RESUMEN
Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs-clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.
Asunto(s)
Ritmo Circadiano/genética , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Regiones no Traducidas 3'/metabolismo , Animales , Conducta Animal/fisiología , Sitios de Unión , Proteínas CLOCK , Línea Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Evolución Molecular , Expresión Génica , Cabeza/fisiología , Masculino , MicroARNs/biosíntesis , MicroARNs/genética , ARN Mensajero/metabolismo , Complejo Silenciador Inducido por ARN/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The efficacy of synaptic transmission depends, to a large extent, on postsynaptic receptor abundance. The molecular mechanisms controlling receptor abundance are poorly understood. We tested whether abundance of postsynaptic glutamate receptors (GluRs) in Drosophila neuromuscular junctions is controlled by microRNAs, and provide evidence that it is. We show here that postsynaptic knockdown of dicer-1, the endoribonuclease necessary for microRNA synthesis, leads to large increases in postsynaptic GluR subunit messenger RNA and protein. Specifically, we measured increases in GluRIIA and GluRIIB but not GluRIIC. Further, knockout of MiR-284, a microRNA predicted to bind to GluRIIA and GluRIIB but not GluRIIC, increases expression of GluRIIA and GluRIIB but not GluRIIC proportional to the number of predicted binding sites in each transcript. Most of the de-repressed GluR protein, however, does not appear to be incorporated into functional receptors, and only minor changes in synaptic strength are observed, which suggests that microRNAs primarily regulate Drosophila receptor subunit composition rather than overall receptor abundance or synaptic strength.
