Kinetic Analysis of Yeast Pre-mRNA Degradation

酵母前体 mRNA 降解的动力学分析

基本信息

批准号：
7426334
负责人：
PATRICIA J HILLEREN
金额：
$ 18.11万
依托单位：
SKIDMORE COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-06-01 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7426334
关键词：
3&apos Untranslated Regions Binding Sites Boxing Cell Nucleus Cells Chimeric Proteins Clear Cell Collection Complex Confocal Microscopy Data Degradation Pathway Engineering Ensure Enzymes Eukaryotic Cell Event Exhibits Failure Frequencies Gene Expression Genes Genetic Transcription Goals Grant Heterogeneous Nuclear RNA Human Introns Kinetics Lesion Link Locales Mammalian Cell Measures Messenger RNA Mutation Nuclear Nuclear Decay Nuclear RNA Pathway interactions Physiologic pulse Process Proteome Pulse taking Quality Control RNA RNA Decay RNA Splicing RNA analysis Rate Reaction Relative (related person)Reporter Reporter Genes Research Research Personnel Role Saccharomyces cerevisiae Series Signal Transduction Site Speed Spliceosome Assembly Pathway Spliceosomes Staging Surveys System Temperature Tetracycline Tetracyclines Transcript Work Yeasts base computerized data processing in vivo mRNA Precursor mutant novel programs research study

项目摘要

DESCRIPTION (provided by applicant): The long term objective of this research is to understand the mechanism and pathways by which eukaryotic cells identify and eliminate defective RNA molecules as a way to ensure accuracy in gene expression. Alternative pre-mRNA processing is a fundamental means through which the complexity of the human proteome is enriched. However, pre-mRNA processing steps can be error prone due to reaction speed and the subtlety of processing signals. To handle these errors, cells have evolved multiple RNA quality control systems that minimize the expression of defective RNAs. Recent analysis in the yeast, Saccharomyces cerevisiae indicate that spliceosome assembly can be non-productive, and that the RNAs within these stalled complexes are targeted for degradation. In mammalian cells, the rate of non-productive spliceosome assembly may be high because of the complex processing of mammalian pre-mRNA. The goal of this work is to understand the how splice-defective pre-mRNAs are cleared from the cell. The specific aims are: 1. To determine the in vivo kinetics of synthesis, processing and decay of reporter pre-mRNAs and intermediates stalled at distinct stages of spliceosome assembly and function. Toward this end we will exploit a representative set of conditional yeast splicing mutants blocked at various stages of spliceosome assembly/function in transcription pulse chase reactions using an inducible intron reporter system. 2. To determine the mechanism, pathway and locale of degradation of splice defective pre-mRNA and intermediates. Toward this end we will combine lesions in known components of the RNA degradation pathways with conditional pre-mRNA splicing mutants and use these strains to measure RNA decay rates to determine those components responsible for degradation of the reporter pre-mRNA. In addition, we will verify the locale in which aberrant RNAs decay using confocal microscopy.