Elucidation of posttranscriptional regulation on circadian rhythm affected by proteins binding to clock genes 3′UTR

DoctorThe mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, a master pacemaker, but also throughout the peripheral tissues. Circadian mRNA oscillations are the main feature of core clock genes. Among them, period 2 (per2) and cryptochrme 1 (cry1) are key components in negative-feedback regulation, showing robust oscillation in both mRNA and protein, as well as their protein heterodimer actively translocate from cytosol to nucleus for the feedback inhibition. However, little is known about post-transcriptional regulation of these genes. During the course of degree, I have been focused on the role of 3′ untranslated region (UTR)-dependent mRNA decay involved in the regulation of circadian oscillation of per2 or cry1 mRNA. I could successfully identify both responsible cis-acting 3′UTR elements (CAEs) as binding flatforms and trans-acting factors (TAFs) as functional players for respective genes mRNA stability and degradation kinetics. Interestingly, during the circadian oscillations of per2 or cry1, cytoplasmic fraction of TAFs showed reciprocal expression profiles compared with respective mRNAs implying their action in cytoplasm. In addition, their peak amplitudes were modulated when TAFs were reduced by RNAi. Because circadian genes have been also found to have a physiological role in the cell cycle or the tumor suppression, elucidating precise molecular mechanism of these genes is important. This thesis on the regulation of per2 and cry1 proposes that post-transcriptional mRNA decay mediated by TAFs is a fine-tuned regulatory mechanism that includes dampening-down effects during circadian mRNA oscillations. The mRNA degradation control is a fundamental step in gene expression regulation. Eukaryotic mRNA degradation mechanisms including mRNA surveillance pathways such as nonsense-mediated mRNA decay (NMD), or nonstop decay (NSD) are mediated by cytoplasmic exosome which is a multi-ribonuclease complex. Exosome components and their interaction are well characterized in yeast. In this research, we speculated the functional protein associating with exosome and target mRNAs. Guanosine triphosphates binding protein 1 (GTPBP1) was identified as one of proteins bound to rat Aanat mRNA, and interestingly it showed nocturnal expression profile in pineal gland. For other class of mRNAs, like Periods or Tnf-α, GTPBP1 binding was also observed. Overexpression of GTPBP1 accelerates target mRNA decay and reduction by RNA interference (RNAi) stabilizes them. It has putative GTP binding domain like other G-proteins or ski7p which is well known core factor of exosome mediated mRNA decay in yeast. Experiments to examine protein interaction or mRNA decay using guanine-nucleotide analogues showed mRNA degradation mechanism mediated by GTPBP1 is modulated by its GTPase activity. Moreover, in comparison with wildtype mice, GTPBP1 knockout model showed not only enhanced level of Aanat mRNA in pineal gland during night but also retarded degradation of cytokine mRNA, such as Tnf-α in lipopolysaccharide (LPS) induced splenocytes. This thesis on the regulation of mRNA degradation suggest that GTPBP1 should be a key regulator and adaptor for exosome mediated mRNA degradation

Post-transcriptional Regulation in Circadian Mouse Period3 Expression

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Circadian Post-transcriptional Control of mouse Period 3: Cooperative Function of 5' and 3' Untranslated Regions

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MicroRNA-185 regulates time-dependent expression ofmouse CRYPTOCHROME1

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Rhythmic interaction between Period1 mRNA and hnRNP Q leads to circadian time-dependent translation

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HnRNP Q Mediates a Rapid Induction of p53 via IRES-dependent Translation

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Circadian Amplitude of Cryptochrome 1 Is Modulated by mRNA Stability Regulation via Cytoplasmic hnRNP D Oscillation ▿ †

The mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, a master pacemaker, but also throughout the peripheral tissues. Its conserved molecular basis has been thought to consist of intracellular transcriptional feedback loops of key clock genes. However, little is known about posttranscriptional regulation of these genes. In the present study, we investigated the role of the 3′-untranslated region (3′UTR) of the mouse cryptochrome 1 (mcry1) gene at the posttranscriptional level. Mature mcry1 mRNA has a 610-nucleotide 3′UTR and mediates its own degradation. The middle part of the 3′UTR contains a destabilizing cis-acting element. The deletion of this element led to a dramatic increase in mRNA stability, and heterogeneous nuclear ribonucleoprotein D (hnRNP D) was identified as an RNA binding protein responsible for this effect. Cytoplasmic hnRNP D levels displayed a pattern that was reciprocal to the mcry1 oscillation. Knockdown of hnRNP D stabilized mcry1 mRNA and resulted in enhancement of the oscillation amplitude and a slight delay of the phase. Our results suggest that hnRNP D plays a role as a fine regulator contributing to the mcry1 mRNA turnover rate and the modulation of circadian rhythm

hnRNP Q mediates a rapid induction of p53 via IRES-dependent translation and induces apoptosis.

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hnRNP Q mediates a phase-dependent translation-coupled mRNA decay of mouse Period3.

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