Made available in DSpace on 2014-06-12T18:04:28Z (GMT). No. of bitstreams: 2 arquivo6197_1.pdf: 1859004 bytes, checksum: e86e2d8c963670c51f4e1b8266e440a2 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / A proteína de ligação à cauda poli-A (PABP) se liga a seqüência de poli adenosinas presente na extremidade 3 do mRNA e possui uma multiplicidade de funções dentro da célula. Dentre as funções atribuídas a PABP destaca-se sua participação em eventos da síntese de proteínas, tais como iniciação e terminação da tradução e reciclagem dos ribossomos, e seu envolvimento no transporte de alguns mRNAs do núcleo para o citoplasma. O objetivo deste trabalho é caracterizar homólogos de proteínas relevantes à iniciação da tradução em tripanosomatídeos. O estudo da PABP teve início com a busca em bancos de dados de L. major de possíveis homólogos, onde foi encontrado tanto o homólogo previamente descrito (LmPABP1) como mais dois homólogos (LmPABP2 e LmPABP3), sendo que o último não possui ortólogos em Trypanosoma. Os três genes foram amplificados, clonados, as proteínas expressas e utilizadas para produção de soro policlonal. Em seguida foi realizada a quantificação dos níveis intracelulares das três proteínas em extratos da forma promastigota de L. major sendo a LmPABP2 a mais abundante, a LmPABP3 em níveis intermediários e a LmPABP1 a menos abundante. No entanto, as LmPABP2-3 são detectadas com uma única forma, enquanto que a LmPABP1 está presente em duas isoformas, provavelmente uma delas devido a fosforilação. Análises da expressão durante o ciclo evolutivo do parasita mostrou que as três proteínas encontravam-se presentes em todas as formas evolutivas, porém a LmPABP3 mostrou um decréscimo na fase estacionária de crescimento e a LmPABP1 apresenta-se fosforilada apenas nas primeiras horas após o repique. Experimentos de localização subcelular indicam que a LmPABP1 está presente na fração citoplasmática, enquanto que LmPABP2-3 estão presentes tanto na fração citoplasmática quanto na nuclear, porém com predominância da fração nuclear. Estudos adicionais precisam ser feitos pra entender como estas proteínas diferem funcionalmente e quais seus papéis na síntese protéica

Gametogenesis is a highly complex process that requires stringent control of gene expression, in which translational regulation plays an essential role. Deleted in Azoospermia-like (DAZL) belongs to the DAZ family of RNA-binding proteins, which are restricted to germ cells, and regulate mRNA translation. Importantly, loss of function of these proteins results in infertility in both males and females in a wide variety of organisms. A model for the mechanism by which DAZL stimulates translation has been proposed based on work in Xenopus laevis (X. laevis) oocytes. In this model, DAZL functions by recruiting the translation initiation factor poly(A)-binding protein (PABP) to the 3’ untranslated region (UTR) of messenger RNAs. Simultaneous binding of PABP to Dazl and factors at the 5’ end confers a “closed-loop” mRNA conformation, which promotes translation initiation. To examine whether DAZL plays a similar role in mammals, co-expression of Dazl and PABP family members was investigated in fetal and adult mouse gonads. In contrast to X. laevis, mammals encode four cytoplasmic PABPs which share a similar domain organisation: PABP1, tPABP, ePABP and PABP4, of which PABP1 and PABP4 appear to be expressed in a wide range of tissues. Immunohistochemistry revealed that Dazl, Pabp1 and Pabp4 are all expressed in primordial germ cells (PGCs) but these show different expression patterns following germ cell sex differentiation. In adult testes Dazl is expressed in spermatogonia and spermatocytes, coinciding with the peak of Pabp4 expression. In contrast, the peak of Pabp1 expression occurs later than that of Dazl, with these proteins only being co-expressed in late pachytene and secondary spermatocyte phases. In adult ovaries, Pabp1, Pabp4 and Dazl are all expressed in the oocytes of primordial and primary follicles. Since both PABP family members are co-expressed with Dazl, the ability of DAZL to interact with PABP1 and PABP4 was investigated in vitro and in vivo. Surprisingly, these studies showed that DAZL discriminates between different PABP family members, only interacting with PABP1, providing the first report of a PABP-specific protein partner. Several putative DAZL mutations have been identified in patients with impaired fertility. Two of these mutations, I37A and R115G, are located in the RNA recognition motif (RRM), a domain which is found in many RNA-binding proteins and mediates both RNA and protein interactions. Thus, the role of these mutations in the ability of DAZL to stimulate translation was investigated. To this end, a translational target of human DAZL (hDAZL) was sought. The 3’UTR of growth differentiation factor 9 (hGDF9) mRNA was found to confer regulation by hDAZL and thus the ability of mutant DAZLs to stimulate reporter mRNAs containing this 3’UTR was examined. This revealed that both mutations compromised the ability of hDAZL to stimulate hGDF9 translation, suggesting a causative effect. These results were further confirmed in assays in which hDAZL is artificially tethered to mRNAs. The ability of mutant hDAZLs to stimulate translation in this assay was compromised suggesting that loss of function is, at least in part, due to impaired protein-protein interactions rather than altered RNA-binding. This work provides insights into the molecular mechanism by which DAZL stimulates the translation of specific mRNAs during mammalian gametogenesis and provides evidence that this function may play an important physiological role in human reproduction.

