Molecular Recognition during pre-mRNA Splicing

描述（由申请人提供）：前体mRNA的剪接为细胞分化和发育提供了转录物多样性的主要来源。剪接的过程需要一个被称为剪接体的剪接机器，它包含约100个蛋白质和5个小的核（sn）RNA。前mRNA剪接位点上剪接体组装的早期阶段是人类遗传疾病和癌症中经常出错的关键调控步骤。然而，剪接体究竟如何从数千个相互竞争的前mRNA序列中选择并切除正确的剪接位点，在分子水平上仍然知之甚少。该项目的总体目标是了解由U2 AF 65、U2 AF 35和SF 1蛋白组成的基本复合物如何准确靶向3'剪接位点，从而导致剪接体组装。假设#1：我们对3'剪接位点RNA的U2 AF 65识别的“全景”理解-从我们完整的U2 AF 65的新结构以及过去对核心结构域的工作中出现-可以用于理解特定人类疾病的前mRNA剪接位点突变。假设#2：我们将扩大我们以前的结果和初步数据，以测试剪接体亚基SF 3b 155作为一个“分子枢纽”协调磷酸化敏感的组件，包括U2 AF 65，癌症相关因子paradoxin CAPERa，和p14亚基，这反过来又接触剪接体的分支位点亲核试剂。假设#3：我们创新的SF 1/U2 AF 65/U2 AF 35/RNA制备使我们能够定位复合物中的蛋白质和RNA亚基，并测试经常导致骨髓增生异常、血液恶性肿瘤和肺癌的U2 AF 35突变的结构和功能影响。我们的总体方法需要使用多学科战略对所有目标进行多方面攻击。我们的核心生物物理技术包括X射线晶体学、荧光各向异性、等温滴定量热法和纯化蛋白质的小角X射线散射。为了迎接挑战并推动该领域向新的方向发展，我们将利用创新方法的强大组合，包括蛋白质和RNA标记，福斯特共振能量转移，小角中子散射和位点特异性光交联，然后进行LC-MS/MS。这些工具通过强有力的合作来补充，以研究核提取物和活细胞中的剪接因子功能。我们的研究基于3'剪接位点识别的基本结构和功能，但将广泛影响该领域对异常剪接的理解，异常剪接是血液疾病，神经肌肉疾病，白血病和癌症的主要原因。

DESCRIPTION (provided by applicant): Splicing of pre-mRNAs provides a major source of transcript diversity for cell differentiation and development. The process of splicing requires a splicing machine known as the spliceosome that comprises ~100 proteins and five small nuclear (sn)RNAs. The early stage of spliceosome assembly on pre-mRNA splice sites is a key regulated step that often goes awry in human genetic diseases and cancers. Yet, exactly how the spliceosome selects and excises the correct splice sites from amidst thousands of competing pre-mRNA sequences remains poorly understood at the molecular level. The overall goal of this project is to understand how an essential complex comprising U2AF65, U2AF35, and SF1 proteins can accurately target 3' splice sites, leading to spliceosome assembly. Specific aims of this proposal will test the following central hypotheses concerning the critical early stages of pre-mRNA splicing: Hypothesis #1: Our 'panoptic' understanding of U2AF65 recognition of 3' splice site RNAs - emerging from our new structures of intact U2AF65 as well as past work on core domains- can be used to understand pre-mRNA splice site mutations from specific human diseases. Hypothesis #2: We will expand our prior results and preliminary data to test the spliceosome subunit SF3b155 as a "molecular hub" coordinating phosphorylation-sensitive assemblies comprising U2AF65, the cancer-related factor paralogue CAPERa, and the p14 subunit, which in turn contacts the branch-site nucleophile of the spliceosome. Hypothesis #3: Our innovative SF1/U2AF65/U2AF35/RNA preparation positions us to locate the protein and RNA subunits in the complex, and to test the structural and functional effects of U2AF35 mutations that frequently cause myelodysplasia, hematological malignancies and lung cancer. Our overall approach entails a multi-front attack on all aims using a multidisciplinary strategy. Our core biophysical technologies entail X-ray crystallography, fluorescence anisotropy, isothermal titration calorimetry, and small-angle X-ray scattering with purified proteins. To meet challenges and propel the field in new directions, we will utilize a powerful combination of innovative methods including protein and RNA labeling, Forster resonance energy transfer, small-angle neutron scattering, and site-specific photo-crosslinking followed by LC-MS/MS. These tools are complemented by strong collaborations to study splicing factor functions in nuclear extracts and in living cells. Our research is grounded in the fundamental structure and function of 3' splice site recognition yet will broadly impact the field's understanding of aberran splicing, which is a dominant cause of blood disorders, neuromuscular diseases, leukemias and cancers.