Acide ribonucléique messager | EPFL Graph Search

vignette|Représentation schématique de la synthèse et de la maturation d'un ARN messager dans une cellule eucaryote. L'acide ribonucléique messager, ARN messager, ou ARNm (en anglais, mRNA, pour messenger ribonucleic acid), est une molécule intermédiaire d'acide ribonucléique (ARN), consistant en une copie transitoire d'une portion de l'ADN correspondant à un ou plusieurs gènes d'un organisme biologique. L'ARNm est utilisé comme intermédiaire par les cellules pour la synthèse des protéines. Le concept d'ARN messager a été émis puis démontré par Jacques Monod, François Jacob et leurs collaborateurs à l'Institut Pasteur en 1961, ce qui leur a valu le prix Nobel en 1965. L'ARNm est une copie simple brin linéaire de l'ADN composée d'ARN, qui comprend la région codant une protéine encadrée de régions non codantes. Il est synthétisé sous forme de précurseur dans le noyau de la cellule lors d'un processus appelé transcription. Il subit alors plusieurs étapes de maturation, ses deux extrémit

RNA-Protein interactions at AU-rich elements

Ramona Pauchard-Batschulat

The expression of many proto-oncogenes, nuclear transcription factors and cytokines is regulated post-transcriptionally by specific interactions between distinct cis-elements within the mRNA and trans-acting factors that bind to these elements and thereby either promote or inhibit the rapid decay of the mRNA. This kind of regulation enables the cell to rapidly respond to changes in the cellular environment. These elements are usually located within the 3’UTR of the mRNA, rich in adenylate and uridylate and called AU-rich elements or AREs. To date, many of these trans-acting factors have been determined and among the best studied are the stabilizing HuR, the rather destabilizing AUF1 and the destabilizing KSRP and TTP. Previous studies have suggested that these ARE-BPs as well as yet unidentified factors might have overlapping target specificities and bind either in a concurrent, concomitant or maybe an even cooperative fashion to their targets to regulate mRNA half-life. However, the exact manner of how these factors might interact and how these interactions might be influenced, is largely unknown. Thus, my first project aimed to study the interactions between AUF1, KSRP and HuR in the context of the 3’UTRs of RANKL and Bcl-6 that were recently identified as AUF1 targets with a short half-life. By combining ARE-BP immunoprecipitations with qRT-PCR analysis of co-precipitated reporter RNAs, we were able to gain interesting insight into the RNA-protein interactions at AU-rich elements. However, this approach is very laborious and can study RNA-protein interactions only within a limited context. Yet, the pools of mRNA targets as well as binding factors are very multifaceted and likely depend on the cell type and condition as well as the cellular environment. Thus, a much broader approach is necessary to reveal the entire network of RNA-protein and protein-protein interactions at AU-rich elements. A promising technique is the combination of ARE-BP immunoprecipitation and high-throughput sequencing to analyze co-precipitated RNA. This approach allows for the extraction of a lot of important data from a single experiment as for example the virtually complete pool of target mRNAs under the studied conditions, the binding sites within these mRNAs as well as the frequency with which these sequences were detected. Based on these data, it would be possible to make suggestions about the binding specificities as well as potential, yet un-identified targets. If accumulating data sets for different cellular or environmental conditions, we might gain insight into the regulation of these RNA-protein interactions. In a similar way, the comparison of data sets for different ARE-binding proteins would give us further insight into the protein-protein interactions at and around AU-rich elements. Therefore, my second project focused on the development of a novel protocol that allows for the rapid, reliable and reproducible generation of high-throughput sequencing samples based on co-immunoprecipitated RNA fragments.

