PROCESSING OF PREMESSENGER RNA

该提案描述了研究加工的实验， 高级真核生物中的前信使RNA。 过去几 多年来，我们对前信使RNA机制的理解 加工量大幅增加， 由于体外系统的发展， 催化简单前体RNA的剪接和聚腺苷酸化。 在加入剪接提取物后，外源前体RNA 迅速组装成一个大的核糖核蛋白复合物（ 剪接体）含有hnRNP和snRNP因子 剪接的切割和连接步骤所需的。 下一 几年的承诺是令人兴奋的拼接领域期间， 其中剪接体组装和参与的规则 将确定剪接反应。 我们计划调查的组装和活动的 剪接体使用体外处理提取物。 七 目前在实验室使用的实验方法将是 用于解决有关剪接体结构的基本问题， 功能，包括：1. snRNP蛋白检测 磷酸化及其与组装和活性的关系 剪接体; 2.二维凝胶的研制 剪接体的检查; 3.剪接提取物的分级 使用基于大小的方法; 4.使用RNP凝胶电泳， 推断剪接体的组装和拆卸途径; 5. 在体外检查基质蛋白的参与 拼接; 6. snRNP组装和亚群的检查 通过在分离的snRNA上组装的复合物的RNP凝胶分析， 和7. 剪接与细胞分裂的关系 通过使用前体RNA的多聚腺苷酸化， 内含子和poly（A）位点。

This proposal describes experiments to investigate processing of pre-messenger RNA in higher eucaryotes. In the last several years, our understanding of the mechanics of pre-messenger RNA processing has increased considerably, in no small measure because of the development of in vitro systems competent to catalyze splicing and polyadenylation of simple precursor RNAs. Upon addition to a splicing extract, an exogenous precursor RNA rapidly assembles into a large ribonucleoprotein complex (the spliceosome) containing hnRNP and snRNP factors known to be required for the cleavage and ligation steps of splicing. The next few years promise to be exciting ones for the splicing field during which the rules of spliceosome assembly and participation in the splicing reaction will be determined. We plan to investigate both the assembly and activity of the spliceosome using in vitro processing extracts. Seven experimental approaches currently in use in the laboratory will be used to address basic questions about spliceosome structure and function, including: 1. Examination of snRNP protein phosphorylation and its relationship to assembly and activity of the spliceosome; 2. Development of two-dimensional gels for the examination of spliceosomes; 3. Fractionation of splicing extracts using size-based approaches; 4. Use of RNP gel electrophoresis to deduce the pathway of spliceosome assembly and disassembly; 5. Examination of the involvement of matrix proteins in in vitro splicing; 6. Examination of snRNP assembly and sub-populations by RNP gel analysis of complexes assembled on isolated snRNAs, and 7. Examination of the relationship between splicing and polyadenylation through the use of precursor RNAs containing introns and poly(A) sites.