Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas.
Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas;
J Vis Exp. 2021 Apr 22(170). doi: 10.3791/62181.
mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5´capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.
mRNA 加工涉及多个同时进行的步骤，以准备 mRNA 进行翻译，例如 5´加帽、多聚 A 添加和剪接。除了组成性剪接外，可变剪接允许从一个基因表达多功能蛋白。由于互作组学研究通常是新的或未知蛋白的第一个分析，诱饵蛋白与剪接因子的关联表明它可以参与 mRNA 剪接过程，但要确定在什么情况下或哪些基因受到调节是一个经验过程。评估此功能的一个良好起点是使用经典的小基因工具。在这里，我们介绍了腺病毒 E1A 小基因的使用，用于评估不同细胞应激刺激后的可变剪接变化。我们评估了在不同应激处理后过表达 Nek4 蛋白的 HEK293 稳定细胞中 E1A 小基因的剪接。该方案包括 E1A 小基因转染、细胞处理、RNA 提取和 cDNA 合成，然后进行 PCR 和凝胶分析以及 E1A 剪接变体的定量。这种简单而成熟的方法与特定处理的结合使用是阐明细胞过程或哪些基因可以通过 mRNA 剪接调节的可靠起点。