L’étude qui vous est présentée dans ce mémoire est réalisée chez l’organisme Schizosaccharomyces pombe et elle porte sur la caractérisation de la protéine Nab2, une protéine liant les queues poly(A) ainsi que sur son implication dans la régulation génique. Tout d’abord, il faut savoir qu’il existe quatre modes de régulation (transcriptionnel, post-transcriptionnel, traductionnel et post-traductionnel) dans lesquels interviennent différents types de protéines, en particulier des protéines liant les queues poly(A) des ARN, aussi connues sous le nom de PABPs. Ces protéines reconnaissent l’ARN à l’aide de différents domaines de liaison et elles se subdivisent en deux catégories : les PABPs nucléaires ou cytoplasmiques, représentées respectivement par PABPN1 et PABPC1 chez les mammifères. Comprendre la fonction des PABPs revêt un intérêt particulier puisqu’elles sont impliquées à différents stades de la régulation génique. Des maladies ont aussi été associées à deux PABPs nucléaires humaines, PABPN1 et ZC3H14, mais aucune association entre leur fonction réciproque et la maladie n’a pu être établie. Une des façons de comprendre leur rôle est d’étudier celui de leurs orthologues respectifs. Chez la levure à fission, un orthologue de PABPN1 a été caractérisé et il s’agit de Pab2. S. pombe possède cependant une seconde PABP nucléaire, Nab2, qui est caractérisée dans ces travaux. Des méthodes in vivo et in vitro ont été utilisées afin de confirmer le statut de la protéine, à savoir qu’il s’agit bel et bien d’une PABP nucléaire et que celle-ci est non essentielle. L’identification de partenaires protéiques de Nab2 par spectrométrie de masse a aussi permis de relier Nab2 avec des processus de régulation génique tels que l’épissage et la dégradation. Puisque Pab2 est aussi associée à des fonctions en lien avec la dégradation, il est possible de faire un parallèle entre ces deux protéines et de supposer qu’elles interagissent ensemble. La deuxième partie de ces travaux porte donc sur l’étude de la relation fonctionnelle entre Nab2 et Pab2 et elle a permis de montrer un mécanisme de régulation opportuniste basé sur la liaison de la cible ARN par l’une ou l’autre de ces PABPs. En effet, l’étude de la régulation du gène modèle RPL30-2 indique que Nab2 et Pab2 ont des rôles opposés puisqu’ils sont respectivement des régulateurs positifs et négatifs de l’expression de son transcrit.

<p>microRNAs (miRNAs) downregulate the expression of numerous mRNAs and are involved in almost every biological process where they have been examined. Inherent sequence or cis-elements located in mRNA termini and 5' and 3' UTRs likewise influence post-transcriptional gene regulation. We delineate the relative importance of the 5' m7G-cap, the 3' poly(A) tail, and Internal Ribosome Entry Sites (IRESs) in miRNA-mediated repression. mRNA targets must contain a m7G-cap to be repressed, are repressed to a greater extent when containing a poly(A) tail, and are not precluded from repression when translating via an IRES. </p><p> miRNAs can inhibit translation and / or induce mRNA decay. While the core effector proteins are established, mechanistic details of how miRNAs interfere with mRNA translation and stability remain elusive. Contrary to the repressive effects of miRNAs, the poly(A)-binding protein (PABP) (through binding to the poly(A) tail and eIF4G) can increase both translation and mRNA stability independently. We elucidate a functional role for the PABP in miRNA repression; manipulation of `active' PABP levels affects repression conversely in part by inhibiting miRNA-induced deadenylation. Furthermore, we find that expression changes in the PABP binding partner PABP interacting protein 2 (Paip2) modulates both miRNA repression and PABP protein complex formation. Additionally, we establish Paip2 as a bona fide miR-128 target, and demonstrate miR-128 de-repression of non-miR-128 target mRNAs through this targeting event.</p> / Dissertation

