Regulation of cellular and viral translation by microRNAs

Les microARN jouent un grand rôle dans la régulation de l'expression des gènes bien que leur mécanisme d'action soit encore sujet à débat. Des premières études chez le vers C. elegans aux différents systèmes in vitro qui ont ensuite été développés, plusieurs modèles ont été proposés, comme l'inhibition de la traduction au niveau de l'initiation ou de l'élongation, et la déstabilisation du transcrit par déadénylation. Cependant, à la lumière des découvertes récentes, un consensus semble apparaître et indique que les miARN inhiberaient d'abord la traduction avant d'induire la déadénylation du transcrit, provoquant ainsi sa dégradation prématurée. D'autre part, le blocage traductionnel semble impliquer à la fois la coiffe en 5' et la queue poly(A) en 3' de l'ARNm ainsi que les facteurs qui s'y lient, c'est à dire le facteur d'initiation de la traduction eIF4F et la Poly(A) Binding Protein (PABP). Ces résultats ont conduit au modèle selon lequel, les miARN seraient capables d'empêcher la liaison entre ces deux facteurs et donc la circularisation du transcrit qui est essentielle à la fois au recrutement de la machinerie traductionnelle et à la stabilité de l'ARNm. Afin de mieux comprendre ce mécanisme, notre laboratoire a développé un système in vitro basé sur l'utilisation du lysat de réticulocytes de lapin qui permet d'étudier l'effet traductionnel des miARN en s'affranchissant de dégradation du transcrit. L'étude de l'effet de drogues et d'enzymes virales, capables de bloquer spécifiquement la fonction de chaque facteur d'initiation dans ce système, a permis de déterminer le rôle clé de eIF4G et PABP dans l'inhibition traductionnelle par les miARN. Cependant, leur interaction n'est pas requise et le blocage s'effectue plutôt au cours de l'étape de balayage de la région 5' non codante par la petite sous-unité ribosomique. En parallèle de cette étude in vitro, un travail sur des lignées cellulaires a permis de déterminer l'influence de la queue poly(A) sur l'effet miARN. De façon très surprenante, l'expression des transcrits non polyadénylés n'est plus inhibée et est même stimulée par les miARN. Cet effet est dépendant de l'association du domaine MIF4G du facteur eIF4G avec le facteur eIF3, ce qui suggère qu'en l'absence de queue poly(A), les miARN seraient capables de stimuler le recrutement de la petite sous-unité ribosomique sur l'ARNm. L'ensemble de ces résultats révèle la complexité de l'effet miARN sur la traduction et ouvre de nouvelles voiesThe mechanism by which microRNAs (miRNAs) can control gene expression has been a great matter of debate. From the first studies in worm to the in vitro systems that are used today, many models have been proposed that include regulation at the level of translation or at the level of mRNA stability by controlling 3' deadenylation and decay. Recent studies provided a consensus model of all these discrepancies and suggested that translation inhibition occured first and is followed by deadenylation and further degradation of the target transcript. Moreover, translation silencing seems to occur at the initiation level, and requires eIF4F and PABP initiation factors. This led to the hypothesis that miRNAs could interfere with the interaction between these two factors thus affecting the circularisation of the mRNA, which is essential for translation efficiency. In order to gain insight into this mechanism, we have used an in vitro system based on the rabbit reticulocyte lysate that fully recapitulates miRNA effects on translation with virtually no effect on deadenylation and decay. Using this system and a wide spectrum of translational inhibitors, we have narrowed down the step of initiation at which repression is exerted and we found that miRNAs affect mainly ribosomal scanning. This effect requires the presence of both eIF4G and PABP but does not rely on their physical interaction. Further analysis of miRNA repression in cells revealed that the poly(A) tail was an absolute requirement for miRNA action. To most of our surprise, we observed that removal of the poly(A) resulted in a shift from repression to stimulation of mRNA expression. This effect seems to require the middle domain of eIF4G and the presence of the Ago proteins. Altogether, these results reveal the complexity of miRNA effect and open new prospects on translation regulatio

INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation.

International audienceThe INT6/EIF3E protein has been implicated in mouse and human breast carcinogenesis. This subunit of the eIF3 translation initiation factor that includes a PCI domain exhibits specific features such as presence in the nucleus and ability to interact with other important cellular protein complexes like the 26S proteasome and the COP9 signalosome. It has been previously shown that INT6 was not essential for bulk translation, and this protein is considered to regulate expression of specific mRNAs. Based on the results of a two-hybrid screen performed with INT6 as bait, we characterize in this article the MIF4GD/SLIP1 protein as an interactor of this eIF3 subunit. MIF4GD was previously shown to associate with SLBP, which binds the stem-loop located at the 3' end of the histone mRNAs, and to be necessary for efficient translation of these cell cycle-regulated mRNAs that lack a poly(A) tail. In line with the interaction of both proteins, we show using the RNA interference approach that INT6 is also essential to S-phase histone mRNA translation. This was observed by analyzing expression of endogenous histones and by testing heterologous constructs placing the luciferase reporter gene under the control of the stem-loop element of various histone genes. With such a reporter plasmid, silencing and overexpression of INT6 exerted opposite effects. In agreement with these results, INT6 and MIF4GD were observed to colocalize in cytoplasmic foci. We conclude from these data that INT6, by establishing interactions with MIF4GD and SLBP, plays an important role in translation of poly(A) minus histone mRNAs

microRNAs stimulate translation initiation mediated by HCV-like IRESes

International audienceMicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by recognizing and hybridizing to a specific sequence generally located in the 3? untranslated region (UTR) of targeted mRNAs. miRNA-induced inhibition of translation occurs during the initiation step, most probably at the level of ribosome scanning. In this process, the RNA-induced silencing complex interacts both with PABP and the 43S pre-initiation complex to disrupt scanning of the 40S ribosome. However, in some specific cases, miRNAs can stimulate translation. Although the mechanism of miRNA-mediated upregulation is unknown, it appears that the poly(A) tail and the lack of availability of the TNRC6 proteins are amongst major determinants. The genomic RNA of the Hepatitis C Virus is uncapped, non-polyadenylated and harbors a peculiar internal ribosome entry site (IRES) that binds the ribosome directly to the AUG codon. Thus, we have exploited the unique properties of the HCV IRES and other related IRESes (HCV-like) to study how translation initiation can be modulated by miRNAs on these elements. Here, we report that miRNA binding to the 3? UTR can stimulate translation of a reporter gene given that its expression is driven by an HCV-like IRES and that it lacks a poly(A) tail at its 3? extremity

miRNA repression of translation in vitro takes place during 43S ribosomal scanning.

International audiencemicroRNAs (miRNAs) regulate gene expression at multiple levels by repressing translation, stimulating deadenylation and inducing the premature decay of target messenger RNAs (mRNAs). Although the mechanism by which miRNAs repress translation has been widely studied, the precise step targeted and the molecular insights of such repression are still evasive. Here, we have used our newly designed in vitro system, which allows to study miRNA effect on translation independently of deadenylation. By using specific inhibitors of various stages of protein synthesis, we first show that miRNAs target exclusively the early steps of translation with no effect on 60S ribosomal subunit joining, elongation or termination. Then, by using viral proteases and IRES-driven mRNA constructs, we found that translational inhibition takes place during 43S ribosomal scanning and requires both the poly(A) binding protein and eIF4G independently from their physical interaction

Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation.

International audienceThe 5'-untranslated region (5'-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5'-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5'-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens

HIV-2 genomic RNA accumulates in stress granules in the absence of active translation

International audienceDuring the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells. This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding protein and stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observed that increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a 'packageable' gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein