 2014
Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarr... more Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile . In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile . Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile , including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The ...
<b>Copyright information:</b>Taken from "Hfq stimulates the activity of the CCA-... more <b>Copyright information:</b>Taken from "Hfq stimulates the activity of the CCA-adding enzyme"http://www.biomedcentral.com/1471-2199/8/92BMC Molecular Biology 2007;8():92-92.Published online 18 Oct 2007PMCID:PMC2175515.es with radioactively labeled yeast tRNAwithout CCA-end as a substrate in the absence or presence of Hfq or BSA, respectively. The reaction products were separated by denaturing polyacrylamide gel electrophoresis. CCA-addition leads to a reduced electrophoretic mobility of the labeled tRNA, and the corresponding signal intensities indicate a dramatic enhancement of the CCA incorporation in the presence of Hfq, while the CCA synthesis without Hfq or BSA addition was only moderate. BSA also led to a considerable stimulation, probably by stabilizing the active CCA-adding enzyme. These results were verified using different tRNA substrates (tRNA, phage T5 tRNA, not shown). M, mock incubation without addition of CCA-adding enzyme; -, activity of CCA-adding enzyme without any additional protein. CCA-addition in the presence of several RNA binding proteins, BSA, Hfq or Hfq variants. Only Hfq and the two variants V43R and K56A lead to a strong increase in CCA-addition, while all other RNA binding proteins show a much weaker stimulating effect, indistinguishable to that of BSA. NusA: transcription elongation factor (); TGT: tRNA guanine transglycosylase (); HU: histone-like protein that also interacts with RNA (); P: RNase P protein subunit ().
Microbiology, 1996
Summary: The rnb gene encodes ribonuclease II (RNase II), one of the two major Escherichia coli e... more Summary: The rnb gene encodes ribonuclease II (RNase II), one of the two major Escherichia coli exonucleases involved in mRNA degradation. In this paper, the rnb transcript is characterized regarding its promoter and terminator regions. The combined results from S1 nuclease protection analysis, DNase I footprinting and gene fusions with IacZ have shown that rnb is expressed from two promoters. S1 nuclease protection analysis and DNA footprinting have shown that rnb has two promoters, P1 and P2. Transcriptional and translational IacZ reporter fusions, constructed to the rnb gene, revealed that P2, the rnb proximal promoter, is stronger than P1. However, P2 is not transcribed in vitro, suggesting that an additional factor is required in vivo. The 3' end of the rnb transcript mapped to a stem-loop structure immediately after the translated region.
The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, bind... more The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AUrich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high speci®city for a 100-nucleotide RNA harboring 18 3¢-terminal A-residues. Structural analysis of the adenylated RNA±Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a singlestranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3¢±5¢ exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.
ABSTRACTNoncoding RNAs (ncRNA) have emerged as important components of regulatory networks govern... more ABSTRACTNoncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found ...
Scientific Reports
Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacte... more Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with th...
Nucleic Acids Research, 2016
Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNA... more Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNAs but relatively few examples have been investigated. We used a combination of 5-tagRACE and RNA-seq to analyze the total RNA content from a wild-type strain and from a poly(A)polymerase deleted mutant. A total of 178 transcripts were either up-or down-regulated in the mutant when compared to the wild-type strain. Poly(A)polymerase up-regulates the expression of all genes related to the FliA regulon and several previously unknown transcripts, including numerous transporters. Notable down-regulation of genes in the expression of antigen 43 and components of the type 1 fimbriae was detected. The major consequence of the absence of poly(A)polymerase was the accumulation of numerous sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the processing of entire transcripts. A new algorithm to analyze the position and composition of post-transcriptional modifications based on the sequence of unencoded 3-ends, was developed to identify polyadenylated molecules. Overall our results shed new light on the broad spectrum of action of polyadenylation on gene expression and demonstrate the importance of poly(A) dependent degradation to remove structured RNA fragments.
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Papers by Eliane Hajnsdorf