PEPseq - Peptide Enhanced Pull-Down for RNA Sequencing

Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal an increase of protein interactions in the coding region of a distinct set of mRNAs, including mRNAs coding for the majority of cytosolic ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.

转录后基因调控（post-transcriptional gene regulation）通过转录组（transcriptome）与RNA结合蛋白（RNA-binding proteins）的相互作用得以实现，该过程会响应细胞环境的改变，以动态方式发生。记录所有结合至转录组的蛋白的联合占据情况，可为探究特定处理是否引发蛋白相互作用改变提供契机，而此类改变可指向RNA中经历转录后调控的位点。本研究建立了一种可通过RNA测序（RNA sequencing）在全转录组层面监测蛋白占据情况的方法。具体而言，肽增强下拉RNA测序（peptide-enhanced pull-down for RNA sequencing，简称PEPseq）采用4-硫尿苷（4-thiouridine，4SU）进行代谢RNA标记，以实现光诱导的蛋白-RNA交联，并借助N-羟基琥珀酰亚胺（N-hydroxysuccinimide，NHS）化学方法，分离所有长RNA生物型中与蛋白交联的RNA片段。我们运用PEPseq对人类细胞在亚砷酸盐诱导的翻译应激起始阶段的蛋白占据变化进行了探究，结果发现一组独特的mRNA的编码区中蛋白相互作用显著增强，其中涵盖编码大多数胞质核糖体蛋白的mRNA。我们通过定量蛋白质组学（quantitative proteomics）证实，在亚砷酸盐应激后的初始恢复阶段的数小时内，这些mRNA的翻译仍处于抑制状态。综上，本研究提出PEPseq可作为一种发现平台，用于无偏倚地探究转录后基因调控。