Analysis of the Ribonucleases of Arabidopsis thaliana

基因表达是一个多步骤的过程，对植物生长和发育的所有阶段都是基本的。到目前为止，这一过程的第一步，即转录，受到了最多的关注，因此在分子水平上也得到了最好的理解。然而，很明显，转录后事件可以对基因表达产生深远的影响。一个显著的转录后事件是信使核糖核酸的降解。在植物和其他真核细胞中，被认为在mRNA稳定水平上受到调控的基因数量正在迅速增加。尽管信使核糖核酸的稳定性在基因表达中很重要，但在植物和其他多细胞真核生物的细胞中催化信使核糖核酸衰变的酶还没有确定。这个项目将利用功能基因组和生化分析来研究拟南芥中几种潜在的mRNA降解酶(MRNase)的功能。假设这些酶在mRNA衰退中催化不同的步骤：AtPARN去掉聚(A)尾巴，AtDCP1与辅因子AtDCP2去掉帽，AtXRN4从一端开始降解RNA的其余部分。虽然DCPS和XRN1促进了酵母中大多数细胞质mRNAs的后两步，但它们在哺乳动物和植物等多细胞真核生物中的功能仍然是一个悬而未决的问题，因为与酵母相比，植物和哺乳动物表现出不同的mRNA衰退。然而，植物和动物中同源基因的存在以及其他观察表明，酵母将为所有真核生物共同的成分提供一些线索。因此，需要对多细胞真核生物中一般的mRNA衰变机制进行仔细的分析，以阐明在复杂系统中相对于酵母如何控制mRNA稳定性的基本原理。拟南芥被选为模型系统，是因为现有的实验工具，以及mRNA稳定性在建立植物基因表达水平方面的重要性。为了进一步了解AtPARN、AtDCP1和AtXRN4及其潜在的底物在全基因组范围内的mRNA衰变作用，本项目将1)对这些酶进行生物化学表征并检测它们在细胞内的位置，2)获得相应基因中的敲除或条件敲除突变体，3)通过DNA微阵列分析和其他RNA分析来分析这些突变体中最具信息量的突变体，以确定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，也没有相应的基因在任何多细胞真核生物中失活。因此，这些实验应该具有广泛的意义。此外，对于这些酶中的一些，已经发现了植物特有的特征；因此，这项工作有可能准确地定位新的和植物特有的mRNA衰退的方面。

Gene 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.