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Principles of RNA-based gene expression control in Vibrio cholerae
Principles of RNA-based gene expression control in Vibrio cholerae
Post-transcriptional control of gene expression by small regulatory RNAs (sRNAs) is a widespread regulatory principle among bacteria. The sRNAs typically act in concert with RNA binding proteins such as the RNA chaperone Hfq to bind mRNA targets via imperfect base pairing. They affect translation initiation and/or transcript stability. Additionally, sRNAs can influence transcription termination of their targets or function indirectly as so-called sponges for other sRNAs. Regulation often involves the major endoribonuclease RNase E, which contributes to both sRNA biosynthesis and function. In the first part of this thesis, we globally identified RNase E cleavage sites in the major human pathogen Vibrio cholerae by employing TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq). We validated the involvement of RNase E in the synthesis and maturation of several previously uncharacterized sRNAs. Two examples, OppZ and CarZ, were chosen for further study due to their unique regulatory mechanism. They are processed from the 3’ untranslated regions (3’ UTR) of the oppABCDF and carAB operons, respectively, and subsequently target mRNAs transcribed from the very same operons by binding to base pairing sites upstream of the second (oppB) or first (carA) cistrons. This leads to translational inhibition and triggers premature transcription termination by the termination factor Rho, thereby establishing an autoregulatory feedback loop involving both the protein-coding genes and the processed sRNAs. In the case of OppZ, the regulation is limited to the oppBCDF part of the operon in a discoordinate fashion due to the position of the OppZ base pairing site. This mechanism of target regulation by Opp and CarZ represents the first report of an RNA-based feedback regulation that does not rely on additional transcription factors. The second study included in the thesis characterizes two sRNAs involved in the envelope stress response (ESR) of V. cholerae. Misfolded outer membrane proteins (OMPs) induce the sigmaE-dependent transcriptional activation of the sRNAs MicV and VrrA, which reduce membrane stress by repressing the mRNAs of several OMPs and other abundant membrane protein. MicV and VrrA share a conserved seed region with their functionally analogous counterpart from Escherichia coli, RybB, indicating that this seed sequence might represent a universally functional RNA domain. To study the involvement of this seed domain in the ESR in an unbiased fashion, we constructed a complex library of artificial sRNAs and performed laboratory selection experiments under membrane-damaging conditions. We isolated the most highly enriched sRNA variants and indeed discovered a strong enrichment of the conserved seed-pairing domain. We were able to pinpoint the repression of ompA as the key factor responsible for the sRNA-mediated resistance to ethanol-induced membrane damage. Taken together, this thesis expanded the knowledge on the mechanisms of sRNA-dependent gene regulation by reporting a novel autoregulatory feedback loop. Additionally, it introduced a synthetic sRNA library as a tool to study complex microbial phenotypes and their underlying sRNA-target interactions.
small RNA, autoregulation, RNase E, synthetic sRNA, Vibrio cholerae
Hoyos, Mona
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Hoyos, Mona (2020): Principles of RNA-based gene expression control in Vibrio cholerae. Dissertation, LMU München: Faculty of Biology
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Abstract

Post-transcriptional control of gene expression by small regulatory RNAs (sRNAs) is a widespread regulatory principle among bacteria. The sRNAs typically act in concert with RNA binding proteins such as the RNA chaperone Hfq to bind mRNA targets via imperfect base pairing. They affect translation initiation and/or transcript stability. Additionally, sRNAs can influence transcription termination of their targets or function indirectly as so-called sponges for other sRNAs. Regulation often involves the major endoribonuclease RNase E, which contributes to both sRNA biosynthesis and function. In the first part of this thesis, we globally identified RNase E cleavage sites in the major human pathogen Vibrio cholerae by employing TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq). We validated the involvement of RNase E in the synthesis and maturation of several previously uncharacterized sRNAs. Two examples, OppZ and CarZ, were chosen for further study due to their unique regulatory mechanism. They are processed from the 3’ untranslated regions (3’ UTR) of the oppABCDF and carAB operons, respectively, and subsequently target mRNAs transcribed from the very same operons by binding to base pairing sites upstream of the second (oppB) or first (carA) cistrons. This leads to translational inhibition and triggers premature transcription termination by the termination factor Rho, thereby establishing an autoregulatory feedback loop involving both the protein-coding genes and the processed sRNAs. In the case of OppZ, the regulation is limited to the oppBCDF part of the operon in a discoordinate fashion due to the position of the OppZ base pairing site. This mechanism of target regulation by Opp and CarZ represents the first report of an RNA-based feedback regulation that does not rely on additional transcription factors. The second study included in the thesis characterizes two sRNAs involved in the envelope stress response (ESR) of V. cholerae. Misfolded outer membrane proteins (OMPs) induce the sigmaE-dependent transcriptional activation of the sRNAs MicV and VrrA, which reduce membrane stress by repressing the mRNAs of several OMPs and other abundant membrane protein. MicV and VrrA share a conserved seed region with their functionally analogous counterpart from Escherichia coli, RybB, indicating that this seed sequence might represent a universally functional RNA domain. To study the involvement of this seed domain in the ESR in an unbiased fashion, we constructed a complex library of artificial sRNAs and performed laboratory selection experiments under membrane-damaging conditions. We isolated the most highly enriched sRNA variants and indeed discovered a strong enrichment of the conserved seed-pairing domain. We were able to pinpoint the repression of ompA as the key factor responsible for the sRNA-mediated resistance to ethanol-induced membrane damage. Taken together, this thesis expanded the knowledge on the mechanisms of sRNA-dependent gene regulation by reporting a novel autoregulatory feedback loop. Additionally, it introduced a synthetic sRNA library as a tool to study complex microbial phenotypes and their underlying sRNA-target interactions.