Wiest D K, O'Day C L, Abelson J
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 1996 Dec 27;271(52):33268-76. doi: 10.1074/jbc.271.52.33268.
Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.
前体mRNA剪接发生在一个大型核糖核蛋白颗粒——剪接体上，剪接体包含五种小核核糖核蛋白（snRNP），即U1、U2、U4、U5和U6。在酿酒酵母中，mRNA剪接因子Prp9、Prp11和Prp21在剪接体组装的早期步骤中，对于将U2 snRNP添加到前体mRNA上是必需的。本文描述了对这些蛋白质之间相互作用及其在剪接体组装中作用的研究。这些蛋白质在大肠杆菌中表达。Prp9和Prp11通过金属亲和层析纯化。Prp21使用溶解/复性方案纯化。我们将这些分别纯化的蛋白质进行组合，并提供了在体外剪接试验中具有功能的Prp9·Prp11·Prp21蛋白质复合物的直接证据。使用体外合成的蛋白质进一步研究了这种Prp9·Prp11·Prp21复合物的特性。此外，我们发现Prp9、Prp11和Prp21以改变U2 snRNA分支点配对区域对寡核苷酸定向核糖核酸酶H切割的可及性的方式影响U2 snRNP的结构。基于本文及随附论文中呈现的数据，我们提出了一个模型，用于说明Prp9、Prp11、Prp21和Prp5在激活U2 snRNP以组装到前剪接体中的联合作用。
