FUNCTION OF PRE-MRNA-PROTEIN COMPLEXES

越来越清楚的是，前mRNA-蛋白质复合物（也 称为异源核RNA-蛋白质复合物或hnRNP） 在RNA剪接中起重要作用。 我们建议， hnRNP在RNA剪接中的作用是螯合RNA 剪接中心和四级结构的稳定性 在RNA合成过程中， 拼接 某些前体mRNA结构， 人为地使交叉点非常接近， 在没有hnRNP形成的情况下在体外剪接， 模拟涉及剪接反应的hnRNP构象 中心 我们认为，仔细分析各种前体mRNA 结构将提供重要的信息， RNA剪接中的hnRNP。 在这个应用程序中，我们将构建 各种前体mRNA结构，其中剪接点位于 鸡b-珠蛋白与小鼠b-珠蛋白的近缘杂交 金属硫蛋白-I前体mRNA与cRNA的结合， 具有互补外显子序列的茎结构。 这样的预- mRNA将在HeLa细胞核提取物中剪接，剪接的mRNA将在HeLa细胞核提取物中剪接。 产品和中间体将通过高分辨率 聚丙烯酰胺凝胶电泳 此外，我们将 研究hnRNP蛋白结合、hnRNP组装 需要snRNP进行剪接，以及可能的螯合 在没有hnRNP形成的情况下的剪接反应。

It is increasingly clear that pre-mRNA-protein complexes (also known as heterogenous nuclear RNA-protein complexes or hnRNP) play an important role in RNA splicing. We propose that one of the functions of hnRNP in RNA splicing is sequestration of RNA splicing center and stabilization of the quaternary structure in which the splice junctions are in a close proximity during RNA splicing. Certain pre-mRNA structures in which the splice junctions are artificially brought into a close proximity appear to be spliced in vitro in the absence of hnRNP formation and perhaps mimic the conformation of hnRNP involving the splicing reaction center. We believe that careful analysis of various pre-mRNA structures will provide important information on the role of hnRNP in RNA splicing. In this application we will construct various pre-mRNA structures in which the splice junctions are in a close proximity by hybridization of chicken b-globin and mouse metallothionein-I pre-mRNAs with cRNA and also by forming a stem structure with complementary exon sequences. Such pre- mRNAs will be spliced in HeLa nuclear extracts and the spliced products and intermediates will be analyzed by high resolution polyacrylamide gel electrophoresis. In addition, we will investigate binding of hnRNP proteins, assembly of hnRNP, requirement of snRNPs for splicing, and possible sequestration of splicing reaction in the absence of hnRNP formation.