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Author:
Date:
2017-05-20
[Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of an RBP to specific regions ...
[摘要] RNA代谢在不同的组织和发育阶段被严格控制，其失调是癌症的分子特征之一。 通过直接结合特定的序列元件，RNA结合蛋白（RBP）在共转录和转录后调控事件中起关键作用。 我们最近证实，在胰腺癌细胞中，获得耐药（DR） - 表型取决于多聚嘧啶区结合蛋白（PTBP1）的上调，其又被引入丙酮酸激酶前mRNA并有利于剪接 致癌性PKM2变体。 在这里，我们描述了紫外（UV）光交联和免疫沉淀（CLIP）方法的逐步方案，以确定RBP与贴壁人细胞系中其目标RNA的特定区域的直接结合。
背景 在细胞核中转录时，新生的RNA立即与被称为RNA结合蛋白（RBP）的反式因子立即组装。这些因子直接与RNA分子中特定的顺式调控序列相互作用，从而形成核糖核蛋白（RNP）复合物（Dreyfuss et al。，2002; ...
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