Analysis of the Ribonucleases of Arabidopsis thaliana

拟南芥核糖核酸酶分析

• 批准号: 0096394
• 负责人: Pamela Green
• 金额: $ 36万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0096394&HistoricalAwards=false
• 关键词: Analysis; Ribonucleases; Arabidopsis; thaliana

0096394 Pam GreenGene expression is a multi-step process fundamental to all phases of plant growth and development. The first step in the process, transcription, has thus far received the most attention and therefore is the most well understood at the molecular level. However, it is clear that post-transcriptional events can have profound effects on gene expression. One prominent post-transcriptional event is that of mRNA degradation. In plant and other eukaryotic cells, the number of genes considered to be regulated at the level of mRNA stability is increasing rapidly. Despite the importance of mRNA stability in gene expression, the enzymes that catalyze mRNA decay in cells of plants and other multicellular eukaryotes have yet to be identified. This project will address the function of several potential mRNA degrading enzymes (mRNases) in Arabidopsis using functional genomic and biochemical analysis. The hypothesis is that these enzymes catalyze different steps in mRNA decay: AtPARN removes the poly(A) tail, AtDCP1 with co-factor AtDCP2 removes the cap, and AtXRN4 degrades the remainder of the RNA starting from one end. Although the DCPs and XRN1 facilitate the latter two steps of most cytoplasmic mRNAs in yeast, their functions in multicellular eukaryotes such as mammals and plants remain an open question because plants and mammals exhibit differences in mRNA decay compared to that in yeast. Nevertheless, the presence of homologous genes in plants and animals, as well as other observations, suggests that yeast will provide some clues about components common to all eukaryotes. Therefore, what is needed is a careful analysis of the general mRNA decay machinery in a multicellular eukaryote to elucidate the fundamentals of how mRNA stability is controlled in complex systems versus yeast. Arabidopsis has been chosen as the model system because of the experimental tools available and the importance of mRNA stability in establishing plant gene expression levels. To further understanding of the mRNA decay roles of AtPARN, AtDCP1, and AtXRN4, and their potential substrates genome-wide, this project will 1) characterize the enzymes biochemically and examine their intracellular locations, 2) obtain knockout or "conditional" knockout mutants in the corresponding genes, and 3) analyze the most informative of these mutants via DNA microarray analysis and other RNA analyses to identify mRNA substrates. The roles and potential targets of these putative mRNases have never been studied on a global scale nor have the corresponding genes been inactivated in any multicellular eukaryote. Therefore, these experiments should have broad significance. Moreover, for some of these enzymes, characteristics unique to plants have been discovered; thus this work has the potential to pinpoint aspects of mRNA decay that are novel and plant specific.

Pam GreenGene表达是植物生长和发育的所有阶段的基础的多步骤过程。这个过程的第一步，转录，迄今为止受到了最多的关注，因此在分子水平上是最好理解的。然而，很明显，转录后事件可以对基因表达产生深远的影响。一个突出的转录后事件是mRNA降解。在植物和其他真核细胞中，被认为在mRNA稳定性水平上受到调控的基因数量正在迅速增加。尽管mRNA稳定性在基因表达中的重要性，但在植物和其他多细胞真核生物细胞中催化mRNA衰变的酶尚未被鉴定。本研究将利用功能基因组学和生物化学分析来研究拟南芥中几种潜在的mRNA降解酶（mRNases）的功能。假设这些酶催化mRNA降解的不同步骤：AtPARN去除poly（A）尾，AtDCP 1与辅因子AtDCP 2去除帽，AtXRN 4从一端开始降解RNA的剩余部分。虽然DCP和XRN 1促进了酵母中大多数细胞质mRNA的后两个步骤，但它们在多细胞真核生物如哺乳动物和植物中的功能仍然是一个悬而未决的问题，因为与酵母相比，植物和哺乳动物在mRNA衰变方面表现出差异。 然而，植物和动物中同源基因的存在，以及其他观察结果，表明酵母将提供一些线索，了解所有真核生物共有的成分。因此，我们需要仔细分析多细胞真核生物中的一般mRNA衰变机制，以阐明mRNA稳定性在复杂系统中与酵母相比如何控制的基本原理。 选择拟南芥作为模型系统是因为实验工具的可用性和mRNA稳定性在建立植物基因表达水平中的重要性。为了进一步了解AtPARN、AtDCP 1和AtXRN 4的mRNA降解作用及其潜在的全基因组底物，本项目将1）对酶进行生物化学表征并检查其细胞内位置，2）获得相应基因的敲除或“条件性”敲除突变体，和3）通过DNA微阵列分析和其它RNA分析来分析这些突变体中信息量最大的，以鉴定mRNA底物。这些假定的mRNase的作用和潜在靶点从未在全球范围内进行过研究，也没有在任何多细胞真核生物中灭活相应的基因。 因此，这些实验应该具有广泛的意义。 此外，对于其中一些酶，已经发现了植物特有的特征;因此这项工作有可能查明mRNA衰变的新方面和植物特异性。