Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

Chez les bactéries à Gram-négatif, l’imperméabilité de la membrane externe est un facteur majeur contribuant au développement de la résistance. Chez Escherichia coli, les porines OmpF et OmpC sont des protéines de la membrane externe qui forment des canaux pour la diffusion de petites molécules hydrophiles tels que les antibiotiques. L’expression des porines est soumise à une régulation fine, et des petits ARN non-codants (sRNAs, small RNAs) jouent un rôle important au niveau post-transcriptionnel. Dans ce cadre, et en utilisant E. coli comme bactérie modèle, les objectifs de mon travail de thèse étaient : (1) de caractériser la régulation du sRNA MicC et la co-régulation putative de la porine quiescente OmpN; (2) d’examiner l'effet global de MicC sur le transcriptome; (3) d’analyser l'impact de l'expression de MicC sur la sensibilité aux antibiotiques. Les résultats obtenus montrent l’induction de MicC en présence d'antibiotiques de la famille des β-lactamines, ou en l’absence du facteur sigma de réponse au stress de l’enveloppe sigmaE. Ces mêmes conditions activent aussi l'activité d'une fusion ompN-lacZ, indiquant une régulation transcriptionnelle commune de micC et ompN. Etant donnée la conservation de MicC chez les entérobactéries, nous avons effectué une étude par RNASeq pour déterminer l'impact de la surexpression de MicC sur le transcriptome d’E. coli et identifié 60 ARNm régulés par MicC en plus de sa cible initiale ompC. L'identification des spectres cibles globaux des sRNAs est importante pour comprendre leur importance dans la physiologie bactérienne, ici celui de MicC dans la résistance aux antibiotiques. / A major factor contributing to antimicrobial resistance is the inability of antibiotics to penetrate the bacterial outer membrane to reach their target. In Escherichia coli, the two abundantly expressed porins OmpF and OmpC form channels for diffusion of small hydrophilic molecules including antibiotics. The expression of porins is under complex regulation and the small regulatory RNAs (sRNAs) fine tune the porin expression level at post-transcriptional level. MicF and MicC are the two major sRNAs that negatively regulate expression of OmpF and OmpC, respectively. Interestingly, these two sRNAs are encoded next to porin gene, i.e. micF-ompC and micC-ompN, suggesting a dual regulation. Our goals in this work were: (1) to characterize the regulation of the sRNA MicC and the putative co-regulation of the quiescent porin OmpN in E. coli; (2) to examine the global effect of MicC on the E. coli transcriptome; (3) to analyze the impact of MicC expression on antibiotic susceptibility. Our work shows that the expression of micC was increased in the presence of carbapenems and cephalosporins and in an rpoE depleted mutant. The same conditions enhanced the expression of OmpN, suggesting a dual regulation of micC and ompN. We also performed RNA sequencing to determine the impact of MicC overexpression on E. coli transcriptome. This identified 60 mRNA targets negatively regulated by MicC apart from its original target. Identification of the global target spectra of MicC is of importance to understand its importance on the overall bacterial physiology, and more specifically on AMR.