Thomas Huw B, O'Keefe Raymond T
Division of Evolution and Genomic Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
Methods Mol Biol. 2019;2049:131-140. doi: 10.1007/978-1-4939-9736-7_8.
Splicing of pre-messenger RNA (pre-mRNA) transcripts is a fundamental process in all eukaryotes that provides a mechanism of increasing the proteomic diversity within a cell that can be tightly regulated in a dynamic manner. While constitutive and alternative splicing are necessary for the correct development and regulation of cells/organisms, aberrant splicing is now associated with an increasingly varied number of human diseases, such as neurological and developmental diseases, and cancer. Studies of splicing mechanisms and regulation are often achieved in nonhuman model organisms such as yeast. Yeasts possess homologs to many of the core spliceosome components of higher organisms, including humans, and as such yeast species are now a well-established model organism for understanding how differential splicing of transcripts can alter the phenotype of a cell or organism. Here we describe methods to investigate pre-mRNA splicing in yeast cells using modern RNA-Seq technology and bioinformatics software. Details of traditional validation methods are also described.
前体信使RNA（pre-mRNA）转录本的剪接是所有真核生物中的一个基本过程，它提供了一种增加细胞内蛋白质组多样性的机制，这种机制可以以动态方式受到严格调控。虽然组成型剪接和可变剪接对于细胞/生物体的正确发育和调控是必需的，但异常剪接现在与越来越多的人类疾病相关，如神经和发育疾病以及癌症。剪接机制和调控的研究通常在非人类模式生物如酵母中进行。酵母拥有许多高等生物（包括人类）核心剪接体成分的同源物，因此酵母物种现在是一种成熟的模式生物，用于理解转录本的差异剪接如何改变细胞或生物体的表型。在这里，我们描述了使用现代RNA测序技术和生物信息学软件在酵母细胞中研究前体mRNA剪接的方法。还描述了传统验证方法的细节。
