The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex-or miRNA-induced silencing complex (miRISC)-can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression. © 2011 European Molecular Biology Organization

GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis

MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis

The histone binding capacity of SPT2 controls chromatin structure and function in Metazoa

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.</p

Détermination des rôles joués par les protéines d'interférence par l'ARN dans la division méiotique chez S. pombe

Les protéines d'interférence par l'ARN (RNAi) chez S. pombe sont responsables de la formation dliétérochromatine aux centromeres durant la mitose via le complexe RTTS -spChpl spTas3 spAgol-. De façon intéressante, la mutation d'une des protéines de RNAi, soit spAgol, spDcrl ou spRdpl, conduit à des aberrations de ségrégation chromosomique en méiose. Bien que ce phénotype puisse être dû à un défaut des centromeres, nous nous sommes intéressés à identifier une possible implication des protéines du RNAi dans un autre mécanisme primordial en méiose : la recombinaison homologue. Nous avons étudié les interactions potentielles entre les protéines de RNAi et les protéines de recombinaison homologue. Nous avons inclus également spArbl, spArb2, spChpl et spTas3, quatre protéines impliquées dans la formation dliétérochromatine aux centromeres via le RNAi. Nous avons ainsi démontré par double-hybride la présence d'interaction entre la protéine méiotique spRecl5 et spRdpl, spArbl et spChpl d'une part, et spRec7 avec spTas3 d'autre part. La deletion de ces gènes conduit à de graves défauts dans la formation des spores, soulignant leur importance dans le processus méiotique. spRec7 et spRecl5 sont deux protéines peu connues, impliquées dans les étapes précoces de la formation des cassures double brin par Spol 1 (spRecl2) à l'origine de la recombinaison homologue. Nous avons analysé par immunoprecipitations de chromatine et par immunofluorescence l'effet des mutations de spChpl et spTas3 sur les étapes précoces de recombinaison homologue, et les résultats préliminaires obtenus à ce jour semblent supporter une forte implication de ces protéines dans la recombinaison homologue méiotique

DCU per a la configurabilitat d'una estació de treball clínic

Gairebé un 50% dels metges de la sanitat catalana realitzen actualment les seves tasques assistencials en entorns SAP. Donada la naturalesa de la seva feina, és fàcil afirmar que les aplicacions que utilitzen habitualment haurien d'haver estat desenvolupades centrant l'atenció en l'usuari i el context en què aquest realitza la seva feina. Però degut a diversos motius organitzatius, això no ha estat així. Aquest projecte pretén demostrar el valor afegit que pot aportar el Disseny Centrat en l'Usuari en el desenvolupament d'aplicacions assistencials en l'àmbit SAP Sanitat. S'ha realitzat una investigació prèvia de les necessitats reals dels usuaris, un anàlisi del context d'ús i dels diferents perfils de usuari, s'ha desenvolupat un prototip amb la nova tecnologia que aporta SAP per al desenvolupament de interfícies tipus web integrades en el sistema (SAP Webdynpro for ABAP), i finalment s'ha realitzat la corresponent avaluació heurística i el test de usuaris. S'ha arribat principalment a tres conclusions: en primer lloc, ha sorprès la bona disposició dels usuaris metges per a participar en aquesta mena de projectes; en segon lloc, ha quedat demostrada la importància de l'anàlisi del context, així com la rellevància que el dissenyador estigui quan més a prop millor de l'usuari final; i finalment, cal destacar que la tecnologia emprada ha limitat qualitativament diverses opcions de disseny.Almost 50% of catalan health care physicians perform currently their work on SAP environment. Given the nature of their work, it's easy to say that the applications you use regularly should have been developed focusing on the user and the context in which it carries out its work. But due to various organizational reasons, this has not happened. This project aims to demonstrate the added value that can be provided in the User Centered Design in the development of SAP Health care applications. In first place, it was done an investigation of the actual user needs, an analysis of use context and different user profiles, a prototype was developed using the new technology offered by SAP to develop web interfaces system integrated (SAP Webdynpro for ABAP), and finally it was performed the corresponding heuristic evaluation and user testing. Three conclusions were mainly reached: first, it was amazing the availability of physician users to participate in these kind of projects; in a second term, it has been demonstrated the importance of the contextual enquiry, and the relevance of the designer proximity to the final user; and finally, should be remarked that the used technology had limited qualitatively several design options.Casi un 50% de los médicos de la sanidad catalana realizan actualmente sus tareas asistenciales en entornos SAP. Dada la naturaleza de su trabajo, es fácil afirmar que las aplicaciones que utilizan habitualmente deberían haber sido desarrolladas centrando la atención en el usuario y el contexto en que éste realiza su trabajo. Pero debido a diversos motivos organizativos, esto no ha sido así. Este proyecto pretende demostrar el valor añadido que puede aportar el Diseño Centrado en el Usuario en el desarrollo de aplicaciones asistenciales en el ámbito SAP Sanidad. Se ha realizado una investigación previa de las necesidades reales de los usuarios, un análisis del contexto de uso y de los diferentes perfiles de usuario, se ha desarrollado un prototipo con la nueva tecnología que aporta SAP para el desarrollo de interfaces tipo web integradas en el sistema (SAP Webdynpro for ABAP), y finalmente se ha realizado la correspondiente evaluación heurística y el test de usuarios. Se ha llegado principalmente a tres conclusiones: en primer lugar, ha sorprendido la buena disposición de los usuarios médicos para participar en este tipo de proyectos, en segundo lugar, ha quedado demostrada la importancia del análisis del contexto, así como la relevancia que el diseñador esté cuanto más cerca mejor del usuario final, y finalmente, cabe destacar que la tecnología empleada ha limitado cualitativamente diversas opciones de diseño

