Nonsense-mediated mRNA decay (NMD) is a cellular mRNA degradation mechanism that inhibits the expression of aberrant mRNAs harboring premature termination codons (PTCs). Recent progress in transcriptome-wide sequencing techniques has revealed that NMD also degrades approximately 5-30% of non-mutated cellular mRNAs in a way that can be regulated in response to various cellular signals. In mammals, NMD is governed by the central NMD factor UPF1, which is activated by phosphorylation after translation terminates at a nonsense codon that triggers NMD. We have found that immunoprecipitation using an antibody that is specific for phosphorylated UPF1 is a useful tool to define not only cellular NMD targets but also the nature of NMD decay intermediates and, thus, the process of NMD. To this end, we describe here a detailed protocol for what we call "NMD degradome sequencing" using high-throughput technology.

中文翻译：

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定义人类细胞中无义介导的 mRNA 衰变中间体

无义介导的 mRNA 衰减 (NMD) 是一种细胞 mRNA 降解机制，可抑制含有过早终止密码子 (PTC) 的异常 mRNA 的表达。转录组范围测序技术的最新进展表明，NMD 还可以通过响应各种细胞信号进行调节的方式降解大约 5-30% 的非突变细胞 mRNA。在哺乳动物中，NMD 受中央 NMD 因子 UPF1 控制，该因子在翻译终止于触发 NMD 的无义密码子后被磷酸化激活。我们发现，使用对磷酸化 UPF1 具有特异性的抗体进行免疫沉淀是一种有用的工具，不仅可以定义细胞 NMD 目标，还可以定义 NMD 衰变中间体的性质，从而确定 NMD 的过程。为此，我们在此描述了我们所谓的“