Role of RNA polymerase IV in the heterochromatic siRNA pathway

描述（由申请人提供）：在人类、苍蝇、线虫、裂殖酵母和植物等多种生物体中，小干扰RNA（siRNA）调节基本过程，包括病毒防御、逆转录转座子沉默、特定mRNA失活和着丝粒维持。siRNA靶向同源RNA以在Dicer和RISC介导的RNA干扰（RNAi）途径中降解的机制已经相当好地理解。相比之下，siRNA指导组蛋白修饰和/或胞嘧啶甲基化事件沉默和维持异染色质基因座的机制知之甚少。在拟南芥异染色质siRNA途径中所需的新发现的活性是核RNA聚合酶IV（pol IV）。pol IV的两个最大亚基与E. coli DNA依赖性RNA聚合酶和真核细胞核RNA聚合酶I、II和III的相应亚基。pol IV亚基的破坏导致异染色质分散，同时在重复基因和反转录转座子处胞嘧啶甲基化和siRNA产生的损失。最简单的假设是pol IV产生Dicer产生的siRNA的前体转录物，其指导异染色质修饰同源序列。有趣的是，pol IV似乎缺乏使用双链DNA作为模板的常规RNA聚合酶活性。Pol IV最大的亚基在推定的活性位点处也具有非规范氨基酸，这表明Pol IV具有新的聚合酶活性的假设。pol IV依赖性siRNA产生也需要RNA依赖性RNA聚合酶2（RDR 2）和胞嘧啶甲基转移酶活性（DRM或MET 1）的发现表明，双链RNA或甲基化DNA可以是由pol IV转录的特化模板。本研究的目的是：1）确定pol IV的模板、产物和亚基; 2）推断pol IV在异染色质siRNA途径中起作用的步骤; 3）检验pol IV的两种不同形式在促进全局异染色质组织中起非冗余作用的假设。染色质修饰越来越多地与siRNA参与有关，并且与许多癌症以及Rett、ICF、Prader-Willi、Beckwith-Wiedemann和脆性X综合征有关。染色体不稳定性，也与着丝粒处的siRNA参与有关，可导致三体或胚胎致死。此外，RNA干扰显示出作为基因治疗工具对抗疾病的希望。因此，我们的研究，利用拟南芥独特的遗传资源，可能会揭示与人类健康相关的siRNA生产和染色质修饰的机制。

DESCRIPTION (provided by applicant): In organisms as diverse as humans, flies, nematodes, fission yeast and plants, small interfering RNAs (siRNAs) regulate essential processes that include viral defense, silencing of retrotransposons, inactivation of specific mRNAs and centromere maintenance. The mechanisms by which siRNAs target homologous RNAs for degradation in Dicer and RISC-mediated RNA interference (RNAi) pathways are fairly well understood. By contrast, the mechanisms by which siRNAs direct histone modifications and/or cytosine methylation events that silence and maintain heterochromatic loci are poorly understood. A newly discovered activity required in the heterochromatic siRNA pathway in Arabidopsis thaliana is Nuclear RNA polymerase IV (pol IV). The two largest subunits of pol IV are similar to the catalytic subunits of E. coli DNA-dependent RNA polymerase and to the corresponding subunits of eukaryotic nuclear RNA polymerases I, II, and III. Disruption of pol IV subunits causes heterochromatin to disperse coincident with losses in cytosine methylation and siRNA production at repetitive genes and retrotransposons. The simplest hypothesis is that pol IV generates precursor transcripts for Dicer-generated siRNAs that direct heterochromatin modifications to homologous sequences. Interestingly, pol IV appears to lack conventional RNA polymerase activity using double-stranded DNA as the template. Pol IV largest subunits also have non- canonical amino acids at the presumptive active site, suggesting the hypothesis that pol IV has a novel polymerase activity. The finding that pol IV-dependent siRNA production also requires RNA-dependent RNA polymerase 2 (RDR2) and cytosine methyltransferase activity (DRM or MET1) suggests that double- stranded RNA or methylated DNA could be specialized templates transcribed by pol IV. The aims of the proposed research are: 1) To determine the templates, products and subunits of pol IV; 2) To deduce the step at which pol IV acts in the heterochromatic siRNA pathway and 3) To test the hypothesis that two distinct forms of pol IV act non-redundantly in contributing to global heterochromatin organization. Chromatin modifications are increasingly linked to siRNA involvement and are implicated in numerous cancers as well as Rett, ICF, Prader-Willi, Beckwith-Wiedemann and Fragile X syndromes. Chromosome instability, also linked to siRNA involvement at centromeres, can lead to trisomies or embryo lethality. Moreover RNA interference shows promise as a gene-therapy tool with which to combat disease. Our studies, which make use of Arabidopsis unique genetic resources, may therefore shed light on mechanisms of siRNA production and chromatin modification that have relevance to human health.