Zamore P D, Green M R
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243-7. doi: 10.1073/pnas.86.23.9243.
Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA branch site is an early step in spliceosome assembly and appears to commit a pre-mRNA to the splicing pathway. We have shown previously that this ATP-dependent binding requires a non-rnRNP factor, U2 snRNP auxiliary factor (U2AF), in addition to U2 snRNP. In this report we have identified U2AF, purified it to homogeneity, and characterized its biochemical properties. Purified U2AF comprises roughly equimolar quantities of two polypeptides, approximately 65 kDa and approximately 35 kDa, which appear to be associated. Measured by ultraviolet crosslinking, the 65-kDa polypeptide binds specifically to the polypyrimidine tract/3' splice site region. U2AF binds rapidly at 4 degrees C in the absence of ATP and remains associated with the pre-mRNA following U2 snRNP binding. Thus, the simple binding of U2AF initiates mammalian spliceosome assembly by facilitating the ATP-dependent binding of U2 snRNP.
U2小核核糖核蛋白（snRNP）与前体mRNA分支位点的结合是剪接体组装的早期步骤，并且似乎使前体mRNA进入剪接途径。我们先前已经表明，这种依赖ATP的结合除了U2 snRNP之外还需要一种非核糖核蛋白因子，即U2 snRNP辅助因子（U2AF）。在本报告中，我们鉴定了U2AF，将其纯化至同质，并表征了其生化特性。纯化的U2AF由大致等摩尔量的两种多肽组成，分别约为65 kDa和约35 kDa，它们似乎是相关联的。通过紫外线交联测量，65 kDa的多肽特异性结合到多嘧啶序列/3'剪接位点区域。U2AF在4℃下于无ATP时快速结合，并且在U2 snRNP结合后仍与前体mRNA相关联。因此，U2AF的简单结合通过促进U2 snRNP的ATP依赖性结合来启动哺乳动物剪接体的组装。
