Center for Integrative Genomics, University of Lausanne, Genopode, 1015 Lausanne, Switzerland.
Department of Dermatology, University Hospital of Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland.
Nucleic Acids Res. 2019 Jun 4;47(10):5193-5209. doi: 10.1093/nar/gkz261.
The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5' UTR features predictive of DENR dependence. Among core clock genes, we identified Clock as a DENR target. Using Clock 5' UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.
非规范起始因子 DENR 促进含有上游开放阅读框（uORF）的 mRNA 重新起始翻译。此外，DENR 耗竭缩短了小鼠成纤维细胞的生物钟周期，表明 uORF 利用和重新起始参与了时钟调控。为了在全转录组范围内鉴定 DENR 调控的翻译事件，特别是受该机制影响的特定核心时钟转录本，我们使用核糖体谱在 DENR 缺陷型 NIH3T3 细胞中进行了研究。我们发现了 240 个翻译速率改变的转录本，并使用线性回归分析提取了预测 DENR 依赖性的 5'UTR 特征。在核心时钟基因中，我们鉴定出 Clock 是 DENR 的靶基因。使用 Clock 5'UTR 突变体，我们通过 DENR 作用来调节 CLOCK 蛋白生物合成的特定 uORF 进行了作图。值得注意的是，这些实验揭示了一个替代的下游起始密码子，可能代表了真正的 CLOCK N 端。我们的研究结果为 uORF 介导的翻译调控提供了新的见解，这种调控可以调节哺乳动物生物钟和整体基因表达。
