Analysis of the Ribonucleases of Arabidopsis thaliana

基因的调控对生长发育的各个阶段都具有根本的重要性。基因的功能是产生信使rna分子，将遗传信息传递给制造蛋白质的机器，而蛋白质是基因的产物。由于mRNA的数量通常与蛋白质产生的数量有关，因此许多基因调控机制控制着mRNA的合成或降解速率。本项目主要研究模式植物拟南芥中降解mRNA的两种核糖核酸酶（RNases）。对其中一种5‘-3’外核糖核酸酶的研究将主要集中在AtXRN4的功能上。先前由NSF资助的研究表明，AtXRN4优先降解特定的mRNA分子，包括被非常小的rna（称为miRNAs）靶向沉默的mRNA子集。为了更好地理解这种特异性的基础，该项目将解决mRNA在细胞内位置和mRNA序列的重要性。死酶是第二种需要研究的rna酶。拟南芥聚(A)核糖核酸酶（AtPARN）和拟南芥Ccr4p死基化酶家族将被检测以确定它们优先降解哪些mrna。这将通过使用缺乏一种或多种这些死酶的突变植物来完成生化和分子生物学实验，如DNA微阵列分析，一种一次性确定数千个基因mRNA水平的技术。这项工作将在植物领域之外产生广泛的影响，因为类似的问题尚未在其他多细胞生物中得到回答。特别是，涉及miRNA的基因调控机制对植物和动物都至关重要。因此，了解为什么AtXRN4降解选定的miRNA靶标对许多系统中的XRN同源物具有重要意义。同样，PARN和多个Ccr4p同源物在酵母中不存在，而在酵母中已经完成了许多mRNA降解的开创性工作，因此对这些deadenylase的研究对其他真核生物也应该具有广泛的意义。除了对基础知识的贡献之外，该项目还将生成数据、突变体和其他工具，这些工具将向公众开放。该项目的实施将有助于包括本科生和邻近机构的各种年轻科学家的教育和职业发展。

The regulation of genes is of fundamental importance for all phases of growth and development. Genes function by producing mRNA molecules that carry genetic information to the machinery that makes proteins, which are the products of genes. Because the amount of mRNA is generally related to the amount of protein produced, many gene regulatory mechanisms control the synthesis or degradation rates of mRNAs. This project focuses on two types of ribonucleases (RNases) that degrade mRNA in the model plant Arabidopsis. Work on one type, the 5'-3' exoribonucleases, will concentrate primarily on the function of AtXRN4. Previous work funded by NSF showed that AtXRN4 preferentially degrades specific mRNA molecules, including a subset of the mRNAs targeted for silencing by very small RNAs called miRNAs. To better understand the basis of this specificity, this project will address the importance of mRNA location within the cell and of sequences within mRNAs. Deadenylases are the second type of RNase to be studied. Both Arabidopsis Poly(A) Ribonuclease (AtPARN) and the Arabidopsis family of Ccr4p deadenylases will be examined to determine which mRNAs they preferentially degrade. This will be accomplished by using mutant plants lacking one or more of these deadenylases for biochemical and molecular biological experiments such as DNA microarray analysis, a technology for determining mRNA levels for thousands of genes at once. This work will have broad impact beyond the plant field because similar questions have yet to be answered in other multicellular organisms. In particular, gene regulatory mechanisms involving miRNA are critical for both plants and animals. Therefore, understanding why AtXRN4 degrades selected miRNA targets has implications for XRN homologs in many systems. Similarly, PARN and multiple Ccr4p homologs are not present in yeast, in which much of the pioneering work on mRNA degradation has been done, so work on these deadenylases should also be of broad significance for other eukaryotes. Beyond its contributions to basic knowledge, this project will generate data, mutants and other tools that will be made publicly available. Execution of this project will contribute to the education and career development of diverse young scientists including undergraduates and those from a neighboring institution.