Terminaison de la transcription. Rho se fixe aux transcrits naissants au niveau d’un site Rut (Rhoutilization) libre à partir duquel il transloque le long de l’ARN (5’→3’) de façon ATP-dépendante pour rattraper le complexe d’élongation de la transcription et induire la dissociation de celui-ci. Il est généralement admis que les sites de fixation de Rho présentent une richesse en Cytosines et une pauvreté en Guanines, ainsi qu’une relative pauvreté en structures secondaires. Les études génomiques ou transcriptomiques n’ont pas dégagé d’éléments consensus ou de règles permettant de prédire les sites de terminaison Rho-dépendants. En combinant approches biochimiques et bioinformatiques, j’ai tenté de comprendre les mécanismes par lesquels Rho reconnait les transcrits.J’ai identifié un ensemble de déterminants de séquence qui, pris ensemble, possèdent un bon pouvoir prédictif et que j’ai utilisé pour construire le premier modèle computationnel capable de prédire la terminaison Rho-dépendante à l’échelle des génomes d’E. coli et Salmonella. J’ai caractérisé in vitro certains de ces terminateurs, en particulier dans les régions 5’UTR, avec l’espoir qu’ils soient impliqués dans des mécanismes de régulation conditionnelle. J’ai identifié des candidats dont l’activité pourrait être sous le contrôle de facteurs comme des petits ARN non codants (sRNA) ou latempérature. J’ai également développé une méthode fluorogénique pour détecter facilement la terminaison Rho-dépendante in vitro et ai commencé à adapter l’approche CLIP-seq à l’étude du transcriptome Rho-dépendant chez Salmonella. Collectivement, mes travaux offrent de nouveaux outils d’analyse et de prédiction de la terminaison Rho-dépendante, une meilleure cartographie des sites d’action de Rho chez E. coli et Salmonella, ainsi que de nouvelles pistes d’étude du rôle de Rhodans l’expression conditionnelle du génome. / Transcripts at a free Rut (Rho-utilization) site from which Rho moves along the RNA in an ATP dependentfashion to catch up with and dissociate the transcription elongation complex. It is generally believed that the Rut sites are, respectively, rich and poor in Cytosines and Guanines as well as relatively poor in secondary structures. Studies at the genomic or transcriptomic scale have notrevealed any stronger consensus features or rules for predicting potential Rho-dependent termination sites. By combining biochemical and bioinformatics approaches, I have explored the mechanisms by which Rho recognizes transcripts to induce transcription termination. I have identified a complex set of sequence determinants which, taken together, have good predictive power and which I used to build the first computational model able to predict Rho-dependent termination at the scale of Escherichiacoli and Salmonella genomes. I have characterized in vitro some of these terminators, particularly in 5'UTRs, with the hope that they will be involved in conditional regulatory mechanisms. I have identified several candidates whose activity may be under the control of factors such as small non-coding RNAs(sRNA) or temperature. I have also developed a fluorogenic method to easily detect Rho-dependent termination in vitro and have begun to adapt the CLIP-seq approach to the study of the Rhodependent transcriptome in Salmonella. Collectively, my work offers new tools for the analysis and prediction of Rho-dependent termination, a better mapping of the sites of probable Rho action in E.coli and Salmonella, as well as several lines of investigation of the role of Rho in the conditional expression of bacterial genomes.

