Günzl A, Cross M, Bindereif A
Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin Dahlem, Germany.
Mol Cell Biol. 1992 Feb;12(2):468-79. doi: 10.1128/mcb.12.2.468-479.1992.
Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.
锥虫中mRNA的成熟涉及剪接前导RNA 5'端与多顺反子前体mRNA外显子的反式剪接，这需要小核核糖核蛋白（snRNP）作为辅助因子。我们通过核糖核酸酶H保护分析、非变性凝胶电泳和CsCl密度梯度离心相结合的方法，绘制了U2和U4/U6 snRNP中的蛋白质结合位点。在U2 snRNP中，蛋白质结合主要发生在3'末端结构域；通过U2 snRNP重组和化学修饰干扰试验，我们确定了布氏锥虫U2 RNA茎环IV内对于蛋白质结合至关重要的离散位置；值得注意的是，其中一些位置与顺式剪接体U2 RNA的共有序列不同。在U4/U6 snRNP中，主要的蛋白质结合区域位于U4 RNA的3'末端一半区域内。总之，虽然U2和U4/U6 snRNP的整体结构域结构在顺式和反式剪接系统之间是保守的，但我们的数据表明，RNA-蛋白质结合也存在反式剪接体特异性决定因素。
