Abstract
Most living creatures have a circadian rhythm that is generated by a precisely regulated transcriptional-translational feedback loop of clock genes. Brain and muscle ARNT-like 1 (BMAL1) is one of the core clock genes and transcription factors that represents a positive arm of this autoregulatory circadian clock system. Despite the indispensable role of BMAL1 in the circadian rhythm, the molecular mechanisms underlying translational control of BMAL1 are largely unknown. Here, using murine NIH-3T3 cells, gene constructs, and a variety of biochemical approaches, including RNAi- and luciferase reporter gene-based assays, along with immunoblotting, in vitro transcription, quantitative real-time PCR, and real-time bioluminescence experiments, we show that translation of Bmal1 is negatively regulated by an RNA-binding protein, heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Interestingly, we found that hnRNP Q rhythmically binds to a specific region of the Bmal1 mRNA 5' UTR and controls its time-dependent expression. Moreover, we demonstrate that knockdown of hnRNP Q modulates BMAL1 protein oscillation amplitude without affecting mRNA rhythmic patterns. Furthermore, hnRNP Q depletion increases the mRNA oscillation amplitudes of BMAL1-regulated target genes. Together, our results suggest that hnRNP Q plays a pivotal role in both Bmal1 translation and BMAL1-regulated gene expression.
Highlights
Most living creatures have a circadian rhythm that is generated by a precisely regulated transcriptional–translational feedback loop of clock genes
Of the three siRNAs, only heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) knockdown caused an increase in the Bmal1 protein level (Fig. 1A)
To determine whether this result comes from the up-regulated Bmal1 mRNA level or stability, we analyzed the Bmal1 mRNA level and decay kinetics after hnRNP Q depletion
Summary
Period; Cry, cryptochrome; hnRNP, heterogeneous nuclear ribonucleoprotein; Fluc, firefly luciferase; qPCR, quantitative PCR; CDS, coding sequence(s); IP, immunoprecipitation; TBS, tris-buffered saline. We have demonstrated previously that various RNAbinding proteins have an important function in translational control of several clock genes [25,26,27,28,29,30]. We hypothesized that a specific RNA-binding protein may have a critical role in Bmal mRNA translation. We determine that an RNA-binding protein, hnRNP Q, has a critical role in translational control of Bmal mRNA. We show that hnRNP Q binds to a specific region of the Bmal mRNA 5Ј UTR and negatively regulates Bmal mRNA translation. We demonstrate that this translation is controlled by rhythmic interaction between hnRNP Q and Bmal mRNA. HnRNP Q–mediated translational repression of Bmal increases the mRNA oscillation amplitude of Bmal target genes, suggesting that this translational control is critical for the circadian clock system
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