Arabidopsis 2010: Functions of Arabidopsis Small RNAs

近年来，通过发现基于RNA的沉默系统，在转录和转录后水平上理解基因调控的主要范式转变已经发生。在植物中，这些系统形成不同类别的小RNA（microRNA、反式作用siRNA、异染色质相关siRNA等），其与含有ARGONAUTE蛋白的效应复合物结合以抑制或调节mRNA、遗传基因座或侵入性元件。这些途径在生长和发育过程中以及在应激和抗病毒反应期间调节基因。它们还与染色质因子相互作用，以引导异染色质在含有重复元件（例如转座子和逆转录元件）的基因座上形成。这个2010年的项目利用新技术在全基因组水平上研究小RNA通路，并以拟南芥为中心模型，将功能分配给生物发生和效应子组件。该研究计划与2010年项目的总体目标“到2010年确定模式植物拟南芥中所有基因的功能”相联系，通过寻找这类特定RNA的功能。 数据将通过拟南芥小RNA项目数据库向公众提供，网址为http://asrp.cgrb.oregonstate.edu/。 该项目的主要目标包括：1。在全基因组水平上分析几种RNA沉默途径的功能、调控和进化。新的使能技术和计算将用于基于测序的A.拟南芥小RNA与一系列沉默缺陷突变体，发育阶段和治疗，以及密切相关的物种。这将为评估植物中小RNA途径的影响、冗余和进化提供关键数据。ARGONAUTE（AGO）家族中与沉默效应蛋白相关的小RNA和小RNA类别的鉴定。将小RNA与效应子相关联将提供全基因组基准以鉴定AGO特异性和功能冗余。更广泛的影响：拟南芥小RNA数据库将扩大为植物生物学研究的主要公共资源。该项目将使本科生参与一线研究体验。此外，与科学教育伙伴关系（SEPS）合作开发一个令人兴奋的高中外展模块。该模块将与更广泛的SEPS活动相结合，利用该项目产生的有趣材料，对教师和学生进行有关基因组科学和基因沉默的新概念培训。该项目包括2010年项目计划说明中所述的所有三个重点或重点领域：1.基准基因功能：每个主要目标都有一个基因功能或活性的基准。小RNA生物发生因子的功能分配将基于小RNA或小RNA类别的定量测量进行。将基于相关小RNA的组成对10种ARGONAUTE蛋白功能中的每一种进行基准测试。通过检测与tasiRNA相互作用位点破坏相关的表型，在确定的遗传背景下对来自每个TAS基因的ta-siRNA的功能进行基准测试。开发用于分析基因功能的新的全基因组实验方法/工具：该提案整合了新的高度并行的测序技术和计算，以在一组小RNA生物发生突变体和其他几种植物中进行无偏见的小RNA分析。该技术允许对小RNA表达水平进行定量的全基因组调查。这些数据将通过我们管理了近四年的一个既定的基于网络的资源向社区展示。探索示例性网络和系统：这种小RNA分析技术也将用于使用平铺阵列整合整个基因组中小RNA的表达和转录活性。综合分析将阐明特定RNA沉默途径在发育过程中的作用。

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.