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）：小型RNA调节动物和植物中对病毒的发育，干细胞身份，基因组完整性和防御。 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链以编程沉默效应。在模型植物拟南芥中，蛋白质的复制和亚功能化导致了不同的生化途径，从而产生具有不同功能的siRNA类别。涉及RDR2和DCL3的一条途径会产生siRNA，该siRNA在可转移元件和串联重复序列上进行程序抑制组蛋白的修饰和DNA甲基化。涉及RDR6和DCL4的另一种途径会产生siRNA，从而裂解编码调节发育蛋白的mRNA。将这些途径分开在生物学上很重要，因为将不正确序列的siRNA载入下游复合物将导致异常基因沉默。一个未解决的中心问题是：通过这些不同途径引导的特定siRNA类别的RNA中间体如何？回答这个问题是我的总体研究目标。从一开始，即前体RNA合成起，siRNA的产生可能是一个高度传播的过程。实际上，已知的RDR6底物衍生自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）。我假设在每种途径中占据相邻步骤的蛋白质特异性相互作用以将RNA中间传递到酶促活性链中。质谱和酵母双杂交系统将用于确定含有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共免疫沉淀测试RDR相互作用。基于这些数据，生化分析将测试每个RDR或DCL相互作用蛋白是否在功能上需要将RNA前体转化为siRNA生物发生途径中的产物。这项工作将发现支持拟南芥中siRNA生物发生的分歧但平行途径的生化机制。公共卫生相关性：因为SiRNA对具有精致特异性的核酸退火和靶向核酸，因此RNA沉默具有基因治疗和抗病毒药物的潜力。 RNA沉默系统在拟南芥中的多样性，关键蛋白质中的功能丧失突变体以及使用标记蛋白营救突变的能力，使我们能够仔细检查产生和传播siRNA的分子相互作用。