Uncovering Mechanisms of 5' Splice Site Fidelity

项目摘要/摘要 在高等真核生物中，前体信使rna(pre-mrna)的剪接是必不可少的，以产生 对蛋白质进行编码的功能性信使RNA。剪接反应是由一个大的 被称为剪接体的大分子机器。剪接体组装在前信使核糖核酸底物上 通过复杂的结合、重排和释放5个小的核RNA和100多个相关的 蛋白质。许多剪接体相关因子的确切作用还知之甚少。我们的实验室 以微小的线虫为模型，通过整合基因， 基因组和生化方法。我们已经建立了敏感的基因筛查来识别因素 对于剪接体在5‘端剪接位的准确组装很重要。我们的数据表明 假设关键剪接体组件在确保准确转移 5‘剪接位点从最初被U1snRNP识别到它被装载到剪接体的活性部位。 我们发现的显性抑制基因突变分为两类：1)那些允许 剪接体保持开放构象，这允许5‘剪接部位位置在两个 2)那些促进内含子使用的区域，该内含子 从一种不同寻常的解脲支原体二核苷酸开始。我们将探讨以下问题： 1.蛋白质因素如何控制剪接体组装过程中5‘端剪接位点的准确选择？ 我们假设拼接的剪接体的成分控制着5‘剪接的最终决定 选址。我们将研究PRPF8、SNRNP27和SNRNP200(Brr2)等位基因如何影响精确的5‘剪接 以蠕虫为单位的站点使用率。因为突变相同的人类遗传病等位基因普遍存在 在我们的基因筛查中探索的那些，我们也计划将我们的研究带入人类细胞来确定 在蠕虫中发现的等位基因可以成为剪接调控的翻译研究的目标。 2.KIN17和PRCC如何保持5‘s保真度？ 我们已经发现了一种现象，在这种现象中，由单个核苷酸分隔的两个5‘都被使用，即使在 存在野生型剪接体。我们已经确定了一类显性的抑制者，它们促进 使用以Uu开头的内含子，而不是标准的Gu二核苷酸。这些等位基因存在于蠕虫体内 人类KIN17和PRCC的同源物；对这两种蛋白在剪接中的作用知之甚少 它们与哺乳动物的BACT剪接体短暂地相互作用。我们现在有了功能剪接分析 这些因素可以用来表征它们在保持剪接位点保真度方面的功能。

PROJECT SUMMARY/ABSTRACT Precursor messenger RNA (pre-mRNA) splicing is essential in higher eukaryotes in order to produce functional messenger RNAs to code for proteins. The splicing reaction is carried out by a large macromolecular machine called the spliceosome. The spliceosome assembles onto pre-mRNA substrates through a complex binding, rearrangement and release of 5 small nuclear RNAs and over 100 associated proteins. The exact roles of many of the spliceosome-associated factors are poorly understood. Our lab uses the microscopic worm C. elegans as a model to explore pre-mRNA splicing through integrated genetic, genomic and biochemical approaches. We have established sensitives genetic screens to identify factors important for the accurate assembly of the spliceosome at the 5' splice site. Our data suggest the hypothesis that key spliceosomal components have an important role in securing the accurate transfer of the 5' splice site from its initial recognition by U1snRNP to its loading into the active site of the spliceosome. The dominant suppressor mutations that we uncovered fall into two classes; 1) those that allow the spliceosome to remain in an open conformation, which allows the 5' splice site position to slide between two regions 23nt apart during loading into the active site and 2) those that promote the usage of an intron that begins with an unusual UU dinucleotide. We will explore the following questions: 1. How do protein factors control the accurate selection of 5' splice sites during spliceosome assembly? We hypothesize that components of the assembled spliceosome control the final determination of 5' splice site selection. We will study how alleles of PRPF8, SNRNP27 and SNRNP200 (Brr2) affect precise 5' splice site usage in worms. Because of the prevalence of human genetic disease alleles with mutations identical to the ones explored in our genetic screen, we also plan to take our studies into human cells to determine if the alleles identified in worms can be targets for translational research into splicing regulation. 2. How do KIN17 and PRCC maintain 5'ss fidelity? We have found a phenomenon in which two 5'ss separated by a single nucleotide are both used even in the presence of a wild type spliceosome. We have identified a class of dominant suppressors that promote usage of introns that begin with UU instead of the canonical GU dinucleotide. These alleles are in the worm homologs of human KIN17 and PRCC; little is known about a role for either protein in splicing other than that they transiently interact with mammalian Bact spliceosomes. We now have functional splicing assays for these factors to exploit in characterizing their function in maintaining splice site fidelity.