Department of Chemistry and Biochemistry, University of Bern, CH-3012, Bern, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, CH-3012, Bern, Switzerland.
Nat Commun. 2020 Aug 17;11(1):4134. doi: 10.1038/s41467-020-17974-z.
Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.
无意义介导的 mRNA 降解 (NMD) 是一种依赖翻译的 RNA 降解途径，对于消除错误的和调节正常的 mRNA 非常重要。NMD 早期步骤的分子细节尚未完全了解，但先前的工作表明，NMD 的激活是核糖体在终止密码子 (TC) 处停滞的结果。为了验证这一假说，我们建立了一种基于人细胞裂解物的体外翻译偶联的足迹分析测定法，该方法允许监测报告 mRNA 上 TC 处的核糖体占有率。与流行的 NMD 模型相反，我们的体外系统显示 NMD 敏感和 NMD 不敏感报告 mRNA 的 TC 处的核糖体占有率相似。此外，核糖体谱分析显示，体内 NMD 敏感和 NMD 不敏感的内源性 mRNA 的 TC 处的核糖体密度相似。总之，这些数据表明 NMD 的激活并不伴随着核糖体在 TC 处的稳定停滞。
