Kinetic Analysis of Yeast Pre-mRNA Degradation

酵母前体 mRNA 降解的动力学分析

• 批准号: 7051409
• 负责人: PATRICIA J HILLEREN
• 金额: $ 18.37万
• 依托单位: SKIDMORE COLLEGE
• 依托单位国家: 美国
• 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/7051409
• 关键词: RNA biosynthesis; RNA splicing; Saccharomyces cerevisiae; cell nucleus; confocal scanning microscopy; eukaryote; gene expression; gene mutation; genetic transcription; green fluorescent proteins; introns; messenger RNA; nucleic acid metabolism; precursor mRNA; reporter genes; spliceosomes; transfection /expression vector

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.

描述（由申请人提供）： 这项研究的长期目标是了解真核细胞识别和消除缺陷 RNA 分子的机制和途径，以确保基因表达的准确性。替代性前 mRNA 加工是丰富人类蛋白质组复杂性的基本手段。然而，由于反应速度和处理信号的微妙性，前 mRNA 处理步骤可能容易出错。为了处理这些错误，细胞进化出了多种 RNA 质量控制系统，最大限度地减少有缺陷的 RNA 的表达。最近对酿酒酵母的分析表明，剪接体组装可能是非生产性的，并且这些停滞复合物内的 RNA 会被靶向降解。在哺乳动物细胞中，由于哺乳动物前 mRNA 的复杂加工，非生产性剪接体组装的比率可能很高。这项工作的目标是了解如何从细胞中清除剪接缺陷的前 mRNA。具体目标是： 1. 确定在剪接体组装和功能的不同阶段停滞的报告基因前体 mRNA 和中间体的合成、加工和衰变的体内动力学。为此，我们将利用诱导型内含子报告系统在转录脉冲追踪反应中在剪接体组装/功能的各个阶段被阻断的一组代表性条件酵母剪接突变体。 2. 确定剪接缺陷前体mRNA和中间体降解的机制、途径和部位。为此，我们将把 RNA 降解途径的已知成分中的损伤与条件前 mRNA 剪接突变体结合起来，并使用这些菌株来测量 RNA 衰减率，以确定负责报告基因前 mRNA 降解的那些成分。此外，我们将使用共聚焦显微镜验证异常 RNA 衰变的区域。