GENETIC ANALYSIS OF PREMRNA SPLICING IN S POMBE

U1 snRNP与其他剪接因子合作，促进 内含子末端和有核体的并置 催化活性剪接体准备加入一对特定的 外显子 这项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 这些事件所需的相互作用，通过利用简单的遗传 操作可在裂殖酵母裂殖酵母粟酒裂殖酵母。 第一个主要目标将是阐明动态snRNA底物 在第一次酯交换反应之前的相互作用， i）确定U1和U 5 snRNA是否 在从承诺复合体向催化性过渡期间相互作用 活性剪接体，它们与剪接的配对相互作用 将单独和组合地调节底物; ii）确定 哪些snRNA在激活非共有5'端转录因子中起决定性作用 剪接位点，将进行补偿碱基分析;和iii）为了 鉴定增强或破坏U1与5'端配对的因子， 3'剪接位点，条件等位基因将被用于抑制 筛选和协同致死筛选; 出现的蛋白质和RNA的突变将提供信息 这些组件如何在物理上和/或功能上与 U1 第二个主要目标是进行结构/功能分析 并分析与两个已知的介导因素相互作用的成分， 用突变分析研究前信使RNA剪接的早期事件 随后进行抑制子选择：i）抑制子的大亚基， 异二聚体剪接因子U2 AF，其结合多聚嘧啶 在早期ATP不依赖复合体中的束，将内含子提交给 剪接并随后促进稳定的U2 snRNP结合至 分支点和ii）Sap 49，多聚体剪接因子的组分 SF 3b被认为在传递和束缚 U2 snRNP到分支点。 这些实验的结果将 促进早期事件的详细模型的构建， 前信使RNA剪接。 因为S.粟酒含有前mRNA， 通过次优剪接信号结合的多个内含子，数据 产生的应该是特别相关的理解， 多细胞生物体中的组成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.