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

Developmental characterization of the microRNA-specific C. elegans Argonautes alg-1 and alg-2.

The genes alg-1 and alg-2 (referred to as "alg-1/2") encode the Argonaute proteins affiliated to the microRNA (miRNA) pathway in C. elegans. Bound to miRNAs they form the effector complex that effects post-transcriptional gene silencing. In order to define biological features important to understand the mode of action of these Argonautes, we characterize aspects of these genes during development. We establish that alg-1/2 display an overlapping spatio-temporal expression profile and shared association to a miRNAs set, but with gene-specific predominant expression in various cells and increased relative association to defined miRNAs. Congruent with their spatio-temporal coincidence and regardless of alg-1/2 drastic post-embryonic differences, only loss of both genes leads to embryonic lethality. Embryos without zygotic alg-1/2 predominantly arrest during the morphogenetic process of elongation with defects in the epidermal-muscle attachment structures. Altogether our results highlight similarities and specificities of the alg-1/2 likely to be explained at different cellular and molecular levels

Caractérisation fonctionnelle de la voie des microARNs chez le nématode caenorhabditis elegans

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2012-2013.Les microARNs sont des petites molécules d'ARN non-codant conservées à travers les espèces qui régulent négativement l'expression génique au niveau post-transcriptionel. Ils figurent parmi les acteurs majeurs du maintien de l'homéostasie cellulaire et leur dérégulation est à l'origine de nombreuses pathologies humaines. Leur biogenèse consiste en deux étapes de maturation successives effectuées par les enzymes Drosha et Dicer, pour générer une molécule effectrice d'ARN d'une longueur de 21-23 nucleotides. La courte molécule produite s'assemble avec une protéine de la famille Argonaute pour former un complexe ribonucléoprotéique capable de cibler spécifiquement un ARNm et d'éteindre son expression. Pour mieux comprendre comment les microARNs régulent l'expression génique, l'objectif principal de mon doctorat a été d'étudier le rôle de la protéine Argonaute ALG-1, en utilisant le nematode Caenorhabditis elegans comme modèle animal. Nous avons dans un premier temps recherché quelles caractéristiques moléculaires étaient importantes pour déterminer la sélection spécifique des protéines Argonautes essentielles à la voie des microARNs parmi les nombreuses Argonautes retrouvées chez le nematode C. elegans. Par une approche génétique, nous avons découvert que la sélection des protéines Argonautes est affectée à la fois par la structure du duplex d'ARN double brins et par les caractéristiques spécifiques de chacune d'elles. Pour étudier plus précisément la fonction de la protéine Argonaute ALG-1, nous avons entrepris un criblage double-hybride pour identifier de nouveaux partenaires protéiques. Parmi eux, nous avons identifié puis prouvé l'importance de la protéine ribosomale RACK1 dans la voie de régulation des microARNs chez le nematode et l'humain. Nous avons démontré que la perte de fonction de RACK1 affecte l'association des microARNs et des protéines Argonaute avec les ribosomes actifs suggérant une contribution de cette protéine dans le recrutement de ces complexes aux sites actifs de traduction. Finalement, nous avons développé une approche génétique systématique permettant d'adresser génétiquement l'implication des partenaires d'ALG-1 dans la voie des microARNs. Collectivement, mes travaux de doctorat nous ont permis de contribuer à l'élargissement des connaissances associées à cette mécanistique complexe de régulation des gènes par les microARNs chez l'animal

Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans

In Caenorhabditis elegans, specific Argonaute proteins are dedicated to the RNAi and microRNA pathways. To uncover how the precise Argonaute selection occurs, we designed dsRNA triggers containing both miRNA and siRNA sequences. While dsRNA carrying nucleotides mismatches can only enter the miRNA pathway, a fully complementary dsRNA successfully rescues let-7 miRNA function and initiates silencing by RNAi. We demonstrated that RDE-1 is essential for RNAi induced by the perfectly paired trigger, yet is not required for silencing by the let-7 miRNA. In contrast, ALG-1/ALG-2 are required for the miRNA function, but not for the siRNA-directed gene silencing. Finally, a dsRNA containing a bulged miRNA and a perfectly paired siRNA can enter both pathways suggesting that the sorting of small RNAs occurs after that the dsRNA trigger has been processed by Dicer. Thus, our data suggest that the selection of Argonaute proteins is affected by two molecular features: (1) the structure of the small RNA duplex; and (2) the Argonautes specific characteristics

A new role for the GARP complex in microRNA-mediated gene regulation.

