Structure-Function Analysis of Spliceosomal ATPases

描述（由申请人提供）：初级转录物的剪接是功能性mrna生成过程中必不可少的调控步骤。许多人类基因通过其主要转录物的选择性剪接表达两种或更多mrna，正常剪接模式的破坏通常与疾病有关。可选剪接位点的使用是通过调节蛋白的作用来实现的，这些调节蛋白促进或阻断构成剪接因子的募集，或影响它们的功能。了解确保有效和准确识别剪接位点的机制需要对核心剪接机制进行详细分析。我们的研究重点是第二步酯交换反应和产物释放。这些步骤取决于DEAH-box NTPases Prp16、Prp22和Prp43的顺序作用。步骤2中分子相互作用的生化解剖对于理解3'剪接位点的选择是如何改变的至关重要，无论是通过选择性剪接中的调节蛋白还是通过致病突变。剪接体拆卸步骤中的缺陷预计会影响后续的剪接体组装和催化，因为一些限制性剪接因子将被隔离在“产物复合物”中而无法再循环。从剪接体释放后，lariant内含子RNA中的2‘,5’磷酸二酯键被一种特殊的酶Dbr1“脱支”。我们正在研究Dbr1特异性识别和切割分支RNA的机制。Dbr1在内含子的周转中起重要作用，内含子构成转录体的很大一部分，并作为非编码小rna的储存库。项目描述：本项目研究mRNA剪接的机制，这是基因表达的一个基本步骤。缺陷和在这个过程中可以改变基因产物的结构和功能，从而导致疾病。了解mRNA剪接的基本机制对于理解缺陷如何导致疾病至关重要。

DESCRIPTION (provided by applicant): Splicing of primary transcript is an essential and regulated step in the generation of functional mRNAs. Many human genes express two or more mRNAs via alternative splicing of their primary transcript, and disruption of normal splicing patterns is often associated with disease. Usage of alternative splice sites is achieved through the action of regulatory proteins, which promote or block the recruitment of constitutive splicing factors, or influence their function. Understanding the mechanisms that ensure efficient and accurate recognition of splice sites demands a detailed analysis of the core splicing machinery. Our studies focus on the second transesterification step and product release. These steps depend on the sequential action of the DEAH-box NTPases Prp16, Prp22 and Prp43. Biochemical dissection of the molecular interactions required for step 2 is essential for understanding how 3' splice site choice can be altered, either by regulatory proteins during alternative splicing or by disease-causing mutations. Defects in spliceosome disassembly steps are expected to influence subsequent rounds of spliceosome assembly and catalysis, insofar as some limiting splicing factors will be sequestered in "product complexes" and fail to recycle. Upon release from the spliceosome, the 2',5' phosphodiester bond in the lariat-intron RNA is "debranched" by a specialized enzyme, Dbr1. We are investigating the mechanism by which Dbr1 specifically recognizes and cleaves branched RNA. Dbr1 plays an important role for the turnover of introns, which comprise a large portion of the transcriptosome and serve as reservoirs for non-coding small RNAs. Project Narrative: This project addresses the mechanism of mRNA splicing, a fundamental step in gene expression. Defects and in this process can alter the structure and function of a gene product thus lead to disease. Understanding the basic mechanism of mRNA splicing is critical to understand how defects can lead to disease.