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”现象涉及从裂殖酵母到人类的保守蛋白质：RNA依赖性RNA聚合酶（RDRs），其将单链RNA转化为双链RNA（dsRNA），Dicer内切核酸酶，其将dsRNA切割成短干扰RNA（siRNA）双链体，以及含有Argonaute的复合物，其使用siRNA链来编程沉默效应。在模式植物拟南芥中，蛋白质复制和亚功能化导致了产生具有不同功能的siRNA类的不同生化途径。一种涉及RDR 2和DCL 3的途径产生siRNA，其在转座元件和串联重复序列处编程抑制性组蛋白修饰和DNA甲基化。另一个涉及RDR 6和DCL 4的途径产生siRNA，其切割编码调节发育的蛋白质的mRNA。保持这些途径的分离在生物学上是重要的，因为将不正确序列的siRNA加载到下游复合物中会导致异常的基因沉默。一个中心的、未解决的问题是：特定siRNA类的RNA中间体是如何通过这些不同的途径传递的？解决这个问题是我的总体研究目标。siRNA的产生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RDR 6底物来源于RNA聚合酶II（Pol II）转录，而重复序列相关的siRNA生物发生需要RDR 2上游的植物特异性RNA聚合酶（Pol IV）。我假设，蛋白质占领相邻步骤，在每个途径，特别是相互作用，通过RNA中间体的酶活性链。质谱和酵母双杂交系统将用于确定含有RDR 2，RDR 6，DCL 3和DCL 4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，生物化学测定将测试每种RDR或DCL相互作用蛋白在功能上是否是将RNA前体转化为siRNA生物合成途径中的产物所需的。这项工作将揭示支持拟南芥中siRNA生物合成的不同但平行的途径的生化机制。 公共卫生相关性：由于siRNA以精确的特异性退火并靶向核酸，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，关键蛋白质中功能丧失的突变体，以及使用标记蛋白质拯救突变的能力，使我们能够仔细研究产生和引导siRNA的分子相互作用。

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.