Messenger RNA localization is a widespread mechanism of posttranscriptional regulation of gene expression in multicellular organisms ranging from yeast to mammals. In Drosophila oocytes, gurken (grk) mRNA is transported by Dynein to produce a local secreted signal to the overlying follicle cells. This signal is responsible for setting up the primary axes in the oocyte. grk mRNA is transcribed in nurse cells and transported into the oocyte where it localizes at two distinct stages of oogenesis, thus targeting the translation of Grk/TGFalpha protein, first to the posterior and later to the dorso-anterior (DA) corner where it is translated. Gurken protein signals to the overlying follicle cells to establish the dorsal fate of the oocyte. grk transcripts are transported by Dynein in EM-dense particles on microtubules. These particles are not associated with vesicles nor membrane-bound and contain many copies of grk mRNA, Dynein and hnRNP Squid. At the DA corner transport particles assemble into large EM-dense cytoplasmic anchoring complexes called Sponge Bodies. In this thesis I present evidence that at their dorso-anterior destination, grk transcripts are statically anchored by Dynein, independently of functional Egalitarian and Bicaudal D, which are required for Dynein transport. I show by the disrupting the protein’s function after it has fulfilled its role in transport that hnRNP Squid is involved in the formation and maintenance of these Sponge Bodies. I provide evidence by EM and fluorescent microscopy that Sponge Bodies share many of components of translational regulation pathways found in Processing Bodies. I show by small RNA interference experiments and by genetic analysis that the structural role of Dynein heavy chain is a unique feature of the Sponge Bodies and that such a function does not occur in Processing Bodies in Drosophila. I show that the localization and anchoring of RNA in Sponge Bodies is not a unique feature of grk mRNA but that I factor RNA is also localized to Sponge bodies. The work presented tries to elucidate the function of Sponge Bodies in translational control of grk mRNA and illustrates by EM the dynamic nature of the Sponge Body structure during oogenesis. My results suggest that Sponge Bodies are RNA granules that are similar to Processing Bodies in a way that they are involved in translational regulation but unlike Processing Bodies depend on Dynein for their structural integrity. I propose that Sponge Bodies are RNA dependent granules that form by the recruitment of proteins involved in the anchoring and translational regulation.

No description available.

Fully grown oocytes undergo their further development in the absence of transcription. Completion of meiosis and early embryo development rely on the maternal mRNAs synthetized and stored during earlier development. Thus, the regulation of gene expression in oocytes during that period is controlled almost exclusively at the level of mRNA stabilization and translation. In the same vein, any mRNA metabolism could play a critical function at this stage of development. RNA localization followed by a local translation is a mechanism responsible for the control of spatial and temporal gene expression in the cell. We focused on visualization of mRNA and in situ translation in the mammalian oogenesis and embryogenesis. We characterized localization of global RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. Additionally we visualized specific ribosomal proteins that contribute to translation in the oocyte and embryo. We have shown that the key player of cap-dependent translation mTOR becomes highly active post nuclear envelope breakdown (NEBD) and in turn its substrate, translational repressor 4E-BP1 becomes inactive. Precise localization of inactivated 4E-BP1 at the newly forming spindle of the oocyte indicates the ongoing translation in this area. Furthermore, from...

