DDX6 decouples translational repression from RNA degradation of miRNA targets [Ribosome Profiling]

Translation and mRNA degradation are intimately connected, yet the mechanisms that regulate them are not fully understood. Here we examine the regulation of translation and mRNA stability in mouse embryonic stem cells (ESCs) and during differentiation. In contrast to previous reports, we found that transcriptional changes account for most of the molecular changes during ESC differentiation. Within ESCs translation level and mRNA stability are positively correlated. The RNA-binding protein DDX6 has been implicated in processes involving both translational repression and mRNA destabilization; in yeast DDX6 connects codon optimality and mRNA stability and in mammals DDX6 is involved in microRNA-mediated repression. We generated DDX6 KO ESCs and found that while there was minimal connection between codon usage and stability changes, the loss of DDX6 leads to the translational depression of microRNA targets. Surprisingly, the translational derepression of microRNA targets occurs without affecting mRNA stability. Furthermore, DDX6 KO ESCs share overlapping phenotypes and global molecular changes with ESCs that completely lack all microRNAs. Together our results demonstrate that the loss of DDX6 decouples the two forms of microRNA induced repression and emphasize that translational repression by microRNAs is underappreciated. Ribosome profilling was performed on mouse embryonic stem cells (ESCs) and Epiblast like cells (EpiLCs).

翻译与mRNA降解紧密关联，但其调控机制尚未完全阐明。本研究针对小鼠胚胎干细胞（mouse embryonic stem cells, ESCs）及其分化过程中的翻译与mRNA稳定性调控机制开展探究。与既往研究报道不同，我们发现转录水平变化是小鼠胚胎干细胞分化过程中绝大多数分子改变的主要成因。在小鼠胚胎干细胞内，翻译水平与mRNA稳定性呈正相关。RNA结合蛋白（RNA-binding protein）DDX6已被证实参与包括翻译抑制与mRNA去稳定在内的多种生物学过程：在酵母中，DDX6可关联密码子最优性与mRNA稳定性；在哺乳动物体内，DDX6则参与微小RNA（microRNA）介导的基因沉默过程。我们构建了DDX6基因敲除（knockout, KO）的小鼠胚胎干细胞，研究发现尽管密码子使用偏好性与稳定性变化之间仅存在微弱关联，但DDX6的缺失会导致微小RNA靶基因的翻译抑制增强。令人意外的是，微小RNA靶基因的翻译去抑制并未对其mRNA稳定性产生影响。进一步分析显示，DDX6基因敲除的小鼠胚胎干细胞与完全缺失所有微小RNA的胚胎干细胞存在重叠的表型特征与全局分子变化。综上，本研究结果表明DDX6的缺失使微小RNA诱导的两种基因抑制形式发生解耦，并揭示了微小RNA介导的翻译抑制此前被低估的重要性。本研究通过核糖体谱分析（ribosome profiling）检测了小鼠胚胎干细胞及上胚层样细胞（epiblast like cells, EpiLCs）的样本。