Kataoka Naoyuki
Medical Innovation Center, Laboratory for Malignancy Control Research, Kyoto University Graduate School of Medicine, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan.
Methods Mol Biol. 2016;1421:45-52. doi: 10.1007/978-1-4939-3591-8_5.
In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.
在真核生物中，前体mRNA剪接是基因表达的一个关键步骤。通过使用从培养细胞中制备的提取物进行体外剪接反应，剪接反应已得到充分研究。在这里，我们描述了从表达带有标签的剪接体蛋白的细胞中制备具有剪接活性提取物的方案。全细胞提取物能够剪接外源添加的前体mRNA，并且体外剪接反应中形成的RNA-蛋白质复合物可以使用针对剪接蛋白上肽标签的抗体通过免疫沉淀进行纯化。这里描述的从全细胞制备剪接活性提取物的方法在研究各种细胞类型中的前体mRNA剪接时特别有用，并且带有标签的剪接体蛋白的表达使得人们能够通过简单的免疫沉淀来纯化和分析RNA-蛋白质复合物。
