Essential role of 3′-untranslated region-mediated mRNA decay in circadian oscillations of mouse Period3 mRNA

Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, we focused on the 3'-untranslated region (3'-UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3'-UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3'-UTR-mediated mRNA decay on circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Interestingly, a stabilizing mutation of 3'-UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3'-UTR began to increase earlier than that in the presence of wild-type 3'-UTR. Consequently, luciferase mRNA with mutant 3'-UTR displayed oscillation patterns with a prolonged rising phase. Our results indicate that mPer3 3'-UTR- mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post- transcriptional control of circadian mRNA oscillations.