Gozani O, Patton J G, Reed R
Department of Cell Biology, Harvard Medical School, Boston, MA 02115.
EMBO J. 1994 Jul 15;13(14):3356-67. doi: 10.1002/j.1460-2075.1994.tb06638.x.
We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.
我们已经分离并确定了剪接体复合物C的蛋白质组成。该复合物中的前体mRNA已经经历了剪接反应的催化步骤I，但未经历步骤II。我们发现一组新的14种剪接体相关蛋白（SAPs）和必需的剪接因子PSF与C复合物特异性相关，表明这些蛋白参与催化步骤II。重要的是，免疫去除和生化互补研究直接证明PSF对于催化步骤II是必需的。已知纯化的PSF可与嘧啶序列进行紫外线交联，我们的数据表明PSF在纯化的C复合物中可与前体mRNA进行紫外线交联。因此，PSF可能替代在剪接体组装过程中不稳定的3'剪接位点结合因子U2AF65。最后，我们表明同时存在于B和C复合物中的SAPs 60和90与U4和U6 snRNPs特异性相关，因此可能在剪接反应期间这些snRNPs的功能中发挥重要作用。
