GENETIC ANALYSIS OF PREMRNA SPLICING IN S POMBE

U1 SNRNP与其他剪接因子合作，促进 内含子末端和核形成的并列 催化活性剪接体准备连接一对特定的 外显子。拟议研究的长期目标是提供一个 RNA-RNA、RNA-蛋白质和蛋白质-蛋白质的分子描述 通过利用简单的基因，这些事件所需的交互 裂殖酵母中可利用的操作方法。 第一个主要目标将是阐明动态的单链RNA底物 在第一次酯交换反应之前的相互作用 研究的三个途径：i)确定U1和U5短链RNA是否 在从承诺复合体到催化剂的过渡过程中进行互动 活性剪接体及其与剪接的配对相互作用 将对底物进行单独和组合调制；ii)确定 哪些小RNA在非共识5‘的激活中起决定性作用 剪接位点，将进行补偿碱基分析；以及iii) 确定加强或不稳定U1与5‘配对的因素和 3‘剪接位点，条件等位基因将被用于抑制 选择和协同致死筛选.表征 出现的蛋白质和RNA的突变将提供信息 关于这些组件如何在物理和/或功能上与 U1。第二个主要目标将是执行结构/功能分析 并分析与两个已知中介因素相互作用的组件 利用突变分析研究前信使RNA剪接的早期事件 接着是抑制子选择：i)该基因的大亚基 异二聚体剪接因子U2AF，与多嘧啶结合 在早期的ATP不依赖的复合体中将内含子连接到 剪接，并随后促进稳定的U2 SnRNP与 分支点和ii)多聚体剪接因子的组成部分Sap49 SF3B，据信在传递和捆绑 U2 SnRNP连接到分支点。这些实验的结果将 促进构建早期事件的详细模型 前信使RNA剪接。因为S.pombe包含带有 由次优剪接信号连接的多个内含子，数据 生成的数据应该与理解两者特别相关 多细胞生物体中的结构性剪接和选择性剪接。

The U1 snRNP, in collaboration with other splicing factors, promotes juxtaposition of intron termini and nucleates formation of a catalytically active spliceosome poised to join a particular pair of exons. The long-term goal of the proposed research is to provide a molecular description of the RNA-RNA, RNA-protein, and protein-protein interactions required for these events by exploiting the facile genetic manipulations available in the fission yeast Schizosaccharomyces pombe. The first major goal will be to illuminate dynamic snRNA-substrate interactions that precede the first transesterification reaction through three avenues of investigation: i) To determine whether U1 and U5 snRNAs interact during the transition from commitment complex to catalytically active spliceosome, their pairing interactions with the splicing substrate will be modulated, singly and in combination; ii) To determine which snRNAs play decisive roles in activation of a non-consensus 5' splice site, compensatory base analysis will be performed; and iii) To identify factors that potentiate or destabilize U1 pairing to the 5' and 3' splice sites, conditional alleles will be used for suppressor selection and synergistic lethal screening; characterization of the mutations in the proteins and RNAs that emerge will provide information about how these components physically and/or functionally interact with U1. The second major goal will be to perform structure/function analysis and analyze components that interact with two factors known to mediate early events in premessenger RNA splicing using mutational analysis followed by suppressor selection: i) the large subunit of the heterodimeric splicing factor U2AF, which binds to the polypyrimidine tract in early ATP-independent complexes that commit an intron to splicing and subsequently promotes stable U2 snRNP binding to the branchpoint and ii) Sap49, a component of the multimeric splicing factor SF3b, which is believed to play a role in delivering and tethering the U2 snRNP to the branchpoint. The results of these experiments will facilitate the construction of detailed models for early events in premessenger RNA splicing. Because S. pombe contains pre-mRNAs with multiple introns bonded by sub-optimal splicing signals, the data generated should be particularly relevant to understanding both constitutive and alternative splicing in multicellular organisms.