講演要旨
MicroRNAs post-transcriptionally regulate gene expression and contribute to numerous life processes, including circadian rhythms. However, whether miRNAs contribute to zebrafish circadian regulation has not yet been investigated. We showed that mature miR-219-5p, and its three pre-miRNAs, mir-219-1, mir-219-2, and mir-219-3, are rhythmically expressed primarily in Tectum opticum (TeO), Corpus cerebelli (CCe), and Crista cerellaris (CC) of the zebrafish brain. While mir-219-1 and mir-219-2 are regulated by the circadian clock through the E-like box, mir-219-3 is regulated by light via the D-box. Deleting mir-219-1, mir-219-2, or mir-219-3 individually or knocking down miR-219-5p all results in a shortened period of locomotor rhythms and up-regulation of bmal1b. RIP assays with Ago2 and miRNA pull-down assays show that miR-219-5p binds to bmal1b in the RISC. Cell transfection and in Vivo assays show that miR219-5p inhibits bmal1b through binding to its 3'UTR. Together, our findings demonstrate that mir-219-1, mir-219-2, and mir-219-3 are controlled directly by the circadian clock; and in turn, miR-219-5p contributes to circadian regulation by targeting bmal1b, highlighting a miR-219-5p-bmal1b negative feedback loop in the zebrafish circadian circuit.
The circadian clock generates and maintains ~24-hour oscillations in almost all organs. The testis, however, remains mysterious without a clear understanding of its circadian functions. We conducted time-series transcriptome analysis and revealed more than 1,000 rhythmically expressed genes in the zebrafish and mouse testes, respectively. Canonical circadian clock genes are rhythmically expressed in Sertoli cells and regulate retinoic acid (RA) production, evidenced by their co-expression with RA synthesis genes in single Sertoli cells. Genetic and pharmacological manipulations and temporal desynchronization revealed that the circadian clock-regulated RA signaling synchronizes spermatogonial differentiation via zbtb16a and promotes fertilization via izumo1 in zebrafish. Our findings indicate that the testicular circadian clock contributes to reproduction in a cell-specific manner through RA signaling, highlighting circadian roles in male fertility.