Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies

In Caenorhabditis elegans, the Mex-3 protein is a translational regulator that specifies the posterior blastomere identity in the early embryo and contributes to the maintenance of the germline totipotency. We have now identified a family of four homologous human Mex-3 genes, called hMex-3A to -3D that encode proteins containing two heterogeneous nuclear ribonucleoprotein K homology (KH) domains and one carboxy-terminal RING finger module. The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway. Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover. Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET

A Novel Role for the SMG-1 Kinase in Lifespan and Oxidative Stress Resistance in Caenorhabditis elegans

The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assays together with expression studies in murine intestine. We demonstrate here that MEX3A has a repressive function by controlling CDX2 levels in gastric and colorectal cellular models. This is dependent on the interaction with a specific binding determinant present in CDX2 mRNA 3'untranslated region. We have further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties. Therefore, we describe a novel CDX2 post-transcriptional regulatory mechanism, through the RNA-binding protein MEX3A, with a major impact in intestinal differentiation, polarity and stemness, likely contributing to intestinal homeostasis and carcinogenesis

Le virus d'Epstein-Barr: identification de genes cellulaires cibles deregules au cours de la latence virale

SIGLEINIST T 73179 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Les proteines Mex-3 humaines (de nouveaux acteurs dans les voies de régulation des ARN)

LKB1 is a tumour suppressor protein kinase controlling energetic metabolism and cell polarity. In order to gain insight into the mechanism by which LKB1 is involved in these functions, we have identified a novel family of four related but distinct human genes (hMex-3A to 3D) orthologous to Caenorhabditis elegans mex-3 gene. hMex-3 genes encode RNA-binding phospho-proteins, two of them (hMex-3A and hMex-3B) being differentially localized in cytoplasmic foci involved in mRNA sorting and decay. Global proteomic approach by affinity purification of protein complexes led to the identification of hMex-3B and 3C interacting proteins amongst which most are known to be involved in mRNA metabolism. We subsequently focused on the binding of the 14-3-3 adaptor proteins, which is specific to hMex-3B, and demonstrated that this interaction regulates hMex-3B stability, mRNA binding and sorting to distinct classes of RNA granules. To conclude, this manuscript presents results concerning the newly described Mex-3 proteins family, which are new regulators of mRNA metabolism. Preliminary results suggest that these proteins are downstream effectors of LKB1, an hypothesis that will be followed-up by further investigations of the functional links between hMex-3 proteins, LKB1 and cell polarityLa protéine oncosuppressive LKB1 est une kinase qui contrôle le métabolisme énergétique et la polarité cellulaire. Afin de comprendre comment LKB1 régule ces processus, nous avons identifié une nouvelle famille de quatre gènes distincts chez l homme (hMex-3A à 3D), homologues au gène mex-3 du nématode Caenorhabiditis elegans, et codant des protéines se liant à l ARN. Les protéines hMex-3 sont phosphorylées et deux d entre elles, hMex-3A et hMex-3B sont recrutées dans des granules cytoplasmiques connus pour être des centres de triage des ARNm. Une approche de purification par affinité des complexes protéiques natifs a permis de caractériser une partie de l interactome des protéines hMex-3B et C, et de montrer que la majorité de leurs partenaires sont des régulateurs connus du métabolisme des ARNm. A la suite de cette analyse, nous avons étudié plus en détail le mode d interaction de la protéine 14-3-3 qui se lie spécifiquement à hMex-3B et montré que cette association contrôle la stabilité, la liaison à l ARN et le routage de hMex-3B dans différents types de granules à ARN. En conclusion, le travail présenté dans ce manuscrit décrit pour la première fois les protéines hMex-3, une nouvelle famille de régulateurs du métabolisme des ARN. Les résultats obtenus à la fin de ce travail indiquent que les protéines hMex-3 pourraient être des effecteurs de LKB1 et les études ultérieures préciseront le lien fonctionnel entre les protéines hMex-3, LKB1 et le contrôle de la polarité cellulaireLYON1-BU.Sciences (692662101) / SudocSudocFranceF