Ro ribonucleoproteins are antigenic protein-RNA particles that are the major targets of the immune reaction in autoimmune disorders like systemic lupus erythematosus. The Ro protein has been implicated in cellular RNA quality control, due to its preference for binding misfolded non-coding RNAs such as pre5S ribosomal RNAs and U2 small-nuclear RNAs besides binding cytoplasmic RNAs called Y RNAs. Although well characterized in eukaryotes, an understanding of Ro in prokaryotes is lacking. To gain structural insight into Ro-RNA interactions we have determined a high resolution crystal structure of Rsr, a Ro ortholog from the bacterium D. radiodurans. The structure of Rsr reveals two domains- a flexible, RNA binding HEAT repeat domain and a cation binding vonWillebrand factor A domain. Structural differences between Rsr and Xenopus laevis Ro at the misfolded non-coding RNA binding site suggest a possible conformational switch in Ro that might enable RNA binding. Structural and biochemical characterization reveals that Ro binds cytoplasmic small RNAs called Y RNAs with low nanomolar affinity, to form ~700kDa multimers. Formation of these multimers suggests one possible mode by which Ro RNAs may be targeted towards downstream processing events. Metal responsive transcriptional regulators sense specific metals in the cells and regulate the expression of specific operons involved in export, import or sequestration of the metal. CsoR is a copper(I) specific transcriptional regulator of the cso operon which consists of a putative copper export pump, CtpV. In copper limiting conditions, CsoR binds the operator/promoter region of the cso operon. In increased concentrations of copper (I), CsoR binds copper (I) with high affinity and is released from the operator/promoter site, causing derepression of the cso operon. To gain structural insight into CsoR function, we have solved the crystal structure of copper(I) bound CsoR. The structure reveals a homodimer with a subunit bridging copper site. The trigonal planar geometry and the presence of cysteine and histidine ligands at the metal site are favorable for copper(I) binding. The structure reveals a novel DNA binding fold in CsoR, making it the founding member of a new structural class of metalloregulators.

Ro ribonucleoproteins are antigenic protein-RNA particles that are the major targets of the immune reaction in autoimmune disorders like systemic lupus erythematosus. The Ro protein has been implicated in cellular RNA quality control, due to its preference for binding misfolded non-coding RNAs such as pre5S ribosomal RNAs and U2 small-nuclear RNAs besides binding cytoplasmic RNAs called Y RNAs. Although well characterized in eukaryotes, an understanding of Ro in prokaryotes is lacking. To gain structural insight into Ro-RNA interactions we have determined a high resolution crystal structure of Rsr, a Ro ortholog from the bacterium D. radiodurans. The structure of Rsr reveals two domains- a flexible, RNA binding HEAT repeat domain and a cation binding vonWillebrand factor A domain. Structural differences between Rsr and Xenopus laevis Ro at the misfolded non-coding RNA binding site suggest a possible conformational switch in Ro that might enable RNA binding. Structural and biochemical characterization reveals that Ro binds cytoplasmic small RNAs called Y RNAs with low nanomolar affinity, to form ~700kDa multimers. Formation of these multimers suggests one possible mode by which Ro RNAs may be targeted towards downstream processing events. Metal responsive transcriptional regulators sense specific metals in the cells and regulate the expression of specific operons involved in export, import or sequestration of the metal. CsoR is a copper(I) specific transcriptional regulator of the cso operon which consists of a putative copper export pump, CtpV. In copper limiting conditions, CsoR binds the operator/promoter region of the cso operon. In increased concentrations of copper (I), CsoR binds copper (I) with high affinity and is released from the operator/promoter site, causing derepression of the cso operon. To gain structural insight into CsoR function, we have solved the crystal structure of copper(I) bound CsoR. The structure reveals a homodimer with a subunit bridging copper site. The trigonal planar geometry and the presence of cysteine and histidine ligands at the metal site are favorable for copper(I) binding. The structure reveals a novel DNA binding fold in CsoR, making it the founding member of a new structural class of metalloregulators.

No description available.

No description available.

