Arabidopsis 2010: Analysis of the Ribonucleases of Arabidopsis Thaliana

智力上的功绩。基因的功能和调控对所有生物体的生长和发育都是至关重要的。为了让基因发挥作用，它的DNA必须被复制到RNA中，然后RNA被翻译成蛋白质来发挥基因的作用，或者在某些情况下，RNA直接发挥作用。这一过程可以通过控制RNA的生产或其降解的机制来调节。这个项目的重点是在拟南芥中降解RNA的蛋白质，称为核糖核酸酶(RNase)。该项目之前的工作研究了一种名为XRN4的RNase，它能降解一端缺少保护帽的RNA。研究发现，XRN4降解的一些RNA是被称为miRNAs的小RNA切割的RNA。尽管确定RNA降解产物的特性对于跟踪RNase功能至关重要，但大多数RNA的衰变产物是难以捉摸的，因为它们是异质性的，并且以非常低的数量存在。该项目之前的工作克服了这些障碍，开发了一种名为RNA末端平行分析(PARE)的新基因组技术，这是一种用于检测部分降解的RNA分子的深度测序技术。这项技术的一个令人兴奋的应用是在全基因组范围内表征被切割的miRNA靶标。该项目的假设是，PARE将有助于检测在RNase突变体中被不适当降解的mRNAs，并且不能用不那么灵敏的方法检测到。这应该会为一般的RNA降解，特别是RNase的降解提供新的见解。为了检验这些想法，PARE将被应用于选定的核糖核酸酶突变体。与正常植物相比，已经改变了降解的RNA将被识别出来。这可以确定使它们对RNase敏感的RNAs的特征，并表明RNase控制的新基因和生物过程。最终，这可能会为农业改良提出新的战略，或者与生物医学相关。该项目的数据将在世界各地的用户友好网站上提供，并配有分析工具，供所有学生、博士后和高级调查人员应用于他们自己的工作。这项比较RNase的工作应该广泛适用于其他植物、动物和微生物，并可能加强对其他基因调控实验的解释，这些实验无法将降解的RNA与完整的RNA分开。该项目将为本科生的博士后助理/助理科学家提供指导和培训，导致新的合作，并使其他科学家和可能的普通公众接触到这项工作。

Intellectual Merit. The function and regulation of genes is of fundamental importance for growth and development in all organisms. For a gene to function, its DNA must be copied into an RNA that is then translated into protein to fulfill the gene's role, or in some cases, the RNA functions directly. The process can be regulated by mechanisms that control the production of the RNA or its degradation. This project focuses on the proteins that degrade RNA, which are called ribonucleases (RNases), in Arabidopsis. Prior work under this project studied an RNase called XRN4 that degrades RNAs that are missing a protective cap on one end. The research discovered that some of the RNAs that XRN4 degrades are RNAs targeted for cleavage by small RNAs called miRNAs. Although characterizing RNA degradation products is critical for tracking RNase function, the decay products of most RNAs can be elusive because they are heterogenous and present in very low amounts. Prior work under the project overcame these obstacles with the development of a new genomic technology called Parallel Analysis of RNA Ends (PARE), a deep sequencing technology for detecting partially degraded RNA molecules. An exciting application of this technology was to characterize cleaved miRNA targets genome-wide. The hypothesis underlying this project is that PARE will facilitate detection of mRNAs that are improperly degraded in RNase mutants and that cannot be detected with less sensitive approaches. This should provide new insights about RNA degradation in general and about RNases in particular. To test these ideas, PARE will be applied to selected RNase mutants. RNAs that have altered degradation compared with those in normal plants will be identified. This could identify features of the RNAs that make them susceptible to RNases, and indicate new genes and biological processes that RNases control. Ultimately, this may suggest new strategies for agricultural improvement or have biomedical relevance.Broader Impacts. The data from this project will be available world-wide on user-friendly websites with analysis tools for all students, postdocs and senior investigators to apply to their own work. This PARE RNase work should be broadly applicable to other plants, animals, and microbes, and may enhance interpretation of other experiments on gene regulation that cannot separate degraded from intact RNA. The project will provide mentoring and training to a postdoctoral associate/associate scientist, an undergraduate student, lead to new collaborations, and expose other scientists and potentially the general public to the work.