The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking [PANQKI_HITS-CLIP]

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3’ end anchored RNA sequencing, we mapped the alternative polyadenylation landscape (APA) following TGF-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3’UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a small subset of events including the length of its own transcript. Analysis of QKI CLIP-seq data identified the binding of QKI within 3’UTRs was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts are altered to produce predominantly shorter 3’UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process. HITS-CLIP was performed on endogenous QKI from using antibodies specific to the QKI-5 isoform (QKI5-Be & QKI5-NM) or all QKI isoforms (panQKI).

上皮间质转化（Epithelial-mesenchymal transition, EMT）在肿瘤进展中发挥关键作用，其进程由基因表达的动态变化统筹调控。尽管目前已明确转录后调控在EMT中具有重要作用，但EMT过程中可变多聚腺苷酸化（alternative polyadenylation, APA）的调控程度尚未得到探索。本研究采用3'端锚定RNA测序技术，对转化生长因子β（transforming growth factor-β, TGF-β）介导的人乳腺上皮细胞EMT诱导模型中的APA景观进行了图谱绘制，发现在该细胞状态转变过程中，APA普遍导致3'非翻译区（3'UTR）延长。对APA潜在调控介质的分析表明，RNA结合蛋白Quaking（QKI）——一种在EMT过程中被诱导表达的剪接因子——可调控一小部分APA事件，包括其自身转录本的长度变化。对QKI紫外交联免疫沉淀测序（CLIP-seq）数据的分析发现，QKI在3'UTR内的结合位点富集于剪切与多聚腺苷酸化位点附近。在QKI被敲低后，众多转录本的APA模式发生改变，主要产生较短的3'UTR，这与基因表达水平降低相关。上述研究结果揭示了EMT过程中APA的动态变化，并明确了QKI在该过程中的潜在调控作用。本研究使用针对QKI-5亚型（QKI5-Be与QKI5-NM）或所有QKI亚型（泛QKI抗体，panQKI）的特异性抗体，对内源QKI开展了高通量测序紫外交联免疫沉淀（HITS-CLIP）实验。