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.
For plants and other organisms to grow, develop, and respond to environmental stimuli, the regulation of numerous genes is required. Monitoring these changes in gene expression at the RNA level on a genome-wide scale has been extremely powerful, especially with the massively parallel deep sequencing approach which RNA-Seq provides. However, resolving the underlying molecular mechanisms can be challenging because usually we do not know if RNA synthesis (transcription) or RNA degradation is changing. Another problem is that we know less about the proteins that degrade mRNA, known as ribonucleases (RNases), than the proteins that synthesize it. This project studied RNases of Arabidopsis to identify which RNAs they degrade within the plant. To do this, we assayed mutants deficient in specific RNases with a technique called Parallel Analysis of RNA Ends (PARE) which captures polyA+ RNA decay intermediates on a global scale. As we had hypothesized, the cytoplasmic 5’ to 3’ exoribonuclease, XRN4, was found to degrade many Arabidopsis RNAs from the 5’ end after a cap structure protecting the end was removed. Accomplishing this necessitated adapting a variation of PARE called Cap-PARE to identify where the decapping site was. Also, a new type of PARE (polyA- PARE) to capture RNA decay intermediates that lacked the protective polyA tail at the 3’ end of the RNA was developed. RNA-Seq was also carried to examine polyA+ and polyA- RNA levels and the data indicated that very few polyA+ RNAs overaccumulated in null mutants of XRN4 (e.g. xrn4-5) compared to the large number of polyA- RNAs that did. Consistently, more RNAs were identified as XRN4 substrates (those that overaccumulated precisely decapped RNA intermediates) in polyA- PARE than in polyA+. The functional associations of some of the substrates led to identification of a new physiological deficiency of xrn4 mutants at the whole plant level. Groups of other substrates were found to share common features, and support the importance of XRN4 in additional RNA decay mechanisms and functions. PARE and RNA-Seq approaches were applied to the study of additional Arabidopsis exoribonucleases, some of which required the development of conditional mutants because the enzymes provide function(s) essential for the plant to survive. Taken together, these studies enhanced understanding of endogenous RNases and the functional impacts of RNases in Arabidopsis. Work under this project had several broad impacts. The project has and will continue to contribute data sets through the Gene Expression Omnibus (GEO) for public use. Homozygous xrn4 mutant seed has been provided to many groups and large quantities were sent to the U.S. and European Arabidopsis stock centers to facilitate distribution. Computational approaches developed to analyze initial PARE data from an Arabidopsis xrn4 mutant provided the foundation for related work in human cells; endogenous substrates of an important human endoribonuclease called SMG6 were identified. Fungi, plants and animals have homologous nuclear and cytoplasmic exoribonucleases including XRN enzymes. Thus, the analysis of polyA- PARE to identify RNase substrates should be broadly applicable to other plants and beyond. Two postdocs, one graduate student, two undergraduates and one technician received training and mentoring under the project. The postdocs and graduate student greatly enhanced their computer programming ability as a result of the project. One postdoc is now part of the workforce of a major plant biotechnology company contributing both computational and laboratory research. Through outreach events, diverse members of the general public of all ages have been encouraged to engage in scientific discussions and/or engage their curiosities.
