Wang Yang, Cheong Cheom-Gil, Hall Traci M Tanaka, Wang Zefeng
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Nat Methods. 2009 Nov;6(11):825-30. doi: 10.1038/nmeth.1379. Epub 2009 Oct 4.
Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.
可变剪接通常由反式作用因子调控，这些因子特异性结合前体mRNA以激活或抑制剪接反应。这种调控对正常基因表达至关重要，剪接失调与人类疾病密切相关。在此，我们通过将人类Pumilio1的序列特异性RNA结合结构域与调控剪接的功能结构域相结合，构建了人工剪接因子。我们应用这些因子来调节选定靶标中不同类型的可变剪接，以检测天然剪接因子效应结构域的活性，并调节内源性人类基因Bcl-X（一个抗癌靶点）的剪接。靶向Bcl-X的设计因子增加了促凋亡Bcl-xS剪接异构体的量，从而促进细胞凋亡并增强癌细胞对常见抗肿瘤药物的化学敏感性。我们的方法能够创建几乎可靶向任何前体mRNA的人工因子，为研究剪接调控和操控与疾病相关的剪接事件提供了一种策略。
