Viscardi Marcus J, Sehgal Enisha, Arribere Joshua A
Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Cruz, Santa Cruz, California 95064, USA.
Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Cruz, Santa Cruz, California 95064, USA;
Genome Res. 2025 Jun 2;35(6):1337-1348. doi: 10.1101/gr.280046.124.
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 biochemical understanding of NMD and hampering therapeutic efforts to control NMD. Here, we modify and deploy single-molecule nanopore mRNA sequencing to clarify the route by which NMD targets are attacked in an animal. We obtain single-molecule measures of splicing isoform, cleavage state, and poly(A) tail length. We observe robust endonucleolytic cleavage of NMD targets in vivo that depends on the nuclease SMG-6. We show that NMD-target mRNAs experience deadenylation and decapping, similar to that of normal mRNAs. Furthermore, we show that a factor (SMG-5) that historically was ascribed a function in deadenylation and decapping 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 we suggest that deadenylation and decapping are normal parts of mRNA (and NMD target) maturation and decay rather than unique facets of NMD.
含有提前终止密码子的信使核糖核酸（mRNA）可产生截短的、具有显性作用的蛋白质，从而对细胞造成损害。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的生化理解，并阻碍了控制NMD的治疗努力。在此，我们改进并应用单分子纳米孔mRNA测序技术，以阐明在动物体内NMD靶标被攻击的途径。我们获得了剪接异构体、切割状态和聚腺苷酸（poly(A)）尾长度的单分子测量结果。我们观察到在体内NMD靶标存在强烈的核酸内切酶切割，这依赖于核酸酶SMG-6。我们表明，NMD靶标mRNA会经历去腺苷酸化和脱帽过程，这与正常mRNA类似。此外，我们表明，一个在历史上被认为在去腺苷酸化和脱帽过程中起作用的因子（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进行核酸内切酶切割来降解动物体内的NMD靶标，并且我们认为去腺苷酸化和脱帽是mRNA（以及NMD靶标）成熟和降解的正常组成部分，而非NMD的独特方面。
