Molecular mechanisms of msl2 mRNA translational regulation

Regulation of msl2 translation is a key step in the modulation of X-chromosome dosage compensation. MSL2 is the limiting subunit of the dosage compensation complex, an assembly that promotes hyper-transcription of the single male X-chromosome to equalize expression of X-linked genes between males and females. In females, dosage compensation must be repressed for viability, and this is achieved in large part by translational repression of msl2 mRNA. The female-specific protein Sex-lethal (SXL) binds to both untranslated regions (UTRs) of the msl2 transcript to inhibit two steps of translation initiation: SXL bound to the 3’ recruits the co-factor UNR and inhibits ribosomal recruitment; SXL bound to the 5’ UTR inhibits ribosomal scanning by promoting the recognition of an upstream AUG. In the lab, we recently found that eIF3d is a target of the 3’UTR repressor complex. In this thesis, we show that eIF3d can be recruited to the mRNA even in the absence of a cap structure by virtue of its binding to msl2 5’ UTR. Our results suggest that recruitment of this factor to the mRNA by multiple routes may sensitize translation of msl2 to inhibition of eIF3d. In addition, we have identified residues of SXL important for 5’ UTR, but not 3’ UTR, -mediated repression. Analysis of SXL variants with mutations in these residues has led us to identify two factors as likely mediators of inhibition via the 5’ UTR. Interestingly, the location of these factors in the ribosome allows us to propose an integrated model for translational repression that explains coordinated inhibition of ribosome recruitment and scanning by SXL.La regulación de la traducción de msl2 es un paso crucial en la modulación de la compensación de la dosis del cromosoma X. MSL2 es la subunidad limitante del complejo de compensación de dosis génica, un complejo que promueve la hiper-transcripción del único cromosoma X en machos para igualar la expresión de sus genes a la de hembras. La viabilidad de las hembras require que la compensación de dosis esté reprimida, y esto se consigue en gran parte por la represión de la traducción del ARNm que codifica para MSL2. La proteína específica de hembras Sex-lethal (SXL) se une a ambas regiones no traducidas (UTRs) del mensajero para inhibir dos etapas del inicio de la traducción: SXL unido al 3’ UTR recluta al co-factor UNR e inhibe el reclutamiento del ribosoma; SXL unido al 5’ UTR inhibe el escaneo del ribosoma al promover el reconocimiento de un uAUG. En el laboratorio, identificamos recientemente eIF3d como una diana del complejo represor unido al 3’ UTR. En esta tesis mostramos que eIF3d puede ser reclutado al ARNm incluso en ausencia del cap, gracias a su unión al 5’ UTR. Nuestros resultados sugieren que el reclutamiento de este factor al ARNm a través de varias rutas sensibiliza la traducción de msl2 a la inhibición de eIF3d. Además, hemos identificado resíduos de SXL importantes para la represión mediada por el 5’ pero no por el 3’ UTR. El análisis de variantes de SXL con mutaciones en estos resíduos nos ha permitido identificar dos factores como posibles mediadores de la inhibición por el 5’ UTR. Curiosamente, la localización de estos factores en el ribosoma nos permite proponer un modelo integrado para la inhibición coordinada del reclutamiento y escaneo del ribosoma por SXL

Molecular mechanisms of msl2 mRNA translational regulation

Regulation of msl2 translation is a key step in the modulation of X-chromosome dosage compensation. MSL2 is the limiting subunit of the dosage compensation complex, an assembly that promotes hyper-transcription of the single male X-chromosome to equalize expression of X-linked genes between males and females. In females, dosage compensation must be repressed for viability, and this is achieved in large part by translational repression of msl2 mRNA. The female-specific protein Sex-lethal (SXL) binds to both untranslated regions (UTRs) of the msl2 transcript to inhibit two steps of translation initiation: SXL bound to the 3’ recruits the co-factor UNR and inhibits ribosomal recruitment; SXL bound to the 5’ UTR inhibits ribosomal scanning by promoting the recognition of an upstream AUG. In the lab, we recently found that eIF3d is a target of the 3’UTR repressor complex. In this thesis, we show that eIF3d can be recruited to the mRNA even in the absence of a cap structure by virtue of its binding to msl2 5’ UTR. Our results suggest that recruitment of this factor to the mRNA by multiple routes may sensitize translation of msl2 to inhibition of eIF3d. In addition, we have identified residues of SXL important for 5’ UTR, but not 3’ UTR, -mediated repression. Analysis of SXL variants with mutations in these residues has led us to identify two factors as likely mediators of inhibition via the 5’ UTR. Interestingly, the location of these factors in the ribosome allows us to propose an integrated model for translational repression that explains coordinated inhibition of ribosome recruitment and scanning by SXL.La regulación de la traducción de msl2 es un paso crucial en la modulación de la compensación de la dosis del cromosoma X. MSL2 es la subunidad limitante del complejo de compensación de dosis génica, un complejo que promueve la hiper-transcripción del único cromosoma X en machos para igualar la expresión de sus genes a la de hembras. La viabilidad de las hembras require que la compensación de dosis esté reprimida, y esto se consigue en gran parte por la represión de la traducción del ARNm que codifica para MSL2. La proteína específica de hembras Sex-lethal (SXL) se une a ambas regiones no traducidas (UTRs) del mensajero para inhibir dos etapas del inicio de la traducción: SXL unido al 3’ UTR recluta al co-factor UNR e inhibe el reclutamiento del ribosoma; SXL unido al 5’ UTR inhibe el escaneo del ribosoma al promover el reconocimiento de un uAUG. En el laboratorio, identificamos recientemente eIF3d como una diana del complejo represor unido al 3’ UTR. En esta tesis mostramos que eIF3d puede ser reclutado al ARNm incluso en ausencia del cap, gracias a su unión al 5’ UTR. Nuestros resultados sugieren que el reclutamiento de este factor al ARNm a través de varias rutas sensibiliza la traducción de msl2 a la inhibición de eIF3d. Además, hemos identificado resíduos de SXL importantes para la represión mediada por el 5’ pero no por el 3’ UTR. El análisis de variantes de SXL con mutaciones en estos resíduos nos ha permitido identificar dos factores como posibles mediadores de la inhibición por el 5’ UTR. Curiosamente, la localización de estos factores en el ribosoma nos permite proponer un modelo integrado para la inhibición coordinada del reclutamiento y escaneo del ribosoma por SXL

Hrp48 and eIF3d contribute to msl-2 mRNA translational repression

Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. Here we identify Hrp48 as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. Our results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.Spanish Ministry of Economy and Competitiveness MINECO and the European Regional Development Fund (ERDF) [BFU2012-37135, BFU2015-68741, Consolider CSD2009-00080]; Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]; La Caixa Foundation (to M.G.-B.); Fondation pour la Recherche Médicale (FRM) (to A.G.). Funding for open access charge: Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2015-68741]