Specification of alternate siRNA biogenesis pathways in Arabidopsis

拟南芥中替代 siRNA 生物发生途径的规范

基本信息

批准号：
7752938
负责人：
Todd Lucas Blevins
金额：
$ 4.72万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2012-09-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7752938
关键词：
Animals Antiviral Agents Arabidopsis Arabidopsis Proteins Binding Biochemical Biochemical Genetics Biochemical Pathway Biogenesis Biological Assay Cleaved cell Co-Immunoprecipitations Complex DNA Methylation DNA Transposable Elements DNA-Directed RNA Polymerase Data Development Double-Stranded RNA Double-Stranded RNA Binding Domain Enzymes Eukaryota Event Fission Yeast Gene Silencing Genetic Genetic Transcription Genetic Translation Genetic screening method Genome Genomics Goals Human Lead Mass Spectrum Analysis Mediating Morphology Mutation Nucleic Acids Pathway interactions Plant Model Plants Polymerase Precursor RNA Process Production Protein Binding Domain Protein Family Proteins RNA RNA Interference RNA Polymerase II RNA Precursors RNA Processing RNA chemical synthesis RNA-Directed RNA Polymerase Research Small RNA Specificity Stem Cell Development System Tandem Repeat Sequences Testing Transcript Virus Work base chromatin modification design ds RNA-Binding Proteins endonuclease gene therapy histone modification human DICER1 protein loss of function mutant novel programs protein complex protein function public health relevance stem yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Small RNAs regulate development, stem cell identity, genome integrity, and defense against viruses in animals and plants. These "RNA silencing" phenomena involve proteins that are conserved from fission yeast to humans: RNA-dependent RNA polymerases (RDRs), which convert single-stranded RNA into double-stranded RNA (dsRNA), Dicer endonucleases that cleave dsRNA into short-interfering RNA (siRNA) duplexes, and Argonaute-containing complexes that use siRNA strands to program silencing effects. In the model plant Arabidopsis, protein duplication and sub-functionalization has resulted in distinct biochemical pathways that generate siRNA classes with different functions. One pathway involving RDR2 and DCL3 produces siRNAs that program repressive histone modifications and DNA methylation at transposable elements and tandem repeats. Another pathway involving RDR6 and DCL4 generates siRNAs that cleave mRNAs encoding proteins that regulate development. Keeping these pathways separate is biologically important, because loading siRNAs of incorrect sequence into downstream complexes would result in anomalous gene silencing. A central, unresolved question is: How are RNA intermediates for specific siRNA classes channeled through these distinct pathways? Answering this question is my overall research goal. Production of siRNAs is likely a highly channeled process from the outset, i.e., precursor RNA synthesis. In fact, known RDR6 substrates derive from RNA polymerase II (Pol II) transcription, whereas repeat- associated siRNA biogenesis requires a plant-specific RNA polymerase (Pol IV) upstream of RDR2. I hypothesize that proteins occupying adjoining steps in each pathway specifically interact to pass RNA intermediates down the chain of enzymatic activities. Mass spectrometry and a yeast two-hybrid system will be used to determine subunit compositions of complexes containing RDR2, RDR6, DCL3 and DCL4; furthermore, I will test RDR interactions by co-immunoprecipitation with candidate proteins. Based on these data, biochemical assays will test whether each RDR or DCL-interacting protein is functionally required for conversion of RNA precursors to products in either siRNA biogenesis pathway. This work will uncover biochemical mechanisms that support divergent, yet parallel pathways of siRNA biogenesis in Arabidopsis. Public Health Relevance: Because siRNAs anneal to and target nucleic acids with exquisite specificity, RNA silencing has potential for gene therapy and antiviral applications. The diversity of RNA silencing systems in Arabidopsis, loss-of-function mutants in key proteins, and ability to rescue mutations using tagged proteins allows us to scrutinize molecular interactions that generate and channel siRNAs.

描述（由申请人提供）：小RNA调节动物和植物的发育、干细胞身份、基因组完整性以及对病毒的防御。这些“RNA沉默”现象涉及从裂殖酵母到人类的保守蛋白质：RNA依赖性RNA聚合酶（RDR），将单链RNA转化为双链RNA（dsRNA），切丁酶核酸内切酶，将dsRNA切割成短干扰RNA RNA (siRNA) 双链体和含有 Argonaute 的复合物，使用 siRNA 链来编程沉默效果。在模型植物拟南芥中，蛋白质复制和亚功能化导致了不同的生化途径，产生具有不同功能的 siRNA 类别。涉及 RDR2 和 DCL3 的一条途径产生 siRNA，对转座元件和串联重复序列处的抑制性组蛋白修饰和 DNA 甲基化进行编程。另一条涉及 RDR6 和 DCL4 的途径产生 siRNA，切割编码调节发育的蛋白质的 mRNA。保持这些途径分开在生物学上很重要，因为将错误序列的 siRNA 加载到下游复合物中会导致异常基因沉默。一个未解决的核心问题是：特定 siRNA 类别的 RNA 中间体如何通过这些不同的途径进行引导？回答这个问题是我的总体研究目标。 siRNA 的生产从一开始就可能是一个高度通道化的过程，即前体 RNA 合成。事实上，已知的 RDR6 底物源自 RNA 聚合酶 II (Pol II) 转录，而重复相关的 siRNA 生物发生需要 RDR2 上游的植物特异性 RNA 聚合酶 (Pol IV)。我假设每个途径中占据相邻步骤的蛋白质特异性相互作用，将 RNA 中间体沿着酶活性链传递。将使用质谱和酵母双杂交系统来确定含有RDR2、RDR6、DCL3和DCL4的复合物的亚基组成；此外，我将通过与候选蛋白的免疫共沉淀来测试 RDR 相互作用。基于这些数据，生化测定将测试每种 RDR 或 DCL 相互作用蛋白是否是 RNA 前体转化为 siRNA 生物发生途径中的产物的功能所必需的。这项工作将揭示支持拟南芥中不同但平行的 siRNA 生物发生途径的生化机制。公共健康相关性：由于 siRNA 以精确的特异性退火并靶向核酸，因此 RNA 沉默具有基因治疗和抗病毒应用的潜力。拟南芥中 RNA 沉默系统的多样性、关键蛋白的功能丧失突变体以及使用标记蛋白挽救突变的能力使我们能够仔细研究生成和引导 siRNA 的分子相互作用。