Big regulation comes in small packages. microRNAs (miRNAs) are 19-24 nucleotide non-coding RNAs that suppress gene expression by blocking protein translation or degrading mRNA. Now Cheng et al. report two miRNAs involved in generating circadian rhythms in a recent article in Neuron.
In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is necessary for circadian rhythms. Here, the expression of several clock genes rhythmically rises and falls throughout the day. Heterodimers of the transcription factors CLOCK and BMAL1 drive expression of period (Per1 and Per2) and cryptochrome (Cry1 and Cry2). Then PER and CRY proteins feed back to inhibit CLOCK and BMAL1.
Clock gene expression is sensitive to light, and light induces CREB-dependent gene expression in the SCN. The authors searched for miRNAs with CREB response elements by serial analysis of chromatin occupancy. Antibodies to CREB and CLOCK immunoprecipitated miR-132 and miR-219, respectively. In cultured cells, CLOCK and BMAL1 increased miR-219 but not miR-132 transcription. In addition to CREB response elements, the miR-219 promoter contained E box response elements for CLOCK and BMAL1. The adenylate cyclase agonist forskolin induced miR-132 transcription, consistent with CREB regulation.
In the mouse SCN, both miR-219 and miR-132 showed rhythmic expression that peaked during the subjective day. However, miRNA expression did not vary in the cortex of wild-type mice or in the SCN of mCry1/mCry2 knockout mice, suggesting that expression of miR-219 and miR-132 is regulated by the SCN clock. A 15-minute light pulse during the night increased expression of miR-132 but not miR219. The MEK1/2 inhibitor U0126 blocked the light-induced increase in miR-132, suggesting that miR-132 is important in light-activated gene expression mediated by MAP kinase and CREB.
miR-219 regulates the length of circadian periods (days), and miR-132 regulates resetting of the clock. The authors reduced miRNA expression with complementary oligoribonucleotides called antagomirs. Mice treated intraventricularly with miR-219 antagomirs had longer circadian periods relative to control mice. In contrast, mice treated with miR-132 antagomirs reset their circadian clocks faster than did control mice in response to a light stimulus.
The authors identified several mechanisms that might mediate miRNA clock regulation. They found 114 targets for miR-219 and 265 for miR-132 with bioinformatics software, several of which were expressed in the SCN. miR-132 increased and miR-219 decreased the excitability of cultured cortical neurons treated with glutamate or NMDA. Both miR-219 and miR-132 increased CLOCK and BMAL1-induced expression of a Per1-luciferase reporter in vitro, and miR-132 antagomirs reduced the light-induced expression of SCN Per1 protein in vivo.
Perhaps in addition to regulating circadian rhythms, miRNAs are important in generating other biological rhythms important in reproductive and seasonal behaviors.