Studying the composition of mRNPs in vitro using splicing-competent cell extracts.
Studying the composition of mRNPs in vitro using splicing-competent cell extracts.
复制标题
使用剪接活性细胞提取物研究体外 mRNP 的组成
DOI：
10.1016/j.ymeth.2013.08.033
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发表时间：
2014
期刊：
影响因子：
4.8
通讯作者：
Gehring NH
中科院分区：
文献类型：
--
作者：
Steckelberg AL;Gehring NH
基因表达过程中mRNA的正确加工和忠实解码依赖于与RNA结合蛋白（RBP）的相互作用。RBP与前体mRNA的结合在RNA聚合酶II的转录过程中开始，并在前体mRNA加工和基因表达的后续步骤中经历不断的重塑。最近开发的高通量方法能够在体内确定RBP结合位点，并在真核细胞中鉴定大量新的RBP。然而，RBP-RNA相互作用的详细表征以及功能性RNP的分析通过良好定义的体外系统大大促进。在这里，我们描述了一种通用的方法来研究组装和剪接依赖的mRNP在体外重塑。该方法采用从转染的人胚肾（HEK）293细胞产生的剪接感受态全细胞提取物（WCE）。WCE中存在的FLAG标记的蛋白质在体外掺入mRNP中，然后用于免疫沉淀底物RNA。我们概述了纯化体外组装的mRNP的原则，并提供了详细的协议，为整个细胞提取物的制备和使用。讨论了替代的纯化策略和RNA底物。
The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP–RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed.