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Principles of RNA-Based Gene Expression Control in Vibrio Cholerae

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Principles of RNA-Based Gene Expression Control in Vibrio Cholerae Principles of RNA-based Gene Expression Control in Vibrio cholerae Dissertation Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Mona Gräfin Hoyos München, August 2020 Diese Dissertation wurde angefertigt im Zeitraum von Februar 2016 bis August 2020 unter der Leitung von Prof. Dr. Kai Papenfort an der Fakultät für Biologie, Department I an der Ludwig-Maximilians-Universität München. Erstgutachter: Prof. Dr. Kai Papenfort Zweitgutachterin: Prof. Dr. Kirsten Jung Tag der Abgabe: 24. August 2020 Tag der mündlichen Prüfung: 21. Dezember 2020 i Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass diese Dissertation von mir selbstständig und ohne uner- laubte Hilfsmittel angefertigt wurde. Die vorliegende Dissertation wurde weder ganz noch teilweise einer anderen Prüfungskommission vorgelegt. Ich habe noch zu keinem früheren Zeitpunkt ver- sucht, eine Dissertation einzureichen oder an einer Doktorprüfung teilzunehmen. I declare that I have authored this thesis independently and that I have not used other than the declared sources. The present dissertation has not been presented to another examination board, neither entirely nor in parts. As well I declare that I have not submitted or defended a dissertation previously without success. München, 24.08.2020 Mona Gräfin Hoyos iii Contents Eidesstattliche Erklärung iii Abbreviations vii Publications and Contributions ix Summary xiii Zusammenfassung xv 1 Introduction 1 1.1 Regulatory RNAs in bacteria . .1 1.2 Various types of sRNAs in bacteria . .2 1.3 Molecular Mechanisms of bacterial small RNAs . .6 1.4 Physiological roles of sRNAs . 14 1.5 Concepts of bacterial gene regulation . 17 1.6 Aim of this work . 21 2 Gene autoregulation by 3’ UTR-derived bacterial small RNAs 23 3 A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria 53 4 Concluding discussion 77 4.1 Studies of the RNA metabolism in a major pathogen . 77 4.2 Autoregulatory sRNAs from 3’ UTRs . 82 4.3 Synthetic sRNAs for targeted gene regulation . 94 4.4 Unbiased selection of sRNAs counter-acting envelope stress . 97 4.5 Summary and Outlook . 105 5 References for Chapters 1 and 4 107 6 Appendix 129 6.1 Figure supplements to chapter 2 . 129 6.2 Appendix to chapter 3 . 156 6.3 Top 50 sRNA variants from chapter 3 . 179 Acknowledgements 181 Curriculum Vitae 183 v Abbreviations asRNA antisense RNA BCM bicyclomycin CDS coding sequence CLASH cross-linking, ligation, and sequencing of hybrids CRISPR clustered regularly interspaced short palindromic repeats CRP cAMP receptor protein CS cleavage site FFL feed-forward loop GFP green fluorescent protein IGR intergenic region IPTG Isopropyl beta-D-1-thiogalactopyranoside kb kilobase miRNA eukaryotic microRNA mRNA messenger RNA NAR negative autoregulation nt nucleotides OMP outer membrane protein ORF open reading frame PNPase polynucleotide phosphorylase RBS ribosome binding site RIL-seq RNA interaction by ligation and sequencing RNA-seq RNA sequencing RNAP RNA polymerase vii Abbreviations rRNA ribosomal RNA rut site Rho utilization site SD sequence Shine-Dalgarno sequence sRNA small regulatory RNA Term-seq 3’ end-specific RNA-seq protocol TF transcription factor TIER-seq transiently inactivating an endoribonuclease followed by RNA-seq TIR translation initiation region tRNA transfer RNA TSS transcriptional start site UTR untranslated region viii Publications and Contributions Publications and Manuscripts Originating from this Thesis Chapter 2: Mona Hoyos, Michaela Huber, Konrad U. Förstner, and Kai Papenfort (2020). “Gene autoregula- tion by 3’ UTR-derived bacterial small RNAs.” eLife 9, e58836 