RESUMEN
The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.
Asunto(s)
Proteínas Argonautas/metabolismo , Elementos Transponibles de ADN/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Empalme del ARN , Ribonucleoproteínas/metabolismo , Animales , Proteínas Argonautas/genética , ADN Complementario/metabolismo , Proteínas de Drosophila/genética , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Intrones/genética , Mutación , Ovario/citología , Ovario/metabolismo , Subunidades de Proteína/metabolismo , Ribonucleoproteínas/genéticaRESUMEN
Multiscale analysis of morphogenesis requires to follow and measure in real-time the in vivo behaviour of large numbers of individual cells over long period of time. Despite recent progress, the large-scale automated tracking of cells in developing embryos and tissues remains a challenge. Here we describe a genetic tool for the random and sparse labelling of individual cells in developing Drosophila tissues. This tool is based on the conditional expression of a nuclear HaloTag protein that can be fluorescently labelled upon the irreversible binding of a cell permeable synthetic ligand. While the slow maturation of genetically encoded fluorescent renders the tracking of individual cells difficult in rapidly dividing tissues, nuclear HaloTag proteins allowed for rapid labelling of individual cells in cultured imaginal discs. To study cell shape changes, we also produced an HaloTag version of the actin-bound protein LifeAct. Since sparse labelling facilitates cell tracking, nuclear HaloTag reporters will be useful for the single-cell analysis of fate dynamics in Drosophila tissues cultured ex vivo.
Asunto(s)
Rastreo Celular , Análisis de la Célula Individual , Animales , DrosophilaRESUMEN
Prions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI(+)] is an aggregated conformation of the translational release factor eRF3. [PSI(+)] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PSI(+)]-controlled expression remain unknown. We used ribosome profiling in isogenic [PSI(+)] and [psi(-)] strains to identify the changes induced by [PSI(+)]. We found 100 genes with stop codon readthrough events and showed that many stress-response genes were repressed in the presence of [PSI(+)]. Surprisingly, [PSI(+)] was also found to affect reading frame selection independently of its effect on translation termination efficiency. These results indicate that [PSI(+)] has a broader impact than initially anticipated, providing explanations for the phenotypic differences between [psi(-)] and [PSI(+)] strains.