Specification of alternate siRNA biogenesis pathways in Arabidopsis

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、基因组完整性和对病毒的防御。这些“RNA沉默”现象涉及从裂变酵母到人类保守的蛋白质：RNA依赖RNA聚合酶（RDRs），将单链RNA转化为双链RNA (dsRNA)， Dicer内切酶将dsRNA切割成短干扰RNA （siRNA）双链，以及使用siRNA链编程沉默效果的含argonaute复合物。在模式植物拟南芥中，蛋白质复制和亚功能化导致了不同的生化途径，产生了具有不同功能的siRNA类。一种涉及RDR2和DCL3的途径产生sirna，在转座元件和串联重复序列上编程抑制组蛋白修饰和DNA甲基化。另一种涉及RDR6和DCL4的途径产生sirna， sirna切割编码调节发育的蛋白质的mrna。保持这些通路的分离在生物学上是重要的，因为将不正确序列的sirna装载到下游复合体中会导致异常的基因沉默。一个尚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？回答这个问题是我总体研究的目标。sirna的产生可能从一开始就是一个高度通道化的过程，即前体RNA合成。事实上，已知的RDR6底物来自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，在每个途径中占据相邻步骤的蛋白质特异性地相互作用，将RNA中间体传递到酶活性链上。质谱法和酵母双杂交系统将用于测定含有RDR2、RDR6、DCL3和DCL4的配合物的亚基组成；此外，我将通过与候选蛋白共免疫沉淀来测试RDR相互作用。基于这些数据，生化分析将测试每种RDR或dcl相互作用蛋白在siRNA生物发生途径中将RNA前体转化为产物的功能上是否必需。这项工作将揭示支持拟南芥中siRNA生物发生的不同但平行途径的生化机制。

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.