Live Imaging of Parental Histone Variant Dynamics in UVC-Damaged Chromatin

International audienc

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA‐guided gene silencing in vivo

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans. In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function in vivo. Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA. Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions

SETDB1 modulates the TGFβ response in Duchenne muscular dystrophy myotubes

International audienceOveractivation of the transforming growth factor-β (TGFβ) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFβ induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFβ signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFβ target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFβ–driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention

The Tryptophan-binding pockets of ALG-1 are essential to mediate the interaction with AIN-1 and AIN-2 proteins <i>in vitro</i> and <i>in vivo</i>.

<p><b>(A and B) Putative Tryptophan-binding pockets of ALG-1.</b> A homology model (pale cyan) of ALG-1 tryptophan-binding pockets 1 (A) and 2 (B) was generated by Swiss-Model from 4OLB and superposed on the tryptophan (pink)-bound hAgo2 structure (PDB code: 4OLB). hAgo2 is not shown for clarity. <b>(C) Schematic view of ALG-1 structured protein domains.</b> The residues forming the tryptophan-binding pocket 1 and 2 are coloured in green and red respectively and mutated into non-polar amino acid alanine to generate an ALG-1 <u>T</u>ryptophan-binding <u>P</u>ockets mutant (hereafter named TPmut). <b>(D) GST pull-down assays using bacterially expressed GST-Tagged wild-type or tryptophan-binding pocket mutants of ALG-1 protein.</b> An AIN-1 peptide fragment containing the AGO Binding Domain (350-641aa) was purified and co-immunoprecipitated with recombinant GST-tagged ALG-1 or ALG-1(TPmut) protein. The upper part of gel was stained with Coomassie Brilliant Blue and the lower blotted with antibody against AIN-1. <b>(E) Co-Immunoprecipitation of ALG-1 wild-type or mutant and AIN-1 and AIN-2 proteins from whole <i>C</i>. <i>elegans</i> extract.</b> Transgenic animals were generated using MosSCI single copy insertion of mCherry-tagged ALG-1 wild-type or TP mutant (TPmut). Animals were then crossed into <i>alg-1(0)</i> genetic background. ALG-1 proteins from young adults animals was co-immunoprecipitated using ALG-1 specific antibody and AIN-1 and AIN-2 proteins were detected by Western Blotting. Inputs represent the equivalent of 20% and 25% of the total protein extracts used for the IPs for detecting AIN-1 and AIN-2, respectively. Numbers indicate the relative level of proteins in input and Co-IP compared to wt for each strain.</p