Arabidopsis 2010: Functions of Arabidopsis Small RNAs

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Major paradigm shifts in understanding gene regulation at the transcriptional and posttranscriptional levels have occurred in recent years through discoveries of RNA-based silencing systems. In plants, these systems form distinct classes of small RNAs (microRNAs, trans-acting siRNA, heterochromatin associated siRNAs, and others) that associate with effector complexes containing ARGONAUTE proteins to suppress or regulate mRNAs, genetic loci or invasive elements. These pathways serve to regulate genes during growth and development, and during stress and antiviral responses. They also interface with chromatin factors to guide heterochromatin formation at loci containing repetitive elements, such as transposons and retroelements. This 2010 Project takes advantage of new technology to examine small RNA pathways at a genome-wide level, and to assign function to biogenesis and effector components, using Arabidopsis thaliana as a central model. This research program is connected to the overall goal of the 2010 Project, "to determine the function of all genes in the model plant Arabidopsis thaliana by the year 2010", by seeking the function of this specific class of RNAs. Data will be made available to the public through the Arabidopsis Small RNA Project database, at http://asrp.cgrb.oregonstate.edu/. The Major Objectives of the project include:1. Analysis of functions, regulation and evolution of several RNA silencing pathways at a genome-wide level. New enabling technology and computation will be used for sequencing-based profiling of A. thaliana small RNAs with a series of silencing-defective mutants, developmental stages, and treatments, as well as with closely related species. This will provide key data to assess the impact, redundancy and evolution of small RNA pathways in plants.2. Identification of small RNAs and small RNA classes that associate with silencing effector proteins in the ARGONAUTE (AGO) family. Associating small RNAs with effectors will provide a genome-wide benchmark to identify AGO specificities and functional redundancies. Broader Impact: The Arabidopsis Small RNA Database will be expanded as a major public resource for plant biology research. The project will engage undergraduates in a front-line research experience. Additionally, an exciting high school outreach module in collaboration with Science Education Partnerships (SEPS) will be developed. This module will be integrated with broader SEPS activities to train teachers and students in new concepts relating to genome-enabled science and gene silencing, using interesting materials generated by this project.This project encompasses all three emphasis or focus areas stated in the 2010 Project Program Description: 1. Benchmarking gene function: Each major objective has a benchmark for gene function or activity. Functional assignments to small RNA biogenesis factors will be made based on quantitative measurements of small RNAs or small RNA classes. Each of the 10 ARGONAUTE protein functions will be benchmarked based on the composition of associated small RNAs. And functions for ta-siRNAs from each TAS gene will be benchmarked in a defined genetic context by detection of phenotypes associated with disruption of tasiRNA interaction sites.2. Developing new genome-wide experimental approaches/tools for analyzing gene function: This proposal integrates new, highly parallel sequencing technology and computation to do non-biased small RNA profiling in a set of small RNA biogenesis mutants and several other plants. This technology allows quantitative, genome-wide surveys of small RNA expression levels. The data will be presented to the community through an established web-based resource that we have managed for nearly four years.3. Exploring exemplary networks and systems: This small RNA profiling technology will also be used to integrate expression of small RNAs and transcriptional activity across the entire genome using tiling arrays. Integrative analyses will illuminate the roles of specific RNA-based silencing pathways during development.