Arabidopsis 2010: Analysis of the Ribonucleases of Arabidopsis Thaliana

智力优势。 基因的功能和调节对于所有生物体的生长和发育都具有根本的重要性。为了使基因发挥作用，它的DNA必须复制成RNA，然后RNA被翻译成蛋白质以完成基因的作用，或者在某些情况下，RNA直接发挥作用。 该过程可以通过控制RNA的产生或其降解的机制来调节。 本项目的重点是拟南芥中降解RNA的蛋白质，称为核糖核酸酶（RNases）。 该项目之前的工作研究了一种名为XRN 4的RNase，它可以降解一端缺少保护帽的RNA。 研究发现，XRN 4降解的一些RNA是被称为miRNA的小RNA切割的RNA。 虽然表征RNA降解产物对于跟踪RNA酶功能至关重要，但大多数RNA的降解产物可能是难以捉摸的，因为它们是异质的并且以非常低的量存在。 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（帕雷）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表征全基因组范围内切割的miRNA靶点。 该项目的假设是，帕雷将有助于检测在RNase突变体中不适当降解的mRNA，并且不能用较低灵敏度的方法检测。这将为RNA降解提供新的见解，特别是关于RNA酶。 为了验证这些想法，帕雷将应用于选定的RNase突变体。 将鉴定与正常植物相比具有改变的降解的RNA。 这可以识别RNA的特征，使它们对RNA酶敏感，并指示RNA酶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，或具有生物医学意义。 该项目的数据将在世界各地的用户友好的网站上提供，并为所有学生，博士后和高级研究人员提供分析工具，以应用于他们自己的工作。这项帕雷RNase的工作应该广泛适用于其他植物、动物和微生物，并可能增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。该项目将为一名博士后助理/副科学家、一名本科生提供指导和培训，促成新的合作，并使其他科学家和潜在的公众了解这项工作。

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.