<p>mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.</p> / Dissertation

<p>RNA localization provides the blueprint for compartmentalized protein synthesis in eukaryotic cells. Current paradigms indicate that RNAs encoding secretory and membrane proteins are recruited to the endoplasmic reticulum (ER), via positive selection of a `signal peptide' tag encoded in the protein. Thus RNA sorting to the ER follows protein sorting and the RNA is considered a passive player. However, RNAs have been shown to access the ER independent of the signal peptide and display a wide range of affinities to the ER that does not correlate with signal peptide strength. How and why mRNAs localize to the ER to varying extents and whether such localization serves a purpose besides protein sorting is poorly understood. To establish the cause and consequence of RNA binding to the ER membrane, I pose three primary questions: 1. How are mRNAs targeted to the ER? 2. Once targeted, how are mRNAs anchored to the ER membrane? 3. Are ER localized mRNAs subject to transcript-specific regulation? </p><p>I address cytosolic mRNA targeting to the ER by comparing the partitioning profiles of cytosolic/nuclear protein-encoding mRNA population (mRNACyto) to that of mRNAs encoding a signal peptide (mRNAER). I show that, at a population level, mRNACyto display a mean ER enrichment that is proportional to the amount of ER-bound ribosomes. Thus, I propose that targeting of mRNACyto to the ER is stochastic and over time, the specific interactions engaged by an individual mRNACyto with the ER determines its steady state partitioning profile between the cytoplasm and the ER. </p><p>To address the modes of direct binding of mRNA to the ER, I examined the association of various RNA populations with the ER after disrupting membrane-bound ribosome's interaction with its ER receptor. mRNACyto and most of mRNAs encoding secretory proteins (mRNACargo) are released upon disruption of ribosome-receptor interactions, indicating no direct mRNA-ER interactions. However, the population of mRNAs that encode resident proteins of the endomembrane organelles such as the ER, lysosome, endosome and the Golgi apparatus (mRNARes) maintain their association with the ER despite the disruption of ribosome-receptor interactions. These results indicate direct binding of mRNARes to the ER, further suggesting that the function of the encoded proteins dictates the mode of association of corresponding mRNA with the ER. </p><p>To uncover the mode of mRNARes binding directly to ER, I performed differential proteomic analysis of cytosolic and membrane bound RNA-protein complexes, which revealed a network of RNA binding proteins that interact uniquely with the ER-anchored mRNAs. The anchoring of endomembrane resident protein-encoding RNAs to the ER through these RNA binding proteins may reflect an imprinting of the ER with the information necessary for the continued biogenesis of the endomembrane organelle system even in situations where translation-dependent ER targeting of an mRNA is compromised. </p><p>Finally, I address whether ER-bound mRNAs can be regulated differentially by comparing the fates of two signal peptide-encoding RNAs, B2M and GRP94, during the unfolded protein response (UPR). I show that in response to ER stress, GRP94 mRNA, but not B2M, relocates to stress-induced RNA granules, thus escaping an RNA decay program that operates at the ER membrane during the UPR. Hence, I propose that the mode of RNA association to the ER is subject to regulation and influences the fate of RNAs during cellular stress. Thus, by demonstrating diverse modes of mRNA localization to the ER and differential regulation of ER bound mRNAs during cellular stress, my work has helped establish an emerging role for the ER as a post-transcriptional gene regulatory platform.</p> / Dissertation

RNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.

mRNA localisation is a widely used mechanism for achieving temporal-spatial restriction of protein expression and is essential during development to establish cell polarity. mRNA localisation is particularly well studied in the Drosophila egg chamber where gurken mRNA is localised to the dorsal-anterior corner of the oocyte in a Dynein-dependent process that establishes the anterior-posterior and dorsal-ventral axes of the future embryo. An RNA stem-loop called the gurken localisation signal is necessary and sufficient to drive gurken localisation through interactions with a specific complement of protein factors. However, the exact RNA sequence and structural features required to promote each stage of gurken localisation are unknown. Using a live-cell injection assay I have dissected regions of the mRNA signal that are responsible for driving gurken transport and anchoring through their association with Egalitarian, Me31B and Squid proteins. I show the structure of an AU-rich stem and a purine stack are essential for gurken transport, and demonstrate that the size of the internal loop between these stems is important. These features of the localisation signal are essential for recruitment of Egalitarian, which links the mRNA to the Dynein transport machinery. I also show that these mRNA sequence and structural elements are present in several other Dynein-transported mRNAs. The bulge at the distal end of the gurken localisation signal is important for anchoring grk at the dorsal-anterior of the oocyte, possibly through Squid binding, and the proximal third of the signal is essential for recruitment of the translation component Me31B. These studies indicate that the role of the gurken localisation signal in controlling gurken transport, anchoring and translation can be mapped to distinct regions of the signal and provide insights into how the signal carries out these numerous functions at a molecular level. Determining the molecular interactions involved in mRNA localisation improves our understanding of how specificity is generated to direct different mRNAs to distinct regions of the cell to restrict protein expression.

