Structure-Function Analysis of Spliceosomal ATPases

描述(由申请人提供)：初级转录本的剪接是产生功能性mRNAs的重要和受调控的步骤。许多人类基因通过其初级转录的选择性剪接表达两个或更多的mRNAs，正常剪接模式的中断通常与疾病有关。选择性剪接位点的使用是通过调控蛋白的作用实现的，调控蛋白促进或阻止结构性剪接因子的招募，或影响其功能。要了解确保高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。我们的研究主要集中在酯交换的第二步和产物的释放上。这些步骤取决于Deah-box NTPase Prp16、Prp22和Prp43的顺序作用。对步骤2所需的分子相互作用的生化解剖对于了解3‘剪接位点的选择如何被选择性剪接过程中的调控蛋白或致病突变所改变是至关重要的。剪接体分解步骤中的缺陷预计会影响随后几轮剪接体的组装和催化，因为一些限制性剪接因子将被隔离在“产品复合体”中，无法回收利用。当从剪接体中释放出来时，套索内含子RNA中的2‘，5’磷酸二酯键被一种特殊的酶DBR1“去分支”。我们正在研究DBR1特异性识别和切割分支RNA的机制。DBR1对内含子的周转起着重要的作用，内含子构成了转录体的很大一部分，是非编码小RNA的储存库。项目简介：该项目阐述了基因表达中的一个基本步骤--信使核糖核酸剪接的机制。在这个过程中，缺陷会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了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.