Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, United States.
Methods. 2014 Feb;65(3):350-8. doi: 10.1016/j.ymeth.2013.08.019. Epub 2013 Aug 22.
The majority of human genes undergo alternative splicing to generate multiple isoforms with distinct functions. This process is generally controlled by cis-acting splicing regulatory elements (SREs) that recruit trans-acting factors to promote or inhibit the use of nearby splice sites. The growing interest in understanding the regulatory rules of splicing necessitates the systematic identification of these SREs and their cognate protein factors using experimental and computational approaches. Here we describe a strategy to identify and analyze both cis-acting SREs and trans-acting splicing factors. This strategy involves a cell-based screen to identify SREs from a random sequences library and a modified RNA affinity purification approach to unbiasedly identify the splicing factors. These methods can be adopted to identify splicing enhancers or silencers in both exons and introns, and can be extended to different cultured cells. The resulting SREs and splicing factors can be further analyzed with a series of computational and experimental approaches. This approach will help us to collect a molecular part-list for splicing regulation, providing a rich data source that enables a better understanding of the "splicing code".
大多数人类基因通过可变剪接产生具有不同功能的多种异构体。这个过程通常由顺式作用剪接调控元件（SREs）控制，这些元件招募反式作用因子来促进或抑制附近剪接位点的使用。为了深入了解剪接的调控规则，人们越来越感兴趣，这就需要使用实验和计算方法系统地识别这些 SREs 及其同源蛋白因子。在这里，我们描述了一种从随机序列文库中识别 SREs 和分析反式剪接因子的策略。该策略涉及一种基于细胞的筛选方法，从随机序列文库中识别 SREs，以及一种改良的 RNA 亲和纯化方法，以无偏地鉴定剪接因子。这些方法可用于鉴定外显子和内含子中的剪接增强子或沉默子，并且可以扩展到不同的培养细胞。然后可以使用一系列计算和实验方法进一步分析所得的 SREs 和剪接因子。这种方法将有助于我们收集剪接调控的分子元件列表，提供丰富的数据源，从而更好地理解“剪接代码”。
