FUNCTIONAL SPLICEOSOMES AND EXTRINSIC SPLICING FACTORS

这项研究项目的广泛、长期目标是 研究RNA代谢和基因表达。RNA等领域 加工、RNA运输以及RNA的稳定性和降解 最终将被探索。最初，前信使核糖核酸的过程 剪接(即从信使RNA中精确剪除内含子 前身)将会被研究。具体地说，体外前信使核糖核酸 我们在酵母中建立的剪接系统 Cerevisiae，将被用于鉴定和表征 拼接机械中的部件(即非本征拼接 因子和功能剪接体)。这项研究的结果 应提供与健康相关的问题的线索 由于RNA剪接缺陷而导致的基因表达异常 进程。 转化前信使核糖核酸所需的三种外源性蛋白质因子 关于功能剪接体到剪接产品的研究 通过分析从几种植物中分离出来的提取物进行鉴定 温度敏感型RNA突变体。我们将努力 通过生化分级提纯这些外在因素 野生型或生产过剩的提取物。抗纯化抗体 蛋白质将会被提升。相应的基因将被分离出来 用抗体或人工合成的抗体筛选酵母DNA文库 寡核苷酸。DNA序列将被确定， 与搜索到的其他基因同源性，以及衍生的蛋白质 序列分析。提纯的外在因子将用于 研究并确定分离的功能性剪接体 不同的条件。三磷酸腺苷在剪接途径中的作用 将会被调查。将尝试识别剪接 可能被磷酸化或腺化的因子 剪接体的转换。两国之间的相互作用 剪接体、外在因子和RNA将被 用免疫沉淀和核糖核酸酶保护进行研究 化验。归根结底，核前mRNA剪接的机制 可能会被揭穿。

The broad, long-term objectives of this research project are to study RNA metabolism and gene expression. Areas such as RNA processing , RNA transport, and RNA stability and degradation will eventually be explored. Initially, the process of pre-mRNA splicing (i.e., the precise excision of introns from messenger RNA precursors) will be studied. Specifically, the in vitro pre-mRNA splicing system that we developed in the yeast, Saccharomyces cerevisiae, will be utilized to identify and characterize the components in the splicing machinery (i.e., the extrinsic splicing factors and the functional spliceosomes). The results of this study should provide clue to health-related problems which are resulted from abnormal gene expression due to defects in the RNA splicing process. Three extrinsic protein factors required to convert the pre-mRNA on a functional spliceosome to spliced products have been identified by analyzing extracts isolated from several temperature-sensitive rna mutants. Efforts will be made to purify these extrinsic factors by biochemical fractionation of wild-type or overproducer extracts. Antibodies against purified proteins will be raised. The corresponding genes will be isolated by screening yeast DNA library with the antibodies or synthetic oligonucleotides. DNA sequences will be determined, the homology with other genes searched, and the derived protein sequences analyzed. The purified extrinsic factors will be used to examine and to define the functional spliceosomes isolated under different conditions. The roles of ATP in the splicing pathway will be investigated. Attempt will be made to identify splicing factors which may be phosphorylated or adenylated during the conversion of the spliceosome. The interactions among the spliceosome, the extrinsic factors, and the RNA will be investigated by using immunoprecipitation and RNase protection assays. Ultimately, the machinery for nuclear pre-mRNA splicing may be revealed.