L?????tude qui vous est pr??sent??e dans ce m??moire est r??alis??e chez l???organisme Schizosaccharomyces pombe et elle porte sur la caract??risation de la prot??ine Nab2, une prot??ine liant les queues poly(A) ainsi que sur son implication dans la r??gulation g??nique. Tout d???abord, il faut savoir qu???il existe quatre modes de r??gulation (transcriptionnel, post-transcriptionnel, traductionnel et post-traductionnel) dans lesquels interviennent diff??rents types de prot??ines, en particulier des prot??ines liant les queues poly(A) des ARN, aussi connues sous le nom de PABPs. Ces prot??ines reconnaissent l???ARN ?? l???aide de diff??rents domaines de liaison et elles se subdivisent en deux cat??gories : les PABPs nucl??aires ou cytoplasmiques, repr??sent??es respectivement par PABPN1 et PABPC1 chez les mammif??res. Comprendre la fonction des PABPs rev??t un int??r??t particulier puisqu???elles sont impliqu??es ?? diff??rents stades de la r??gulation g??nique. Des maladies ont aussi ??t?? associ??es ?? deux PABPs nucl??aires humaines, PABPN1 et ZC3H14, mais aucune association entre leur fonction r??ciproque et la maladie n???a pu ??tre ??tablie. Une des fa??ons de comprendre leur r??le est d?????tudier celui de leurs orthologues respectifs. Chez la levure ?? fission, un orthologue de PABPN1 a ??t?? caract??ris?? et il s???agit de Pab2. S. pombe poss??de cependant une seconde PABP nucl??aire, Nab2, qui est caract??ris??e dans ces travaux. Des m??thodes in vivo et in vitro ont ??t?? utilis??es afin de confirmer le statut de la prot??ine, ?? savoir qu???il s???agit bel et bien d???une PABP nucl??aire et que celle-ci est non essentielle. L???identification de partenaires prot??iques de Nab2 par spectrom??trie de masse a aussi permis de relier Nab2 avec des processus de r??gulation g??nique tels que l?????pissage et la d??gradation. Puisque Pab2 est aussi associ??e ?? des fonctions en lien avec la d??gradation, il est possible de faire un parall??le entre ces deux prot??ines et de supposer qu???elles interagissent ensemble. La deuxi??me partie de ces travaux porte donc sur l?????tude de la relation fonctionnelle entre Nab2 et Pab2 et elle a permis de montrer un m??canisme de r??gulation opportuniste bas?? sur la liaison de la cible ARN par l???une ou l???autre de ces PABPs. En effet, l?????tude de la r??gulation du g??ne mod??le RPL30-2 indique que Nab2 et Pab2 ont des r??les oppos??s puisqu???ils sont respectivement des r??gulateurs positifs et n??gatifs de l???expression de son transcrit.