La protéine SRSF1, aussi appelée ASF/SF2, fait partie de la famille des protéines SR, une famille de protéines liant l’ARN très conservées. Ces protéines jouent un rôle régulateur de l’épissage, également lors de l’épissage alternatif. Une centaine d’ARN cible ont été décrits pour SRSF1 mais la manière dont SRSF1 sélectionne ses cibles parmi tous les pré-ARNm est mal comprise. Des études in vitro et in vivo ont montré que les protéines SR reconnaissent un petit motif dégénéré qui est souvent présent en plusieurs copies dans les ESE («enhancer splicing element »). Bien que les protéines SR lient ces motifs avec une faible spécificité, la définition des exons se fait avec une grande fidélité. Afin de mieux comprendre le mécanisme d’action de SRSF1, j’ai réalisé une étude cinétique des interactions SRSF1-ARN dans les cellules vivantes par des techniques de microscopies avancées. Grâce au système CRISPR, j’ai pu étiqueter la protéine SRSF1 avec la protéine Halo puis j’ai combiné une technique de photo-blanchiment (FRAP) et une technique de suivi de particule unique (« single particle tracking, SPT) pour mesurer la diffusion de SRSF1 et son affinité pour l’ARN. J’ai mesuré la durée de vie des événements de liaison individuellement aussi bien sur le pool global de pré-ARNm que sur des cibles spécifiques. Nos résultats indiquent que la liaison de SRSF1 ne dépasse pas quelques secondes, même sur les cibles de haute affinité. Cette cinétique rapide permet à SRSF1 d’être en contact avec l’ensemble des transcrits naissants qui est produit en permanence dans la cellule. De plus, mon travail apporte une analyse cinétique de la dynamique des snRNP à la résolution de la molécule unique dans le nucléoplasme des cellules vivantes. Nous avons déterminé les coefficients de diffusion des snRNP et la durée de leur association à l’ARN dans ces cellules. / SRSF1, formerly known as ASF/SF2, belongs to the SR protein family, which is a conserved family of RNA-binding protein that plays essential roles as regulators of both constitutive and alternative splicing. Hundreds of RNA targets have been described for SRSF1 but how SRSF1 selects its targets from the entire pool of cellular pre-mRNAs remains an open question. In vitro and in vivo studies have shown that SR proteins recognize short degenerated motifs often present in multiple copies at ESEs. Similar cryptic motifs are however frequently present in pre-mRNAs, and this low specificity of binding contrasts with the great fidelity of exon definition. To better understand the mechanism of action of SRSF1, I performed a kinetic study of SRSF1-RNA interactions in live cells using advanced microscopic techniques. Taking advantage by the CRISPR system, I tagged endogenous SRSF1 with Halo protein, and I combined photobleaching (FRAP) and single particle tracking (SPT) techniques to estimate diffusion and binding rates of SRSF1. I measured the duration of individual binding events, both on the cellular pool of pre-mRNAs and on specific targets. Our results indicate that binding of SRSF1 does not exceed few seconds, even on high-affinity targets. This rapid kinetics allows SRSF1 to rapidly sample the entire pool of nascent RNAs continuously produced in cells. Moreover, we provided a kinetic analysis of snRNP dynamics at a single-molecule resolution in the nucleoplasm of living cells. Our results enabled us to determine diffusion coefficients of snRNPs and their RNA binding duration in vivo.

No description available.

No description available.

Even after their discovery more than 60 years ago, little is known about how messenger RNAs (mRNAs) are packaged inside the cells. To ensure efficient and accurate delivery of the intended message to its proper destination, it is important to package the informational molecule in a way that protects it from premature degradation but also proper decoding at the destination. However, very little is known about the this fundamentally important step of mRNA packaging inside eukaryotic cells. To this end, we developed a novel approach, RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), to capture the 3D architecture of the ribonucleoprotein particles (RNPs) of interest transcriptome-wide. To begin with, we applied RIPPLiT to the exon-junction complex (EJC), a set of proteins stably bound to a spliced RNA. EJCs have been shown to interact with other proteins like SR- and SR-like to form megadalton sized complexes and help protect large regions of mRNAs. Thus, we hypothesized that these RNPs would provide an ideal system to elucidate the higher order organization of mRNPs. Preliminary analysis of data obtained from RIPPLiT consisted of “chimeric reads”, reads with multiple RNA fragments ligated together, which could not be analyzed with any of the existing bioinformatics tools. Thus, we developed a new bioinformatics suite, ChimeraTie, to map, analyze and visualize chimeric reads. Performing polymer analysis on chimeric reads obtained for hundreds of mRNAs, we were able to predict that mRNPs are linearly and densely packed into flexible rod-like structures before they undergo translation. In this thesis, along with the detailed biological conclusion, I have also provided a step-wise manual to perform RIPPLiT experiment and analyze the ensuing data using ChimeraTie.

L’activité RNase P est l'activité essentielle qui élimine les séquences 5' supplémentaires des précurseurs d'ARN de transfert. "PRORP" (PROteinaceous RNase P) définit une nouvelle catégorie de RNase P uniquement protéique. Avant la caractérisation de PRORP, on pensait que les enzymes RNase P étaient universellement conservées sous forme de ribonucléoprotéines (RNP). La caractérisation de PRORP a révélé une enzyme avec deux domaines principaux, un domaine N-terminal contenant plusieurs motifs PPR et un domaine NYN C-terminal portant l’activité catalytique. Nous avons utilisé une combinaison d'approches biochimiques et biophysiques pour caractériser le complexe PRORP / ARNt. La structure du complexe en solution a été déterminée par diffusion des rayons X aux petits angles (SAXS) et les Kd des interactions de différents mutants de PRORP avec l’ARNt ont été déterminées par ultracentrifugation analytique. Notre analyse révèle un cas intéressant d'évolution convergente. Il suggère que PRORP a développé un processus de reconnaissance de l'ARN similaire à celui des RNase P RNP. Par ailleurs, nous avons mis en place une approche de co-immunoprécipitation de PRORP avec l’ARN afin de définir le spectre de substrats des RNase P protéiques. / RNase P is the essential activity that removes 5'-leader sequences from transfer RNA precursors. “PRORP” (PROteinaceous RNase P) defines a novel category of protein only RNase P. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins (RNP). The characterization of PRORP revealed an enzyme with two main domains, an N-terminal domain containing multiple PPR motifs and a C-terminal NYN domain holding catalytic activity. We used a combination of biochemical and biophysical approaches to characterize the PRORP / tRNA complex. The structure of the complex in solution was determined by small angle X-ray scattering and Kd values of the PRORP / tRNA interaction were determined by analytical ultracentrifugation. We also analyzed direct interaction of a collection of PPR mutants with tRNA in order to determine the relative importance of individual PPR motifs for RNA binding. This reveals to what extent PRORP target recognition process conforms to the mode of action of PPR proteins interacting with linear RNA. Altogether, our analysis reveals an interesting case of convergent evolution. It suggests that PRORP has evolved an RNA recognition process similar to that of RNP RNase P. Moreover, we also implemented a PRORP-RNA co-immunoprecipitation approach to determine the full extent of PRORP substrates.

No description available.