DDX54 regulates transcriptome dynamics during DNA damage response [PAR-CLIP]. Homo sapiens

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of posttranscriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing irradiation (IR). Interestingly, more than 260 proteins including many nucleolar proteins showed increased binding to poly(A) RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well defined class of pre-mRNAs which harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Since DDX54 promotes survival after exposure to IR its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR. Overall design: Identification of wild-type and mutant DDX54 binding sites in untreated and DNA-damage induced condition

细胞对遗传毒性应激（genotoxic stress）的响应由一套已被充分阐明的DNA监测通路（DNA surveillance pathways）网络所介导。转录后基因调控网络（posttranscriptional gene regulatory networks）在DNA损伤响应（DNA damage response, DDR）中的作用尚未得到广泛研究。本研究系统鉴定了人乳腺癌细胞暴露于电离辐射（ionizing irradiation, IR）后，与多聚腺苷酸化转录本（polyadenylated transcripts）差异结合的RNA结合蛋白（RNA-binding proteins, RBPs）。有趣的是，超过260种蛋白质（包括众多核仁蛋白（nucleolar proteins））在电离辐射暴露的细胞中与poly(A) RNA的结合水平显著升高。

我们对候选遗传毒性应激响应性RNA解旋酶（RNA helicase）DDX54进行功能分析后发现，该蛋白是一类即时-早期DDR调控因子，对其靶标电离辐射诱导的前体mRNA（pre-mRNAs）的剪接效率至关重要。电离辐射暴露后，DDX54通过两种方式发挥调控作用：一是与一类特征明确的、携带弱剪接受体位点（acceptor splice sites）内含子的前体mRNA结合增强；二是与U2小核糖核蛋白颗粒（U2 snRNP）及剪接体B复合物（spliceosomal B complex）的组分形成蛋白质-蛋白质相互作用，最终降低其靶标转录本的内含子滞留（intron retention）水平并提升其加工速率。鉴于DDX54可促进细胞在电离辐射暴露后的存活，其表达水平及/或突变率可能会影响与DDR相关的病理过程。本研究揭示了众多未被表征的RBPs潜在参与DDR的重要意义。

整体实验设计：在未处理及DNA损伤诱导条件下，鉴定野生型（wild-type）与突变型（mutant）DDX54的结合位点。