EPFL2013

Functional analysis of Bicc1 and novel interacting proteins in renal tubule cell metabolism and polycystic kidneys

Lucía Carolina Leal Esteban

Genetic disorders known as ciliopathies develop polycystic kidneys, including autosomal dominant and autosomal recessive polycystic kidney diseases (ADPKD and ARPKD). Several signaling pathways including the cAMP/PKA pathway are implicated in driving cystic growth. However, the mechanisms underlying cysts formation is still elusive. Renal cysts also arise in patients and in mouse models with mutations in the RNA-binding protein Bicaudal-C (Bicc1). Bicc1 binds specific mRNAs such as adenylate cyclase VI (AC6) mRNA for translational repression. Conducting a structure-function analysis, we demonstrated that Bicc1 self-polymerizes through its sterile alpha motif (SAM domain) to form cytoplasmic clusters, which specifically localize and silence bound mRNAs. Altered glucose and lipid metabolism also sustain cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Here, we found that Bicc1 is essential for normal expression of the gluconeogenic enzymes FBP1 and PEPCK specifically in kidneys, and to maintain euglycemia. In a proteomic interactome screen using a knock-in cell line, we found that TAP-tagged Bicc1 binds the C-Terminal to Lis-Homology domain (CTLH) complex, the mammalian orthologue of the glucose-induced degradation (Gid) complex that triggers degradation of FBP1 in S. cerevisiae. Bicc1 stimulates FBP1 expression in vitro in cultured murine inner medullary collecting duct (mIMCD3) cells, albeit independently of its interaction with the CTLH complex. Instead, Bicc1 and CTLH complex seem to mutually downregulate each other. Bicc1 interactome is also enriched with metabolic related proteins. Here we found that Bicc1 coimmunoprecipitates with rate-limiting metabolic enzymes and contributes to maintain their protein levels, and that deletion of Bicc1 significantly alters the renal metabolome while enhances autophagy. Altogether, these results implicate Bicc1 as an important metabolic regulator in the etiology of polycystic kidneys.

EPFL2017

Characterizing the localization and role of lin-5 and era-1 mRNAs in early C. elegans embryos

Zoltán Péter Spiró

Asymmetric cell division is essential for the generation of diversity during development and the function of stem cell lineages. The Caenorhabditis elegans zygote is an attractive model to investigate the mechanisms of spindle positioning during asymmetric cell division. In this polarized cell, the asymmetric distribution of cortical force generators along the antero-posterior axis and pulling on astral microtubules leads to the unequal cleavage of the one-cell embryo. The mechanisms underlying such cortical force generation are thought to act strictly at the protein level. In this thesis work we report that the mRNA encoding the cortical force generator component LIN-5 is enriched around centrosomes in early embryos, in a manner that depends on microtubules and dynein. We established that the lin-5 coding sequence is necessary and sufficient for mRNA enrichment around centrosomes in C. elegans. In addition, we found that lin-5 mRNA is mislocalized in lin-5(ev571) mutant embryos, which harbor a 9 nucleotide insertion in the coding sequence. Moreover, an intragenic revertant of lin-5(ev571), lin-5(ev571he63), also exhibits mislocalized lin-5 mRNA distribution. We demonstrated that this is accompanied by diminished pulling forces on the posterior spindle pole, suggesting that centrosomal localization of lin-5 mRNA is important for robust pulling forces. We found also that lin-5 mRNA centrosomal enrichment is slightly asymmetric during anaphase, with more transcripts present on the anterior side. We developed a novel FRAP-based assay, which revealed that lin-5 is translated/folded preferentially in the cytoplasm compared to centrosomes. Furthermore, we found that morpholino-mediated inhibition of lin-5 translation diminishes pulling forces on the posterior side during anaphase. Together, these findings lead us to propose that preferential translation/folding of lin-5 in the posterior cytoplasm following release of the mRNA from the posterior centrosome contributes to asymmetric cortical distribution of force generators, and thus to proper spindle positioning. Moreover, we found that the mRNA of an uncharacterized gene, era-1 is enriched on the anterior side of the zygote and is inherited by the anterior blastomeres. Similar to era-1 mRNA, a YFP fusion of ERA-1 protein is also asymmetrically distributed. Moreover, asymmetric distribution of both era-1 mRNA and YFP-ERA-1 protein requires the era-1 3'UTR. Furthermore, the RNA-binding protein MEX-5 is needed for both asymmetric era-1 mRNA localization and for its translational activation. Furthermore, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain normal levels of CHC-1. We demonstrate that CHC-1 is necessary for proper organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network results in excess centrosome movements. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension.

EPFL2014