RNA helicases are found in all kingdoms of life, functioning in all aspects of RNA biology mainly through modulating structures of RNA and ribonucleoprotein (RNP) complex. RNA structures have fundamental impacts on steps in gene expression, including transcription, pre-mRNA processing, and translation. However, the precise roles and regulatory mechanisms of RNA structures in co- and post-transcriptional processes remain elusive. By probing genome-wide RNA structures in vivo, a recent study suggested that ATP-dependent factors, such as RNA helicases, maintain the actively unfolded state of RNAs. Among all RNA helicases, DEAD-box proteins form the largest family in eukaryotes, and have been shown to remodel RNA/RNP structures both in vitro and in vivo. Nevertheless, for the majority of these enzymes, it is largely unclear what RNAs are targeted and where they modulate RNA/RNP structures to regulate co-transcriptional processes. To fill the gap, my research focused on identification of the RNAs and structures targeted by the DEAD-box protein Dbp2 in S. cerevisiae to uncover the cellular processes that Dbp2 is involved in.<br><div><div>My studies revealed a role of Dbp2 in transcriptional termination. Dbp2 binds to ~34% of yeast mRNAs and all snoRNAs, and loss of DBP2 leads to a termination defect as evidenced by RNA polymerase II (RNAPII) accumulation at 3’ ends of these genes. In addition, the binding pattern of Dbp2 in mRNAs is highly similar to Nrd1 and Nab3 in the Nrd1-Nab3-Sen1 (NNS) termination complex, and deletion of DBP2 leads to reduced recruitment of Nrd1 to its target genomic loci. In Dbp2 and NNS targeted 3’ UTRs, RNA structural changes resulted from DBP2 deletion also overlap polyadenylation elements and correlate with inefficient termination, and loss of stable structure in the 3’ UTR bypasses the requirement for Dbp2. These findings lead to a model that Dbp2 promotes efficient termination of transcription through RNA structure remodeling.</div><div>Interestingly, my research also revealed the requirement of DBP2 for efficient splicing, as loss of DBP2 leads to accumulation of unspliced pre-mRNAs. Moreover, this function is dependent on the helicase activity of Dbp2. Further studies are needed to characterize the molecular mechanism of how Dbp2 facilitates splicing in cells. Overall, my research demonstrated that DEAD-box RNA helicases remodel mRNA structure in vivo and that structural alteration can be essential for proper gene expression.</div></div>

Female adults of many mosquito species possess distinct physiological features adapting to blood feeding for successful reproduction. The disease pathogens that are transmitted by mosquitoes have evolved to take advantages of the indispensable blood feedings to complete their transmission cycles and to survive attacks from the mosquito's innate immune system. Normal egg development and mosquito immunity are tightly controlled by tissue- and stage-specific gene expression and coordinated by many signal molecules in the mosquito. Understanding gene regulation affecting mosquito reproduction and malaria parasites infection is of paramount importance for developing novel malaria control strategies. A growing body of evidence indicates that microRNAs (miRNAs) are involved in egg maturation and immune reactions against invading pathogens in mosquitoes. However, the molecular mechanisms by which specific miRNAs selectively modulate reproduction and the survival of pathogens are largely unknown. The miRNA-induced gene-silencing pathway in mosquitoes was mostly extrapolated from the studies of flies. To explore the dynamics of miRNAs in reproduction, I used small RNAs sequencing to monitor miRNAs expression and their association with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) in the malaria mosquito Anopheles gambiae (An. gambiae) during the 72-h period immediately after blood feeding. I found the abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. I confirmed that miR-309 is essential for normal egg development by depletion of endogenous miR-309 with a specific antagomir. In addition, my results showed that the Ago association of some miRNAs was not proportional to their cellular abundance implying additional regulation at miRNA integration. To investigate the functional roles of miRNAs and define context-dependent miRNA-mRNA interactions during the reproductive process, I have applied an innovative experimental approach to study miRNA-mRNA interactome. CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP can generate miRNA-mRNA chimeras from UV-irradiation stabilized Ago-miRNA-mRNA complex. My results have defined tens of thousands of miRNA-mRNA interactions in mosquitoes, including novel targets for mosquito-specific miRNAs. Verification of the predicted interactions using mRNA-seq, ribosome-profiling, and luciferase reporter assay revealed a reliable miRNA-mRNA interaction network. Based on the detected interactions, I refined the paring rules for mosquito miRNAs and illustrated the dynamic pairing between different regions of miRNAs with their targets in vivo. The miRNA-mRNA interactions were compared using this approach at multiple time points before and after blood feeding. Importantly, this study showed that the interactions were dynamic and enriched in genes that are involved in metabolisms, supporting the proposed functions of miRNAs in coordinating the gene regulation in mosquito reproduction. Plasmodium falciparum (P. falciparum) is a major human malaria parasite. To understand the functions of miRNAs in the mosquito resistance to Plasmodium infection, we analyzed the miRNA-mRNA interactions after female mosquitoes taking a P. falciparum-infected blood meal or an uninfected blood meal. Comparison of the interactions revealed enhanced miRNA-mRNA interactions after P. falciparum infection involving a group of immunity-related genes. In summary, this study has provided a systematic view and significantly advanced our understanding of the miRNA functions in mosquito reproduction and P. falciparum infection. / PHD

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