Au sein des cellules, les ARNms sont liés par de nombreuses protéines, générant ainsi des particules appelées mRNPs (Ribonucléoparticules de messagers). Leur formation est cotranscriptionnelle, et leur composition va réguler l’ensemble des étapes du métabolisme des ARNms : stabilité, maturation, export, localisation et traduction. Au vu de l’importance de ces mécanismes dans la physiologie cellulaire, le contenu protéique des mRNPs est finement régulé dans le temps et l’espace et fait l’objet de nombreux remodelages. Ces changements de composition dépendent notamment des hélicases, ainsi que des modifications post-traductionnelles ; cependant, ces mécanismes demeurent à caractériser de façon plus approfondie. Une modification post-traductionnelle susceptible de moduler ces remaniements depuis la levure S. cerevisiae jusqu’aux métazoaires est la sumoylation. En effet, la SUMO-protéase Ulp1/SENP2, une enzyme clé de la machinerie de sumoylation, est localisée au panier des pores nucléaires, à proximité d’une plateforme d’ancrage des mRNPs destinées à l’export. Par ailleurs, il a été rapporté chez la levure que des mutants affectant la localisation et la stabilité d’Ulp1 présentent des défauts d’export et de localisation des mRNPs. Au vu de ces données, le laboratoire s’est intéressé aux rôles potentiels de la sumoylation dans le métabolisme de ces particules d’ARNm. Dans ce but, un crible protéomique a été réalisé chez la levure S. cerevisiae afin de comparer la composition des mRNPs entre des cellules sauvages ou mutantes pour Ulp1. Ce crible a mis en évidence un rôle d’Ulp1 dans le recrutement de deux composants des mRNPs, le complexe THO et l’hnRNP Hek2. Le complexe THO est un facteur multiprotéique qui participe à la prévention de l’instabilité génique et contribue à la transcription des ARNms, à l’assemblage des mRNPs et à leur export. L’hnRNP Hek2 est une protéine aux rôles multiples, dont l’association à un ARNm est susceptible de moduler sa stabilité, sa traduction et/ou sa localisation. Des analyses biochimiques nous ont permis de mettre en évidence l’existence de formes sumoylées de la sous-unité Hpr1 du complexe THO ainsi que de l’hnRNP Hek2. Toutes deux sont Ulp1-dépendantes, et interviennent sur la partie C-terminale de ces protéines. Nous avons également mis en évidence que chacune de ces sumoylations contrôle le recrutement de son substrat au sein des mRNPs. L’analyse fonctionnelle d’un mutant affectant la sumoylation d’Hpr1 a identifié cette modification comme nécessaire au recrutement du complexe THO sur une population d’ARNms impliqués dans la résistance au stress acide, autrement dégradés par l’exosome. Ainsi, l’absence de sumoylation d’Hpr1 diminue fortement la viabilité cellulaire en conditions de stress, un phénotype supprimé par l’inactivation de l’exosome. L’étude des effets de la sumoylation d’Hek2 suggère une modulation par SUMO de certaines de ses fonctions, notamment dans la localisation cellulaire des ARNms. L’ensemble de ces données fournit donc les deux premiers exemples de régulation du métabolisme des mRNPs par des événements de sumoylation intervenant au niveau du pore nucléaire. / Within the cells, mRNAs are associated to proteins, thereby generating particles called mRNPs (messenger ribonucleoproteins). mRNPs form in a cotranscriptional manner and their composition defines the fate of mRNAs by modulating the different steps of their metabolism, including their stability, their processing, their export, their localisation and their translation. In view of the importance of such mechanisms for cell physiology, several mechanisms ensure a tight spatio-temporal control of mRNPs composition through multiple mRNP remodelling events. These changes in the protein content of mRNPs depend on helicases and post-translational modifications, but remain to be further investigated. Sumoylation is one of the modifications that could contribute to mRNPs remodelling from yeast (S. cerevisiae) to metazoans. Indeed, it has been reported that the SUMO-protease Ulp1/SENP2, a key enzyme of the sumoylation machinery, is localized at the basket of nuclear pore complexes, in close vicinity with mRNPs committed for export. This particular localization, together with the reported defects in mRNPs export and localisation of yeast mutants affecting Ulp1, prompted the lab to ask whether sumoylation could contribute to mRNP biogenesis. In order to investigate this hypothesis, our lab compared mRNPs composition between wild-type and ulp1 mutant S. cerevisiae yeast strains using a proteomic approach. This screen identified two mRNP components that depend on Ulp1 for their recruitment onto these particles: the THO complex and the hnRNP Hek2. The THO complex is a multi-subunit factor that prevents genome instability and contributes to transcription, mRNP assembly and export. Hek2 has multiple functions in mRNA stability, translation and/or localization. Using biochemical approaches, we have been able to visualize sumoylated versions of the Hpr1 subunit of the THO complex and of the hnRNP Hek2. In both cases, this modification depends on Ulp1 activity and occurs on the C-terminal part of the protein. We further showed that these sumoylation events control THO and Hek2 recruitment onto mRNPs. Functional analysis of a mutant impairing Hpr1 sumoylation revealed that this modification is required for proper recruitment of the THO complex onto a subset of mRNAs involved in acidic stress resistance, which are otherwise degraded by the exosome. Decreased Hpr1 sumoylation results in a strong reduction of viability in acid stress conditions, a phenotype that is rescued by inactivation of the exosome. The investigation of the role of Hek2 sumoylation in mRNPs metabolism suggests that this modification regulates some of Hek2 functions, especially in mRNA localisation. All together, these results provide the two first examples of mRNPs components whose functions are regulated by sumoylation events occurring at the level of nuclear pores.

