Essential Role of 3′-Untranslated Region-mediated mRNA Decay in Circadian Oscillations of Mouse Period3 mRNA

Abstract

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.Circadian rhythms, defined as daily behavioral and physiological oscillations, have been observed in a variety of organisms. These oscillations are driven by a self-sustained timekeeping system, the intracellular clock (1, 2). Over the past several years, great effort has been focused on understanding the molecular mechanisms that underlie the circadian clock and indispensable components, called clock genes, have been elucidated (3-8). In addition, it was revealed that the intracellular molecular clock consists of interacting positive and negative transcriptional and translational feedback loops that involve these components (9 -12).Daily oscillations in protein and/or mRNA levels are central features of clock genes (1, 12). As for the underlying mechanism of mRNA cycling, a number of studies have revealed that mRNA oscillations largely depend on transcriptional regulation (10, 13-17). In Drosophila, nevertheless, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations. First, despite the absence of rhythmic transcription, period (per) mRNA showed circadian cycling in transgenic flies carrying promoter-less per gene (18). Second, results of a nuclear run-on assay suggested that post-transcriptional regulation is responsible for mRNA cycling (19).In mammals, however, much less is known about the relevance of post-transcriptional control to the circadian oscillation...

CIRCADIAN CLOCK ASSOCIATED1 Transcript Stability and the Entrainment of the Circadian Clock in Arabidopsis

“…The combination of these two events, induction of transcription and transcript degradation, will result in a sharper peak of CCA1 mRNA accumulation, allowing a more precise detection of early morning, irrespective of seasonal changes. Consistent with the idea that mRNA stability is important for the pattern of oscillator transcript accumulation, experiments carried out in mice have shown that increasing the stability of a chimeric construct based on Period3, an oscillator gene, significantly broadens the peak of expression (Kwak et al, 2006).…”

Rhythmic Interaction between Period1 mRNA and hnRNP Q Leads to Circadian Time-Dependent Translation

“…The circadian oscillation of NIH 3T3 cells was synchronized by treatment with 100 nM dexamethasone. After 2 h, the medium was replaced with complete medium (4,29,46,47). To block the translation system, NIH 3T3 cells were treated with 20 nM rapamycin or 100 g/ml cycloheximide (CHX) and then harvested at the indicated times.…”

“…These regulatory steps must be coordinated properly for the fine-tuning of both amplitude and 24-h periodicity. In particular, posttranscriptional regulation plays an important role, although its mechanism is less well understood (17,26,29,46,47). One of the core clock genes in mammals, Period1 (Per1), was originally identified as a structural homologue of the Drosophila melanogaster circadian clock gene per (42).…”

Post-transcriptional control of circadian rhythms

“…A later study demonstrated that an element in the 3Ј-UTR of Per1 promotes its instability, thereby contributing to this destabilization (Kojima et al, 2003). The 3Ј-UTRs of other clock genes, such as Drosophila Per, rat Aanat, and mouse Per2, Per3 and Cry1, also contribute to regulating their rhythmic expression, thereby leading to an accurate circadian period (Chen et al, 1998;Kim et al, 2005;Kim et al, 2007a;Kwak et al, 2006;Woo et al, 2010;Woo et al, 2009).…”