?4 phosphoprotein (also known as IGBP1) is a component of the mammalian target-of-rapamycin (mTOR) pathway that controls the initiation of translation and cell-cycle progression in response to nutrients and growth factors. Aberrant signaling of the mTOR pathway has been reported in many cancers. ?4 interacts with the catalytic subunit of protein phosphtase 2A (PP2Ac) to mediate the dephosphorylation of eukaryotic initiation factor 4E-binding protein1 (4E-BP1) and p70S6 kinase (p70S6K). Our laboratory has reported that EDD E3 ubiquitin ligase (EDD/UBR5) and poly(A)-binding protein (PABP) are novel binding partners of ?4 phosphoprotein. In the present study, the interaction of EDD and PABP with ?4 was confirmed in human MCF-7 breast cancer and African green monkey COS-1 kidney cell lines, using immunoprecipitation and immunoblotting (IP/IB) analysis. However, co-IP of total MCF-7 cell lysates with anti-EDD antibodies revealed that EDD does not physically interact with PP2Ac. Several ?4 deletion constructs, that contained either the N-terminal or C-terminal regions of ?4, were transfected into MCF-7 and COS-1 cells. Co-IP studies with anti-EDD and PABP antibodies revealed that EDD interacts with the C-terminal region of ?4 whereas PABP, like PP2Ac, binds to the N-terminal region. EDD and PABP were found to interact with ?4 in both quiescent and actively growing cells. EDD is known to ubiquitinate poly(A)-binding protein-interacting protein 2 (Paip2), targeting it for proteosomal degradation. Paip2 is an antagonist of PABP activity. When ?4 levels in MCF-7 cells were knocked down using small interfering RNA (siRNA), there was no effect on EDD protein levels. There was also no effect on Paip2 levels, indicating that ?4 is not involved in the EDD- mediated ubiquitination of Paip2. Knockdown of EDD gene expression by siRNA did not alter mono-ubiquitination of ?4, indicating that ?4 is not a substrate of EDD. However, knockdown of EDD gene expression decreased poly-ubiquitination of PP2Ac and increased the overall cellular levels of PP2Ac, suggesting PP2Ac as a novel substrate of EDD. The present study suggests a potential role for ?4 in PABP-mediated initiation of mRNA translation. Furthermore, this study suggests a role for EDD in regulating PP2Ac levels through its interaction with ?4. In summary, the ?4 partners EDD, PABP and PP2Ac interact at specific regions of ?4. PP2Ac, but not ?4, is a substrate of EDD. The interaction of PABP with ?4 suggests a potential role for ?4 in PABP-mediated initiation of mRNA translation.

Gene expression at the level of mRNA translation is critical for cells to respond to external signals; it allows changes in protein synthesis without triggering transcription of a new set of genes. Control of mRNA translation and stability is important in several cellular processes including cell growth and differentiation. Thus regulation of the cellular machinery involved in mRNA translation is crucial. Poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) are important members of the cellular mRNA translation machinery, the mRNAs that encode these proteins belong to the terminal oligo pyrimidine tract (TOP) containing family. Translation of the TOP mRNAs is regulated by growth signals and usually codes for several proteins involved in mRNA translation. Our laboratory has previously reported up regulation of PABP1 mRNA translation during recovery from heat shock. It was also shown that the terminal oligopyrmidine tract (TOP) cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation; however the mechanism for achieving this is unknown. In the studies reported here, we showed that translation of eEF1A and RPS6 expression was similarly enhanced during recovery from heat shock. Analyses of samples of in vivo cross linked RNA– protein complexes, immunoprecipitated by ZNF9 antibody, for the presence of specific mRNAs showed that the cellular nucleic acid binding protein ZNF9 binds not only to TOP mRNAs but also mRNA that lack the TOP element such as to β-actin mRNA. To elucidate the mechanism of activation of TOP mRNA translation, as a candidate trans acting factor, siRNA was used to deplete the cellular level of ZNF9 from heat shocked HeLa cells to examine its potential role in stimulation of TOP mRNA translation during recovery from heat shock. Results show that the knock down of ZNF9 disallowed the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect on the constitutive expression of either β-actin or PABP1, eEF1A and RPS6 in exponentially growing HeLa cells. These results suggest that binding of ZNF9 to TOP mRNAs per se does not inhibit translation, but more likely it acts as a general facilitator of mRNA translation. It is possible that modification of the interaction between ZNF9 with other unknown protein factors is responsible for its preferred effect on all three TOP mRNAs studied here. Additionally, results also suggest that a different TOP sequences amongst the observed TOP mRNAs responds similarly to ZNF9.

No description available.

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 voies / The 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 regulation

