Analysis of the Ribonucleases of Arabidopsis thaliana

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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.