Kinetic Analysis of Yeast Pre-mRNA Degradation

酵母前体 mRNA 降解的动力学分析

• 批准号: 6901646
• 负责人: PATRICIA J HILLEREN
• 金额: $ 20.67万
• 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/6901646
• 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）： 这项研究的长期目的是了解真核细胞识别并消除有缺陷的RNA分子的机制和途径，以确保基因表达的准确性。替代性mRNA加工是一种基本手段，通过它丰富了人类蛋白质组的复杂性。但是，由于反应速度和处理信号的微妙之处，前MRNA处理步骤可能是错误的。为了处理这些误差，细胞已经发展出多个RNA质量控制系统，以最大程度地减少RNA缺陷的表达。在酵母中，酿酒酵母的最新分析表明，剪接体组装可能是非生产力的，并且这些停滞的复合物中的RNA是针对降解的。在哺乳动物细胞中，由于哺乳动物前MRNA的复杂处理，非生产性剪接体组装的速率可能很高。这项工作的目的是了解如何从细胞中清除剪接缺陷的前MRNA。具体目的是：1。确定记者前MRNA的合成，处理和衰减的体内动力学和中间体在剪接体组装和功能的不同阶段停滞不前。为此，我们将利用可诱导的内含子报告器系统在转录脉冲追逐反应中的各个阶段封闭的有条件酵母剪接突变体一组代表性的条件酵母剪接突变体。 2。确定剪接缺陷前MRNA和中间体降解的机理，途径和位置。为此，我们将在RNA降解途径的已知成分中结合病变与条件MRNA剪接突变体，并使用这些菌株测量RNA衰减速率，以确定那些负责降解记者前MRNA降解的成分。此外，我们将验证使用共聚焦显微镜的异常RNA衰减的位置。