Jablonski Joseph, Clementz Mark, Ryan Kevin, Valente Susana T
Department of Infectious Diseases, The Scripps Research Institute.
Department of Chemistry, City College of New York.
J Vis Exp. 2014 May 3(87):51309. doi: 10.3791/51309.
The 3' end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3' end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5' cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3' end processing of HIV-1 mRNAs.
哺乳动物mRNA的3'末端并非由RNA聚合酶II（RNPII）转录突然终止形成。相反，RNPII会在成熟RNA末端之外合成前体mRNA，并且需要在特定位点进行内切核酸酶活性的活跃过程。前体RNA的切割通常发生在CA二核苷酸之后，在共有多聚腺苷酸化位点（AAUAAA）下游10 - 30个核苷酸处。切割复合物中的蛋白质（一种约800 kDa的多因子蛋白质复合物）完成这种特定的核酸酶活性。多聚腺苷酸化位点上游和下游的特定RNA序列控制着切割复合物的募集。切割后，前体mRNA立即被多聚腺苷酸聚合酶（PAP）进行多聚腺苷酸化，以产生成熟稳定的RNA信息。可以使用含有特定放射性标记RNA底物的细胞核提取物来研究RNA转录本3'末端的加工过程。总之，将一个长的32P标记的未切割前体RNA与细胞核提取物在体外孵育，并通过凝胶电泳和放射自显影评估切割情况。当发生正确切割时，会检测到并定量一个较短的5'切割产物。在这里，我们以HIV-1 mRNA的3'末端加工为例详细描述切割分析。
