Regulation of alternative polyadenylation by the C2H2-zinc finger protein Sp1 [CLIP-seq]. Regulation of alternative polyadenylation by the C2H2-zinc finger protein Sp1 [CLIP-seq]

About 70% of human genes carry multiple polyadenylation signals, and mRNA 3’end formation is dynamically regulated under different physiological conditions. Global 3’end shortening through alternative polyadenylation (APA) correlates with enhanced cellular proliferation, and 3’ untranslated region (UTR) shortening is a widespread phenomenon in tumour cells, where it appears to enhance tumorigenic properties. However, the mechanisms responsible for this dynamic APA regulation remain incompletely understood. Here we show that transcription factor Sp1 binds directly to RNA in vivo and is a common repressor of distal poly (A) site usage. RNA-sequencing (RNA-seq) analysis identified 2344 genes (36% of total mapped mRNA transcripts) with lengthened 3’UTRs upon Sp1 depletion. Sp1 preferentially binds in vivo within the 3’UTRs of many of these lengthened transcripts and inhibits cleavage at distal sites by interacting physically with subunits of the core cleavage and polyadenylation (CPA) machinery. The 3’UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings reveal an important mechanism for dynamic APA regulation by unraveling a novel function of Sp1. Overall design: iCLIP-seq of GFP-tagged and endogenous proteins in HEK293 cells

约70%的人类基因携带多个多聚腺苷酸化信号（polyadenylation signals），mRNA的3'端形成过程在不同生理条件下受到动态调控。通过可变多聚腺苷酸化（alternative polyadenylation, APA）实现的全局3'端缩短与细胞增殖增强密切相关；而3'非翻译区（3' untranslated region, UTR）缩短是肿瘤细胞中的普遍现象，该现象似乎可增强肿瘤发生相关特性。然而，介导这种动态APA调控的分子机制仍未完全阐明。本研究证实，转录因子Sp1（transcription factor Sp1）可在体内直接结合RNA，并作为远端多聚腺苷酸化位点使用的普遍抑制因子。RNA测序（RNA-sequencing, RNA-seq）分析显示，在Sp1敲低后，共有2344个基因（占总比对上的mRNA转录本的36%）的3'UTR发生延长。Sp1在体内优先结合于这类延长转录本的3'UTR区域，并通过与核心剪切与多聚腺苷化复合物（core cleavage and polyadenylation, CPA）的亚基发生物理相互作用，抑制远端位点的剪切过程。在乳腺癌患者的RNA-seq数据中，Sp1靶基因的3'UTR长度与Sp1的表达水平呈显著相关，这提示Sp1介导的APA调控可参与调节肿瘤发生特性。综上，本研究通过揭示Sp1的全新功能，为动态APA调控提供了重要的分子机制。整体实验设计：在HEK293细胞中对绿色荧光蛋白（GFP）标记蛋白及内源性蛋白开展紫外交联免疫沉淀测序（iCLIP-seq）。