YTHDC1 m6A-dependent and m6A-independent functions converge to preserve DNA damage response (RIP-seq)

Cells have evolved a robust and highly regulated DNA damage response to preserve their genomic integrity. Although increasing evidence highlights the relevance of RNA regulation, our understanding of its impact on a fully efficient DNA damage response remains limited. Here, through a targeted CRISPR-knockout screen, we identified RNA binding proteins and modifiers that participate in mediating the p53 response. Among the top hits, m6A reader YTHDC1 was identified as a master regulator of p53 expression. YTHDC1 binds to the transcription start sites ofTP53and other genes involved in DNA damage response, promoting their transcriptional elongation. YTHDC1 deficiency leads to reducedTP53expression, and also retention of introns leading to aberrant protein production of key DNA damage factors. While intron retention is dependent on m6A, YTHDC1 favoursTP53transcriptionalpause-release independently of m6A. Depletion of YTHDC1 causes genomic instability and aberrant cancer cell proliferation mediated by genes regulated by YTHDC1. Our results uncover YTHDC1 as an orchestrator of the DNA damage response through distinct mechanisms of co-transcriptional mRNA regulation. Overall design: A CLIP experiment was performed to identify YTHDC1 binding sites

细胞已演化出一套强健且受高度调控的DNA损伤应答（DNA damage response）机制，以维持其基因组完整性（genomic integrity）。尽管越来越多的研究证据凸显了RNA调控的重要性，但我们对其在保障DNA损伤应答完全高效运行中所发挥作用的认知仍十分有限。本研究通过靶向CRISPR敲除筛选（CRISPR-knockout screen），鉴定出了参与介导p53应答（p53 response）的RNA结合蛋白（RNA binding protein）及其调控修饰因子。在筛选得到的核心候选基因中，m6A阅读器（m6A reader）YTHDC1被鉴定为p53表达（p53 expression）的核心调控因子。YTHDC1可结合至TP53以及其他参与DNA损伤应答的基因的转录起始位点（transcription start site），进而促进这些基因的转录延伸（transcriptional elongation）。YTHDC1的缺失会降低TP53的表达水平，同时还会引发内含子滞留（intron retention），导致关键DNA损伤因子产生异常蛋白产物。尽管内含子滞留依赖于m6A修饰，但YTHDC1却可通过不依赖m6A的途径，促进TP53的转录暂停释放（transcriptional pause-release）。YTHDC1的敲除会引发基因组不稳定性（genomic instability），并通过受其调控的基因介导异常的癌细胞增殖（cancer cell proliferation）。本研究结果揭示，YTHDC1可通过多种共转录mRNA调控（co-transcriptional mRNA regulation）机制，作为DNA损伤应答的协调调控因子发挥功能。实验整体设计：本研究通过CLIP实验（CLIP experiment）鉴定YTHDC1的结合位点（binding site）。