No description available.

La dystrophie myotonique de type 1 (DM1) est une maladie dégénérative impliquant des symptômes d’atrophie musculaire et de myotonie. Au niveau moléculaire, elle est caractérisée par une expansion aberrante de CUG dans la région 3’UTR de l’ARNm de DMPK (Dystrophia Myotonica protein kinase). Ces répétitions CUG forment des agrégats toxiques (appelés foci) majoritairement nucléaires dans les cellules de patients DM1 et causent la séquestration anormale de ribonucléoprotéines (RBP), tel que le facteur «Muscleblind-like 1» (MBNL1), qui lieraient normalement les motifs CUG d’autres ARN. Les fonctions normales de ces RBPs seraient alors perturbées. En plus de leur rôle dans l’épissage alternatif, MBNL a récemment été caractérisé pour son implication dans la localisation intracellulaire de ses ARN cibles. Ceci suggèrerait que la pathogénèse de la DM1 pourrait résulter de l’effet perturbateur des répétitions CUG sur la localisation d’ARN précis et de protéines RBPs. À cet effet, un criblage basé sur de la microscopie fluorescente de 322 RBPs dans des myoblastes de patients DM1 a permis d’identifier des nouveaux facteurs qui colocaliseraient avec les expansions pathogéniques CUG. De plus, ces myoblastes DM1 ont été fractionnés et un séquençage-ARN a par la suite permis l’identification de transcrits délocalisés. Les deux banques de données ainsi générées, tant par le criblage que par le fractionnement/séquençage-ARN, pourraient ouvrir des nouvelles avenues de recherches dans la compréhension des anomalies moléculaires associées à la DM1, et potentiellement d’autres maladies à expansions microsatellites. / Myotonic dystrophy of type 1 (DM1) is a degenerative disorder implicating symptoms of muscular atrophy and myotony. In a molecular level, it is caused by the aberrant expansion of CUG repeats in the 3’-UTR region of the DMPK mRNA (Dystrophia Myotonica protein kinase). Excessive CUG repeats then form toxic aggregates (foci) enriched within the nucleus of DM1 patient cells. These RNA foci cause the abnormal sequestration of RNA Binding Proteins (RBP), in particular members of the Muscleblind-like protein 1 (MBNL), that normally bind the CUG motif of other target RNAs, and will hence alter their normal functions. In addition to their role in alternative splicing, MBNL1 has recently been implicated in the intracellular localisation of its RNA targets. It remains elusive whether the pathogenesis of DM1 could result from the deregulating effect of CUG repeats on the localisation of specific RNAs and RBP proteins. In this thesis, a fluorescent imaging-based screening of 322 RBPs in DM1 patient’s myoblasts has been conducted and this had led to the identification of new factors that may colocalize with pathogenic CUG expansions. Moreover, these DM1 myoblasts have been fractionated and subsequent RNA-sequencing has permitted the identification of transcripts that are delocalised between subcellular compartments. From the two large datasets generated from the RBP imaging-based screening and fractionation/RNA-sequencing, new avenues of research can be initiated to further understand not only DM1, but perhaps also other disorders that implicate microsatellite expansions.