Structural Biology of RNA Splicing

描述（由申请人提供）： RNA剪接的结构生物学。RNA剪接是每个真核生物生存所必需的基本生物学机制。它涉及从前信使RNA中切除非编码内含子并连接相邻外显子以产生用于核糖体翻译的成熟mRNA。剪接体内含子占人类基因组的约30%，并且关于剪接体催化的剪切机制的精确结构细节知之甚少。第二组内含子被认为是剪接体内含子的祖先，因此我们使用第二组内含子作为一个易于处理的模型系统，以获得详细的分子洞察真核剪接。在此之前，PI致力于确定II组内含子的第一个晶体结构。该结构揭示了内含子通过双金属离子机制剪接。然而，这种结构是一个水解的线性II组内含子，缺少一个称为结构域6的重要区域。这个关键的区域含有凸出的腺苷，负责形成被称为lactase的分支RNA。内含子由一个不寻常的2 '-5'磷酸二酯键组成，该键位于凸起的腺苷的核糖和内含子的5'端之间。剪接体内含子中类似的基序称为分支位点。分支位点的突变导致脂质形成缺陷，这是几种人类疾病的原因。剪接体的剪接位点选择是剪接体选择剪接位点的关键，也是真核生物剪接的特征。我们的长期目标是通过研究形成lariat的II组内含子来获得真核剪接机制的知识。我们将使用生物化学和结构的方法：1）确定lactamins形成的结构基础。2)分析剪接途径中的催化中间体，以确定与剪接进展相关的构象变化。这与真核生物中剪接体催化的RNA剪接有直接的相似之处。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT Structural biology of RNA splicing. RNA splicing is a fundamental biological mechanism essential for the survival of every eukaryotic organism. It involves the excision of non-coding introns from pre-messenger RNAs and ligation of the adjacent exons to generate mature mRNA for translation by the ribosome. Spliceosomal introns comprise ~30% of the human genome and there is relatively little known about the precise structural details of their mechanism of excision catalyzed by the spliceosome. Group II introns are considered to be the ancestors of spliceosomal introns, therefore we are using the group II intron as a tractable model system to gain detailed molecular insight into eukaryotic splicing. Previously, the PI worked to determine the first crystal structure of a group II intron. This structure revealed that introns splice via a two-metal ion mechanism. However, this structure was of a hydrolytic, linear group II intron that was missing an important region called domain 6. This crucial domain contains the bulged adenosine responsible for forming the branched RNA known as a lariat. The lariat consists of an unusual 2'-5' phosphodiester bond between the ribose sugar of the bulged adenosine and the 5' end of the intron. The analogous motif in spliceosomal introns is called the branch site. Mutations in the branch site lead to defects in lariat formation that are the cause of several human diseases. Lariat formation is essential for proper splice site selection by the spliceosome and is considered to be a characteristic hallmark of eukaryotic splicing. Our long-term goal is to gain knowledge of eukaryotic splicing mechanisms by studying a lariat-forming group II intron. We will use biochemical and structural approaches to: 1) Determine the structural basis for lariat formation. 2) Analyze catalytic intermediates in the splicing pathway to determine the conformational changes that are associated with the progression of splicing. This is expected to have direct parallels with RNA splicing catalyzed by the spliceosome in eukaryotes.