Viscardi Marcus J, Arribere Joshua A
Department of Molecular, Cellular and Developmental Biology, University of California at Santa Cruz, Santa Cruz, CA, USA.
bioRxiv. 2023 Sep 29:2023.09.29.560204. doi: 10.1101/2023.09.29.560204.
Premature stop codon-containing mRNAs can produce truncated and dominantly acting proteins that harm cells. Eukaryotic cells protect themselves by degrading such mRNAs via the Nonsense-Mediated mRNA Decay (NMD) pathway. The precise reactions by which cells attack NMD target mRNAs remain obscure, precluding a mechanistic understanding of NMD and hampering therapeutic efforts to control NMD. A key step in NMD is the decay of the mRNA, which is proposed to occur via several competing models including deadenylation, exonucleolytic decay, and/or endonucleolytic decay. We set out to clarify the relative contributions of these decay mechanisms to NMD, and to identify the role of key factors. Here, we modify and deploy single-molecule nanopore mRNA sequencing to capture full-length NMD targets and their degradation intermediates, and we obtain single-molecule measures of splicing isoform, cleavage state, and poly(A) tail length. We observe robust endonucleolytic cleavage of NMD targets that depends on the nuclease SMG-6 and we use the occurence of cleavages to identify several known NMD targets. We show that NMD target mRNAs experience deadenylation, but similar to the extent that normal mRNAs experience as they enter the translational pool. Furthermore, we show that a factor (SMG-5) that historically was ascribed a function in deadenylation, is in fact required for SMG-6-mediated cleavage. Our results support a model in which NMD factors act in concert to degrade NMD targets in animals via an endonucleolytic cleavage near the stop codon, and suggest that deadenylation is a normal part of mRNA (and NMD target) maturation rather than a facet unique to NMD. Our work clarifies the route by which NMD target mRNAs are attacked in an animal.
含有提前终止密码子的信使核糖核酸（mRNA）可产生对细胞有害的截短型显性作用蛋白。真核细胞通过无义介导的mRNA降解（NMD）途径降解此类mRNA来保护自身。细胞攻击NMD靶标mRNA的精确反应仍不清楚，这妨碍了对NMD的机制理解，并阻碍了控制NMD的治疗努力。NMD的一个关键步骤是mRNA的降解，据推测其通过几种相互竞争的模型发生，包括去腺苷酸化、核酸外切酶降解和/或核酸内切酶降解。我们着手阐明这些降解机制对NMD的相对贡献，并确定关键因子的作用。在此，我们改进并应用单分子纳米孔mRNA测序来捕获全长NMD靶标及其降解中间体，并获得剪接异构体、切割状态和聚腺苷酸（poly(A)）尾长度的单分子测量值。我们观察到NMD靶标的强大核酸内切酶切割依赖于核酸酶SMG - 6，并且我们利用切割的发生来鉴定几个已知的NMD靶标。我们表明，NMD靶标mRNA会经历去腺苷酸化，但程度与正常mRNA进入翻译池时的情况相似。此外，我们表明一个历史上被认为在去腺苷酸化中起作用的因子（SMG - 5），实际上是SMG - 6介导的切割所必需的。我们的结果支持一种模型，即NMD因子协同作用，通过在终止密码子附近的核酸内切酶切割来降解动物体内的NMD靶标，并表明去腺苷酸化是mRNA（和NMD靶标）成熟的正常部分，而不是NMD特有的一个方面。我们的工作阐明了动物体内NMD靶标mRNA被攻击的途径。
