Hong W, Bennett M, Xiao Y, Feld Kramer R, Wang C, Reed R
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore.
Nucleic Acids Res. 1997 Jan 15;25(2):354-61. doi: 10.1093/nar/25.2.354.
In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mRNA. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snRNP first binds tightly to the 5'splice site in the E complex and U2 snRNP first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mAb 4G8) directed against SAP 62, a component of U2 snRNP and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-SAP 62 antibody also immunoprecipitates U2 snRNP and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-SAP 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snRNP components are associated with the E complex, we used antibodies to the U2 snRNP proteins B"and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snRNP is associated with the E complex. However, we find that U2 snRNP is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snRNP with the E complex is discussed.
在多细胞动物中，E复合物在操作上被定义为一种不依赖ATP的剪接体复合物，它在凝胶过滤柱上以单一峰洗脱，并且在存在过量竞争性前体mRNA的情况下可以追踪到剪接产物中。A复合物是第一个依赖ATP的功能性剪接体复合物。U1 snRNP首先紧密结合到E复合物中的5'剪接位点，U2 snRNP首先紧密结合到A复合物中的分支位点。在本研究中，我们产生并鉴定了一种针对SAP 62的单克隆抗体（mAb 4G8），SAP 62是U2 snRNP的一个组分，也是必需的哺乳动物剪接因子SF3a的一个亚基。我们表明，该抗体对SAP 62具有高度特异性，在蛋白质印迹上仅检测到SAP 62，并且仅从核提取物中免疫沉淀出SAP 62。抗SAP 62抗体也免疫沉淀U2 snRNP和A复合物。然而，重要的是，我们发现E复合物也能被抗SAP 62抗体有效地免疫沉淀。该抗体不与任何E复合物特异性组分发生交叉反应，表明SAP 62本身与E复合物相关。为了确定其他U2 snRNP组分是否与E复合物相关，我们使用了针对U2 snRNP蛋白B"和SAP 155的抗体。这些抗体也特异性地免疫沉淀E复合物。这些观察结果表明U2 snRNP与E复合物相关。然而，我们发现U2 snRNP在E复合物中的结合不如在A复合物中紧密。讨论了U2 snRNP与E复合物弱结合的可能意义。
