Furman E, Glitz D G
Department of Biological Chemistry, UCLA School of Medicine, University of California 90024-1737, USA.
J Biol Chem. 1995 Jun 30;270(26):15515-22. doi: 10.1074/jbc.270.26.15515.
Pre-mRNA splicing occurs on spliceosomes, a family of ribonucleoprotein particles. Spliceosome assembly on exogenous adenovirus pre-mRNA was blocked at the A-complex (or pre-spliceosome) stage, either by destruction of the small nuclear ribonucleoproteins (snRNPs) that comprise the U4/U5/U6 tri-snRNP complex, or by interference in tri-snRNP assembly and interactions. The A-complex was isolated by size exclusion chromatography; homogeneity was shown by electrophoresis in nondenaturing polyacrylamide gels, gradient sedimentation, and electron microscopy. Northern hybridization showed U1 and U2 snRNAs to be present in the preparation, but not U4, U5, or U6. Antibodies specific for a component of the U1 snRNP or for a component that is common to all snRNPs (except U6) each precipitated an A-complex containing pre-mRNA, U1 and U2 snRNPs. Electron micrographs showed 230 x 270-A particles whose two components appear similar to individual U1 and U2 snRNPs. Electron micrographs of an A-complex-5'-biotinyl oligonucleotide-streptavidin-gold composite allowed identification of the U2 snRNP within the structure and the localization of the 5'-segment of U2 snRNA at a unique site in the A-complex. This region of U2 RNA is adjacent to the developing catalytic center of the spliceosome.
前体mRNA剪接发生在剪接体上，剪接体是一类核糖核蛋白颗粒。外源性腺病毒前体mRNA上的剪接体组装在A复合体（或前剪接体）阶段被阻断，这要么是通过破坏构成U4/U5/U6三小核核糖核蛋白复合体（tri-snRNP）的小核核糖核蛋白（snRNP），要么是通过干扰三小核核糖核蛋白的组装和相互作用来实现的。通过尺寸排阻色谱法分离出A复合体；通过非变性聚丙烯酰胺凝胶电泳、梯度沉降和电子显微镜显示其均一性。Northern杂交显示制备物中存在U1和U2 snRNA，但不存在U4、U5或U6。针对U1 snRNP的一个组分或所有snRNP（U6除外）共有的一个组分的特异性抗体，各自沉淀出一个包含前体mRNA、U1和U2 snRNP的A复合体。电子显微镜照片显示出230×270埃的颗粒，其两个组分看起来类似于单个的U1和U2 snRNP。A复合体-5'-生物素化寡核苷酸-链霉亲和素-金复合物的电子显微镜照片使得能够在结构内鉴定U2 snRNP，并将U2 snRNA的5'-片段定位在A复合体中的一个独特位点。U2 RNA的这个区域与剪接体正在形成的催化中心相邻。
