Department of Chemistry and Biochemistry, University of Bern, Bern 3012, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern 3012, Switzerland.
RNA. 2018 Apr;24(4):557-573. doi: 10.1261/rna.063719.117. Epub 2018 Jan 18.
The term "nonsense-mediated mRNA decay" (NMD) originally described the degradation of mRNAs with premature translation-termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%-10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5-SMG7 or SMG5-PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5'-3' exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.
“无意义介导的 mRNA 降解”（NMD）一词最初描述的是具有过早翻译终止密码子（PTC）的 mRNA 的降解，但最近其含义已扩展为一种翻译依赖性的基因表达后转录调控因子，影响所有 mRNA 的 3%-10%。NMD 靶 mRNA 的降解涉及哺乳动物细胞中的外切核酸酶和内切核酸酶途径。虽然后者是由内切核酸酶 SMG6 介导的，但前者途径已被报道需要 SMG5-SMG7 或 SMG5-PNRC2 复合物与 UPF1 结合。然而，这些外切核酸酶途径的存在、优势和机制细节存在分歧。因此，我们通过使用功能测定和相互作用图谱组合，研究了 NMD 中 mRNA 降解的可能外切核酸酶模式，以检查 UPF1、SMG5、SMG7 和 PNRC2 的作用。与之前的工作一致，我们检测到 SMG5 和 SMG7 之间的相互作用，并且该复合物在 NMD 中也具有功能需求。相比之下，我们没有发现 SMG5 和 PNRC2 之间存在物理或功能相互作用的证据。相反，我们表明 UPF1 与 PNRC2 相互作用，并且它在牵引测定中触发报告转录物的 5'-3'外切核酸酶降解。PNRC2 主要与脱帽因子相互作用，其敲低不影响 NMD 报告物的 RNA 水平。我们得出结论，PNRC2 可能是一种重要的 mRNA 脱帽因子，但它似乎不是 NMD 所必需的。
