FUNCTIONAL SPLICEOSOMES AND EXTRINSIC SPLICING FACTORS

RNA剪接，导致内含子移除的生化过程 从前体RNA，是基因表达的重要步骤， 信息. 与自催化的自剪接反应不同， 核前mRNA需要ATP，并以复杂的动态结构发生 称为剪接体，由小的核核糖核蛋白组成。 一 含有未剪接的前mRNA的剪接体已从 酵母prp 2突变体。 这种功能性剪接体可以被分离， 在加入ATP和外源性剪接后被激活进行剪接 因素（ESF）。 ESF之一是RNA依赖性ATP酶，由 酵母PRP 2基因，它与剪接体结合并触发剪接 在ATP的存在下。 因此，PRP 2可能是一种关键成分， 在校对步骤中催化中心或功能。 本研究项目的长期目标是了解 核前体mRNA剪接的生化和遗传机制 剪接体成分和ESF的分析。 具体而言是 构成剪接体中催化中心的组分将 追究 目前的研究集中在识别 由PRP 2和激活ATP酶的RNA结合的剪接体组分 PRP 2的活性。 放射性PRP 2蛋白将在体外合成 或从酵母细胞中分离;剪接提取物将从 具有或不具有放射性标记的PrP 2突变体。 相互作用 蛋白质或RNA可以通过使用UV交联来鉴定， 免疫学和凝胶电泳技术。 遗传方法 还分离了一个基因外抑制基因SRP 2， 抑制prp 2的温度敏感表型，并具有冷- 敏感表型 本身 SRP 2基因将被分离;其 在剪接中的作用及其与PRP 2的关系将通过 分子遗传学技术 具体的、重要的 将分析剪接催化步骤中的snRNA序列 通过在分离功能性多肽之前切割这些序列， 剪接体 热稳定性ESF-BN的表征和热稳定性 对PrP 2校正突变体的搜索将被给予较低的优先级。 剪接体的重要成分及胚胎干细胞的研究 酵母可能揭示了前mRNA剪接的催化中心， 更好地理解选择性和调节性剪接， 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.