Analysis of the Ribonucleases of Arabidopsis thaliana

基因表达的变化对植物的生长发育和抗病起着至关重要的作用。更好地了解基因表达是如何调控的，是植物工程过程的基础，因此也是提高农业生产的基础。植物在多个水平上调节基因的表达，该项目的目的是表征被称为内切核酸酶的蛋白质，它在mRNA降解的水平上调节基因的表达。这个项目的一个重要方面是确定这些内切核酸酶如何影响植物的生物学，包括植物的生长和发育。该项目将对年轻科学家进行基因调控基本方面的培训。这位年轻的科学家将通过与系统生物学和化学工程专家的合作伙伴进行互动，接受跨学科培训。科学家的影子计划将使中学生有机会与科学家见面，参观研究实验室，并观看正在进行的这个项目的实验。其他外联活动的目的是让公众参与，并激发人们对各种背景和年龄段的人的科学兴趣。该项目的长期目标是阐明核糖核酸酶(RNase)在拟南芥中参与mRNA降解的作用。研究内切核糖核酸酶在基因转录后控制中的作用是一个不发达的研究领域，但这是一个及时的领域。这项研究的假设是，阐明内切核酸酶的靶标和转录后影响将有助于我们了解植物mRNA衰退的关键角色，如外切体，以及鲜为人知或假定的内切核酸酶，其作用尚未被发现。为了测试这个想法和其他想法，该项目将首先创建一组双重突变体来稳定内切核酸酶产生的mRNA衰退中间产物，以便能够对它们进行深度测序；为了实现这一点，将在xrn4缺失突变背景中引入单个内切核酸酶突变。其次，为了确定内切核酸酶的底物，将使用RNA末端平行分析和RNA-Seq数据在全球范围内分析双突变体和对照中的RNA衰变中间体和RNA水平。如果任何一种内切核酸酶作用于无意义介导的信使核糖核酸酶(NMD)靶标，这将是耐人寻味的，因为植物缺乏后生动物NMD内切核酸酶SMG6。第三，内切核酸酶的新生物学影响将根据其裂解底物的功能关联在单个突变体中进行假设和测试。第四，将研究涉及内切核酸酶的转录后机制。内切核酸酶的定位、与它们相互作用的蛋白质的鉴定以及其他转录后数据将完善表型假说和模型。除了使用虚拟植物来实现这一目标外，该项目还将通过新的转录后组件来增强其能力。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Changes in gene expression are crucial for plant growth, development and resistance to pathogens. A better understanding of how gene expression is regulated is fundamental to the process of plant engineering and hence to improved agricultural production. Plants regulate gene expression at multiple levels and the aim of this project is to characterize proteins called endoribonucleases, which regulate gene expression at the level of mRNA degradation. An important aspect of this project is to determine how these endoribonucleases impact the biology of plants, including plant growth and development. This project will train young scientists on fundamental aspects of gene regulation. The young scientist will receive interdisciplinary training through interactions with collaborators who are experts in systems biology and chemical engineering. A Shadow a Scientist program will give middle school students the chance to meet scientists, tour a research laboratory, and to see experiments for this project in progress. Other outreach activities aim to engage the public and to inspire scientific interest in people from a broad range of backgrounds and age groups. The long-term goal of this project is to elucidate the roles of ribonucleases (RNases) that participate in mRNA degradation in Arabidopsis. Examining the roles of endoribonucleases in post-transcriptional control of genes is an underdeveloped area of research, but a timely one. The hypothesis underlying this proposed research is that elucidating the targets and post-transcriptional impact of endoribonucleases will enhance our understanding of key players in plant mRNA decay, such as the exosome, as well as lesser known or putative endoribonucleases whose role is yet to be discovered. To test this idea and others, the project will first create a set of double mutants to stabilize mRNA decay intermediates produced by endoribonucleases so they can be deeply sequenced; single endoribonuclease mutations will be introduced into an xrn4 null mutant background to achieve this. Second, to identify substrates of the endoribonucleases, RNA decay intermediates and RNA levels will be analyzed on a global scale in the double mutants and controls using Parallel Analysis of RNA Ends and RNA-Seq data. Should any of the endoribonucleases act on nonsense-mediated mRNA decay (NMD) targets, that would be intriguing given that plants lack the metazoan NMD endoribonuclease, SMG6. Third, new biological impacts of the endoribonucleases will be hypothesized and tested in the single mutants on the basis of the functional associations of their cleaved substrates. Fourth, post-transcriptional mechanisms that involve the endoribonucleases will be investigated. Localization of the endoribonucleases, the identification of proteins interacting with them and other post-transcriptional data will refine phenotypic hypotheses and models. Beyond making use of The Virtual Plant for this pursuit, this project will enhance its power with new post-transcriptional components.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.