Uncovering Mechanisms of 5' Splice Site Fidelity

项目总结/摘要 前体信使RNA（pre-mRNA）剪接在高等真核生物中是必不可少的，以便产生 编码蛋白质的信使RNA。剪接反应是由一个大的 一种叫做剪接体的大分子机器。剪接体在前体mRNA底物上组装 通过复杂的结合，重排和释放5个小的核RNA和超过100个相关的 proteins.许多剪接体相关因子的确切作用知之甚少。我们实验室 使用的是微型蠕虫C线虫作为一个模型，探索前mRNA剪接通过整合的遗传， 基因组和生物化学方法。我们已经建立了敏感的遗传筛选，以确定因素 这对于剪接体在5 ′剪接位点的精确组装是重要的。我们的数据表明， 假设关键的剪接体成分在确保准确转移中起重要作用， 5 '剪接位点从其最初被U1snRNP识别到其装载到剪接体的活性位点。 我们发现的显性抑制基因突变分为两类：1）那些允许基因突变的突变。 剪接体保持开放构象，这允许5 '剪接位点位置在两个剪接体之间滑动。 在加载到活性位点期间间隔23nt的区域，和2）促进内含子使用的那些， 由一个不寻常的UU二核苷酸开始我们将探讨以下问题： 1.剪接体组装过程中蛋白质因素如何控制5 '剪接位点的准确选择？ 我们假设组装的剪接体的组成部分控制5 '剪接的最终决定 选址我们将研究PRPF 8、SNRNP27和SNRNP200（Brr 2）的等位基因如何影响精确的5 '剪接 网站使用蠕虫。由于人类遗传疾病等位基因的流行， 除了在我们的基因筛选中探索的那些，我们还计划将我们的研究带入人类细胞，以确定是否 在蠕虫中鉴定的等位基因可以作为剪接调节的翻译研究的目标。 2. KIN17和PRCC如何保持5'ss保真度？ 我们已经发现了这样一种现象，其中被单个核苷酸分开的两个5 'ss即使在 野生型剪接体的存在。我们已经确定了一类主要的抑制因子， 使用开始于UU而不是典型的GU二核苷酸的内含子。这些等位基因在蠕虫中 人KIN17和PRCC的同源物;除了 它们能与哺乳动物的细菌剪接体短暂相互作用。我们现在有功能性剪接测定， 利用这些因素来表征它们在维持剪接位点保真度中的功能。

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.