Les microARN (miARN) sont de petits ARN non-codants qui contrôlent l’expression génique, en s’hybridant, le plus souvent, de manière imparfaite à des séquences spécifiques qui se trouvent généralement dans la région non traduite en 3' (3’UTR) de transcrits cibles. Les miARN guident sur l’ARN messager (ARNm) un complexe protéique appelé RNA-induced Silencing Complex (RISC), composé des protéines Argonaute et TNRC6, qui perturbe l’initiation de la traduction et provoque la déadénylation et la dégradation du transcrit. C'est l’interaction entre le RISC et le complexe de pré-initiation de la traduction 43S (composé de la petite sous-unité ribosomique 40S et des facteurs d’initiation associés) qui entraîne la répression traductionnelle de l’ARNm ciblé. Des résultats récents ont démontré que le RISC perturbe le balayage de la région non traduite en 5’ (5’UTR) par le ribosome, étape qui requiert la présence de 2 facteurs d’initiation qui sont eIF4F qui reconnaît et lie la coiffe ainsi que la protéine PABP, fixée le long de la queue poly(A). Toutefois, les miARN peuvent également induire la stimulation de la traduction des transcrits cibles dans les cellules quiescentes, dans un lysat d’embryons de drosophiles ou encore dans les ovocytes de Xénope. Le mécanisme moléculaire de stimulation de l’expression par les miARN est encore mal connu mais requiert l’absence de queue poly(A) en 3’ des ARN cible et de TNRC6 au sein du complexe RISC. Le Virus de l’Hépatite C (VHC) possède en 5’ de son ARNg un site d’entrée interne du ribosome (IRES) qui recrute la petite sous-unité ribosomique 40S, sans nécessiter la reconnaissance de la coiffe par eIF4F, ni la protéine PABP, ni le balayage de la 5’UTR par le ribosome. Ces caractéristiques singulières nous ont conduits à rechercher l'impact du complexe RISC fixé en 3’ de l’ARNm sur l’initiation de la traduction du VHC. Pour cela, nous avons utilisé des transcrits contenant l'IRES du VHC en 5' et des sites d’hybridation du miARN let-7 en 3’. Ces ARNm ont ensuite été transfectés dans des lignées cellulaires hépatocytaires, ou non. A notre grande surprise, nous avons observé que la fixation du miARN let-7 sur la région 3' du transcrit stimulait fortement l’expression dirigée par l’IRES de VHC. Toutefois, l’augmentation de l’expression n’est pas due à la stabilisation du transcrit mais bien à une hausse significative de la synthèse protéique indépendamment d’un quelconque effet de miR-122. En utilisant d’autres IRES dites 'HCV-like', nous avons pu confirmer ces résultats et démontrer que, l’ajout d’une queue poly(A) en 3’ du transcrit, capable de fixer la PABP, annule cet effet stimulateur suggérant que l’absence de cette protéine est nécessaire pour que le complexe RISC stimule la traduction du VHC. / MicroRNAs (miRNAs) are small non coding RNAs which control gene expression by recognizing and hybridizing to a specific sequence generally located in the 3’UTR of targeted messenger RNA (mRNA). miRNAs serve as a guide for the RNA-Induced Silencing Complex (RISC) that is composed by, at least, the Argonaute proteins and TNRC6. Recent studies have suggested that translation inhibition occurs first and is then followed by deadenylation and degradation of the targeted transcript. The miRNA-induced inhibition of protein synthesis occurs at the level of translation initiation during the ribosomal scanning step and it requires the presence of both the initiation factor eIF4G and the poly(A) Binding Protein (PABP). In this process, the RISC interacts with both PABP and 43S pre-initiation complex (composed by initiation factors and ribosome) and it results in the disruption of linear scanning of the ribosome along the 5’ Untranslated Region (5’UTR). In some specific cases, the binding of miRNAs to their target sequences can upregulate translation initiation. This has notably been demonstrated in G0 quiescent cells, drosophila embryos and Xenopus oocytes. Although the molecular mechanism by which upregulation occurs remains to be precisely determined, it appears that the absence of a poly(A) tail and the lack of availability of the TNRC6 proteins are amongst the major determinants. In the particular case of the Hepatitis C Virus (HCV), the genomic RNA is uncapped and non polyadenylated and harbors an Internal Ribosome Entry Site (IRES) which directly binds to the ribosome with no need for cap-recognition, PABP binding and ribosome scanning. These peculiar features of the HCV IRES prompted us to investigate how viral translation can be regulated by the miRNA machinery. In order to do that, we have used a mRNA that contains the HCV IRES in 5’ and 4 let-7 binding sites in its 3’ extremity. To most of our surprise, we have observed a strong stimulation of the expression of the HCV IRES when the construct is bearing the let-7 sites. This effect is not due to any interference with the miR-122 binding sites although the magnitude of stimulation reached the same level. Our data show that it is the presence of the RISC on the 3' end of the transcript that can stimulate internal ribosome entry at the 5' end. By using other HCV-like IRESes, we could confirm these data and further showed that the absence of a poly(A) tail was an absolute requirement for the stimulation to occur. These effects are not due to an increase of mRNA stability and are rather exerted at the level of translation.