Molecular Recognition during pre-mRNA Splicing

描述(申请人提供)：前mRNAs的剪接为细胞分化和发育提供了转录多样性的主要来源。剪接的过程需要一个被称为剪接体的剪接机，它由大约100种蛋白质和5个小的核(Sn)RNA组成。剪接体在前信使核糖核酸剪接位点上组装的早期阶段是一个关键的调控步骤，在人类遗传性疾病和癌症中经常出错。然而，剪接体如何从数千个相互竞争的Pre-mRNA序列中选择和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U2AF65、U2AF35和SF1蛋白组成的基本复合体如何准确地靶向3‘剪接位点，从而导致剪接体组装。这一提议的具体目的将检验以下关于前mRNA剪接关键早期阶段的中心假设：假设1：我们对U2AF65识别3‘剪接位点RNA的“全景”理解--来自我们完整的U2AF65的新结构以及过去在核心域上的工作--可用于理解特定人类疾病的前mRNA剪接位点突变。假设2：我们将扩展我们先前的结果和初步数据，以测试剪接体亚单位SF3b155作为协调磷酸化敏感组件的“分子枢纽”，包括U2AF65、癌症相关因子辅助体CAPERa和p14亚基，后者进而接触剪接体的分支部位的亲核者。假设3：我们创新的SF1/U2AF65/U2AF35/RNA制剂使我们能够定位复合体中的蛋白质和RNA亚基，并测试U2AF35突变对结构和功能的影响，这些突变经常导致骨髓发育不良、血液系统恶性肿瘤和肺癌。我们的总体方法需要使用多学科战略对所有目标进行多战线攻击。我们的核心生物物理技术包括X射线结晶学、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。为了迎接挑战并将该领域推向新的方向，我们将利用强大的创新方法组合，包括蛋白质和RNA标记、Forster共振能量转移、小角中子散射和随后的LC-MS/MS定点光交联法。这些工具与这些工具相辅相成，研究核提取物和活细胞中剪接因子的功能。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.