Chapter 3: Nikolai Peschek, Mona Hoyos, Roman Herzog, Konrad U. Förstner, and Kai Papenfort (2019). “A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacte- ria.” The EMBO Journal 38.16, e101650 Not presented in this Thesis Benjamin R. Wucher, Thomas M. Bartlett, Mona Hoyos, Kai Papenfort, Alexandre Persat, and Carey D Nadell (2019). “Vibrio cholerae filamentation promotes chitin surface attachment at the expense of competition in biofilms.” PNAS 116.28, pp. 14216–14221 Manuscripts in preparation Michaela Huber, Mona Hoyos, Anne Lippegaus, and Kai Papenfort. “A Qrr sponge modulates quorum sensing dynamics in Vibrio cholerae.” Mona Hoyos*, Kavyaa Venkat*, Liam Cassidy, Andreas Tholey, David Grainger, and Kai Pa- penfort. “The dual function regulator VcdR controls carbohydrate uptake and TCA cycle activity in Vibrio cholerae.” *Authors contributed equally ix Publications and Contributions Contributions to Publications Presented in this Thesis Chapter 2: MHo and KP initialized and conceptualized the study. MHo constructed the majority of plasmids and strains and performed the majority of experiments and data analyses (Fig. 1A, Fig. 1-S1, Fig. 1-S4, Fig. 1-S5, Fig. 2A-C, Fig. 2-S1, Fig. 3B; D, Fig. 3-S1C-D, Fig. 3-S2C, Fig. 3-S3, Fig. 4, Fig. 4-S1, Fig. 5, Fig. 6, Fig. 7, Fig. 7-S1A-B, Fig. 7-S2, Fig. 8, Fig. 8-S1, Fig. 9). MHu contributed by plasmid and strain construction, by analyzing Hfq dependence of OppZ and CarZ through Co-IP and sRNA stability experiments, by measuring OppZ repression strength, by probing Northern blots and by initial work on CarZ regulation (Fig. 1S6, Fig. 2D, Fig. 2-S2, Fig. 3C, Fig. 5D, Fig. 7-S1C). MHo and MHu performed OppZ pulse expression and analyzed sequencing data (Fig. 3A, Fig. 3-S1A-B). KUF contributed by analyzing the TIER-seq data (Fig. 1B-D, Fig. 1-S2, Fig. 1-S3). MHo had technical assistance from Andreas Starick (Fig. 3-S2A-B, Fig. 7-S1C-D). MHo constructed the figures, KP and MHo wrote the manuscript. Mona Gräfin Hoyos Prof. Dr. Kai Papenfort x Publications and Contributions Chapter 3: NP and KP initiated and conceptualized the study of MicV and VrrA. NP, MH and KP concep- tualized the experiments employing the synthetic sRNA library. NP constructed the majority of plasmids and strains and performed the majority of experiments and data analyses on MicV and VrrA (Fig. 1, Fig. 2, Fig. 3, Fig. 4A; B; D, Fig. 7, Fig. EV1A-E, Fig. EV2, Appendix Fig. S1B-C, Appendix Fig. S2, Appendix Fig. S3). MH constructed and validated the synthetic sRNA library and performed the selection experiments, MH and NP analyzed and validated the resulting sequencing data (MH: Fig. 5B-C, Fig. EV4A-C, MH and NP: Fig. EV3, Fig. EV5A-B, NP: Fig. 5A, Fig. EV4D). NP and MH identified and validated OmpA as the key target for ethanol resistance (NP: Fig. 6D, Fig. EV5C-E, MH: Fig. 6C, Fig. EV4A, NP and MH: Fig. 6A; B). RH contributed by measuring PmicV promoter activity in E. coli and performing analyses of OMP composition in V. cholerae and E. coli cells carrying sRNA over-expression plasmids (Fig. 4C, Fig. EV1F, Appendix Fig. S1A). KUF contributed by analyzing the transcriptional start sites identified in (46) for sigma factor binding motifs. NP constructed the figures, KP, NP, MH, and RH wrote the manuscript. NP was assisted by the research students: Roman Herzog and Raphaela Götz. Mona Gräfin Hoyos Prof. Dr. Kai Papenfort xi Summary 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 pro- teins 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 tran- scription 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 sE-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 Es- cherichia 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 xiii Summary 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.
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