Many core components of the microRNA pathway have been elucidated and knowledge of their mechanisms of action actively progresses. In contrast, factors with modulatory roles on the pathway are just starting to become known and understood. Using a genetic screen in Caenorhabditis elegans, we identify a component of the GARP (Golgi Associated Retrograde Protein) complex, vps-52, as a novel genetic interactor of the microRNA pathway. The loss of vps-52 in distinct sensitized genetic backgrounds induces the enhancement of defective microRNA-mediated gene silencing. It synergizes with the core microRNA components, alg-1 Argonaute and ain-1 (GW182), in enhancing seam cell defects and exacerbates the gene silencing defects of the let-7 family and lsy-6 microRNAs in the regulation of seam cell, vulva and ASEL neuron development. Underpinning the observed genetic interactions, we found that VPS-52 impinges on the abundance of the GW182 proteins as well as the levels of microRNAs including the let-7 family. Altogether, we demonstrate that GARP complex fulfills a positive modulatory role on microRNA function and postulate that acting through GARP, vps-52 participates in a membrane-related process of the microRNA pathway

PTB association with the <i>let-7</i> bead depends of the <i>let-7</i> seed complementary sequences.

<p>(A) <i>let-7</i> seed mutant oligo could not inhibit <i>let-7</i> mediated gene repression. Renilla luciferase expressing plasmid containing a part of the 3′ UTR of human HMGA2 that carries four <i>let-7</i> target sites were transfected into HeLa cells together with Firefly expressing plasmid, as internal control, and the indicated 2′-<i>O</i>-methyl oligos. The graph shows the result of the dual-luciferase assay normalized to the control oligo. The error bars represent the standard error of three experiments. (B) <i>let-7</i>, hAgo2 and PTB are sensitive to the presence of the seed sequence of the <i>let-7</i> oligo. The quantity of <i>let-7</i> miRNA associated with the indicated oligos was quantified using Northern hybridization and normalized to the amount of miRNA pulled down with the wild-type <i>let-7</i> oligo. The presences of hAgo2 and PTB on the indicated beads were monitored by Western hybridization. (C) PTB association with the <i>let-7</i> column does not depend on the presence of the canonical PTB site in the oligo. Affinity purifications were carried out with the indicated oligos and the association of miRNAs, hAgo2 and PTB with these oligos was monitored by Northern hybridization and Western blotting. sup.: supernatant.</p

Affinity purification of <i>let-7</i> associated complexes.

<p>(A) Biotinylated 2-<i>O</i>-methylated oligos used in this study. Sequences highlighted with red are complementary to <i>let-7a</i>. Blue nucleotides indicate changes generated from the original <i>let-7</i> oligo. (B) Northern hybridization (top panel) and Western blot (bottom panel) show that <i>let-7</i> oligo specifically purifies <i>let-7</i> miRNA and hAgo2 protein. sup.: supernatant; c and cont.: control oligo. (C) Proteins co-purify with <i>let-7</i> oligo. Right and left panels show the results of the independent affinity purifications. Proteins that are specifically pulled down with the <i>let-7</i> oligo are labeled next to the stained gels.</p

PTB and <i>let-7</i> miRNA contribute together to regulate gene expression in <i>C. elegans</i>.

<p>(A) Synchronized L1 animals were placed at semi-permissive temperature (20°C) and adult animals were scored after seventieth-two hours. The animal sterility observed in the population is caused by either a vulval bursting at the L4-adult transition or by a severe gonadal defect. Error bars represent the 95% confidence interval from independent experiments (n) where between 20 and 40 animals have been scored. ***: p<0.0001 (B) <i>let-7</i> level remained unchanged in the <i>let-7ts</i>/<i>ptb-1</i> animals. RNAs were purified from the indicated genotypes and probed for <i>let-7</i> and U6 RNAs. The amount of RNA was used for Northern blotting is indicated on the top of the panel and the U6/<i>let-7</i> ratios are presented at the bottom of the panel.</p