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Structure-Function Analysis of Spliceosomal ATPases

剪接体 ATP 酶的结构功能分析

基本信息

批准号：
8069334
负责人：
BEATE SCHWER
金额：
$ 36.94万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-01-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8069334
关键词：
3&apos Splice Site 5&apos Splice Site ATP phosphohydrolase Address Affect Alternative Splicing Binding Biochemical Boxing Catalysis Cleaved cell Complex Defect Deoxyribonucleotides Deposition Disease Dissection Elements Ensure Enzymes Event Exons Freezing Functional RNA Gene Expression Generations Genes Goals Human Hydrolysis Introns Joining Exons Lead Link Messenger RNA Methods Modeling Modification Nuclear Pattern Play Positioning Attribute Precursor RNA Process Proteins RNA RNA Binding RNA Splicing Reaction Recycling Research Proposals Role Site Small Nuclear Ribonucleoproteins Small RNA Spliceosome Assembly Pathway Spliceosomes Staging Structure Substrate Specificity Testing Transcript U5 Small Nuclear Ribonucleoprotein Vertebral column Work disease-causing mutation genetic regulatory protein helicase mRNA Precursor novel phosphodiester phosphoric diester hydrolase research study ribose phosphate

项目摘要

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.

描述（由申请人提供）：初级转录物的剪接是产生功能性mRNA的必要和受调控的步骤。许多人类基因通过其初级转录物的选择性剪接表达两种或更多种mRNA，并且正常剪接模式的破坏通常与疾病相关。可变剪接位点的使用是通过调节蛋白的作用实现的，所述调节蛋白促进或阻断组成型剪接因子的募集，或影响它们的功能。了解机制，确保有效和准确的识别剪接位点需要详细分析的核心剪接机制。我们的研究集中在第二个酯交换步骤和产品释放。这些步骤取决于DEAH盒NTPases Prp 16、Prp 22和Prp 43的顺序作用。步骤2所需的分子相互作用的生物化学解剖对于理解3'剪接位点的选择如何被改变是必不可少的，无论是通过可变剪接过程中的调节蛋白还是通过致病突变。预期剪接体拆卸步骤中的缺陷会影响随后几轮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”中并且不能再循环。当从剪接体释放时，lariat-内含子RNA中的2 '，5'磷酸二酯键被一种专门的酶Dbr 1“去分支”。我们正在研究Dbr 1特异性识别和切割分支RNA的机制。Dbr 1在内含子的周转中起着重要作用，内含子构成转录体的大部分，并作为非编码小RNA的储存库。项目叙述：该项目涉及mRNA剪接的机制，这是基因表达的基本步骤。在这一过程中，基因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理解缺陷如何导致疾病至关重要。