PROCESSING OF PREMESSENGER RNA

这项建议描述了研究加工过程的实验 高等真核生物中的前信使RNA。在过去的几年中 多年来，我们对前信使RNA机制的理解 加工量大大增加，在不小的程度上 因为体外系统的发展有能力 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 快速组装成一个大的核糖核蛋白复合体( 剪接体)含有已知的hnRNP和SnRNP因子 剪接的切割和连接步骤所需的。下一个 对于拼接领域来说，几年有望成为令人兴奋的几年 剪接体的组装和参与的规则 将确定剪接反应。 我们计划调查该组织的集会和活动。 剪接体采用体外加工提取液。七 目前在实验室中使用的实验方法将是 用于解决剪接体结构的基本问题和 功能包括：1.SnRNP蛋白的检测 磷酸化及其与组装和活性的关系 剪接体；2.二维凝胶的开发 剪接体的检查；3.剪接体提取物的分级 使用基于大小的方法；4.使用RNP凝胶电泳法 推测剪接体的装配和拆解途径； 基质蛋白参与体外实验的研究 剪接；6.SnRNP组装和亚群的检测 通过对组装在分离的SnRNA上的复合体进行RNP凝胶分析， 7.研究剪接和剪接之间的关系 通过使用含有以下成分的前体RNA进行多腺苷基化 内含子和多聚(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.