FUNCTIONAL SPLICEOSOMES AND EXTRINSIC SPLICING FACTORS

该研究项目的广泛和长期目标是 研究RNA代谢和基因表达。 RNA等领域 加工、RNA转运、RNA稳定性和降解 最终将被探索。 最初，前mRNA的过程 拼接（即，从信使RNA中精确切除内含子 前体）进行研究。 具体来说，体外前mRNA 我们在酵母中开发的剪接系统， 酿酒酵母，将用于识别和表征 拼接机器中的部件（即，外源性剪接 因子和功能性剪接体）。 本研究结果 应提供线索，以健康相关的问题， 由于RNA剪接缺陷导致的异常基因表达 过程 转化前mRNA所需的三种外源蛋白因子 在功能性剪接体上， 通过分析从几个 温度敏感的RNA突变体。 将努力 通过生物化学分离纯化这些外源因子， 野生型或超量生产者提取物。 抗纯化的 蛋白质会增加。 相应的基因会被分离出来 通过用抗体或合成抗体筛选酵母DNA文库， 寡核苷酸 DNA序列将被确定， 与其他基因的同源性，以及衍生的蛋白质 序列分析。 纯化的外源因子将用于 检查并定义分离的功能性剪接体， 不同条件 ATP在剪接途径中的作用 将被调查。 将尝试识别拼接 这些因子可能在细胞凋亡过程中被磷酸化或腺苷化， 剪接体的转化。 之间的交互 剪接体、外源因子和RNA将被 通过使用免疫沉淀和RNase保护进行研究 测定。 最终，核前体mRNA剪接的机制 可能会被揭露。

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.