Kurosaki Tatsuaki, Hoque Mainul, Maquat Lynne E
Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA.
Center for RNA Biology, University of Rochester, Rochester, NY, USA.
Methods Mol Biol. 2018;1720:175-186. doi: 10.1007/978-1-4939-7540-2_13.
Recent progress in the technology of transcriptome-wide high-throughput sequencing has revealed that nonsense-mediated mRNA decay (NMD) targets ~10% of physiologic transcripts for the purpose of tuning gene expression in response to various environmental conditions. Regardless of the eukaryote studied, NMD requires the ATP-dependent RNA helicase upframeshift 1 (UPF1). It was initially thought that cellular NMD targets could be defined by their binding to steady-state UPF1, which is largely hypophosphorylated. However, the propensity for steady-state UPF1 to bind RNA nonspecifically, coupled with regulated phosphorylation of UPF1 on an NMD target serving as the trigger for NMD, made it clear that it is phosphorylated UPF1 (p-UPF1), rather than steady-state UPF1, that can be used to distinguish cellular NMD targets from cellular RNAs that are not. Here, we describe the immunoprecipitation of p-UPF1 followed by RNA sequencing (p-UPF1 RIP-seq) as a transcriptome-wide approach to define physiologic NMD targets.
转录组范围的高通量测序技术的最新进展表明，无义介导的mRNA降解（NMD）会靶向约10%的生理转录本，以便在响应各种环境条件时调节基因表达。无论研究的真核生物是什么，NMD都需要ATP依赖性RNA解旋酶移码1（UPF1）。最初人们认为，细胞中的NMD靶点可以通过它们与主要处于低磷酸化状态的稳态UPF1的结合来定义。然而，稳态UPF1非特异性结合RNA的倾向，再加上作为NMD触发因素的NMD靶点上UPF1的磷酸化受到调控，这表明可以用于区分细胞NMD靶点与非NMD细胞RNA的是磷酸化的UPF1（p-UPF1），而不是稳态UPF1。在此，我们描述了通过RNA测序进行磷酸化UPF1免疫沉淀（p-UPF1 RIP-seq），这是一种在转录组范围内定义生理NMD靶点的方法。
