FUNCTIONAL SPLICEOSOMES AND EXTRINISIC SPLICING FACTORS

RNA剪接，导致内含子移除的生化过程 从前体RNA，是基因表达中必不可少的一步 信息。与自动催化的自剪接反应不同，剪接 核前mRNA需要三磷酸腺苷，以复杂、动态的结构存在 称为由小核核糖核蛋白组成的剪接体。一个 含有未剪接的Pre-mRNA的剪接体已从 酵母prp2突变体。这种功能性剪接体可以被分离出来并 在添加ATP和外在剪接时激活剪接 因素(ESFS)。其中一个ESF是由编码的RNA依赖的ATPase 酵母PrP2基因，它与剪接体结合并触发剪接 在有ATP存在的情况下。因此，PRP2可能是一个关键组件 在校对步骤中催化中心或功能的。 这项研究项目的长期目标是了解 核前-mRNA剪接的生化和遗传学机制 剪接体成分和ESFS的分析。具体地说， 在剪接体中构成催化中心的组件将 被调查。目前的研究主要集中在鉴定上。 PRP2与激活ATPase的RNA结合的剪接体成分 PRP2的活性。放射性PRP2蛋白将在体外合成 或从酵母细胞中分离出来；剪接提取物将从 带或不带放射性标记的prp2突变体。互动中的 蛋白质或RNA可以通过使用UV交联来识别， 免疫学和凝胶电泳法。一种遗传方法 也被用来分离一种基因外抑制物，SRP2；它 抑制对温度敏感的prp2表型，并患有感冒- 敏感的表型本身。SRP2基因将被分离；其 在剪接中的作用及其与PRP2的关系将通过 分子遗传学技术。参与具体的、重要的 将分析剪接催化步骤中的SnRNAs序列 通过在分离功能基因之前切割这些序列 剪接体。热稳定性ESF-BN的表征及热稳定性 搜索prp2校对突变体将被给予较低的优先级。 银杏剪接体和ESFS重要成分的研究 酵母可能揭示前mRNA剪接的催化中心并导致 更好地理解Higher中的替代剪接和规范剪接 有机体。

RNA splicing, the biochemical process leading to the removal of introns from precursor RNA, is an essential step in the expression of genetic information. Unlike auto-catalyzed self-splicing reactions, splicing of nuclear pre-mRNA requires ATP and occurs in a complex, dynamic structure called the spliceosome composed of small nuclear ribonucleoproteins. A spliceosome containing unspliced pre-mRNA has been isolated from the yeast prp2 mutant. This functional spliceosome can be isolated and activated to splice upon the addition of ATP and extrinsic splicing factors (ESFs). One of the ESFs is an RNA-dependent ATPase encoded by the yeast PRP2 gene, which binds to the spliceosome and triggers splicing in the presence of ATP. Therefore, PRP2 may either be a key component of the catalytic center or function in a proofreading step. The long-term objectives of this research project are to understand the mechanism of nuclear pre-mRNA splicing through biochemical and genetic analysis of the spliceosomal components and the ESFs. Specifically, the components which constitute the catalytic center in the spliceosome will be investigated. Current studies focus on the identification of the spliceosomal components bound by PRP2 and the RNA activating the ATPase activity of PRP2. Radioactive PRP2 protein will be synthesized in vitro or isolated from yeast cells; splicing extracts will be prepared from prp2 mutants with or without radioactive labeling. The interacting proteins or RNAs can be identified by using UV cross-linking, immunological, and gel electrophoresis techniques. A genetic approach has also been taken to isolate an extragenic suppressor, SRP2; it suppresses the temperature-sensitive phenotype of prp2 and has a cold- sensitive phenotype by itself. The SRP2 gene will be isolated; its role in splicing and its relation with PRP2 will be investigated by molecular genetic techniques. The involvement of specific, important sequences of snRNAs in the catalytic steps of splicing will be analyzed by cleaving these sequences prior to the isolation of the functional spliceosome. The characterization of the heat stable ESF-bn and the search for prp2 proofreading mutants will be given a lower priority. Studies of the important constituents of the spliceosome and the ESFs in yeast may reveal the catalytic center of pre-mRNA splicing and lead to a better understanding of alternative and regulated splicing in higher organisms.