Role of RNA polymerase IV in the heterochromatic siRNA pathway

描述(申请人提供)：在各种生物中，如人类、苍蝇、线虫、分裂酵母和植物，小干扰RNA(SiRNAs)调节基本过程，包括病毒防御、反转录转座子沉默、特定mRNAs失活和着丝粒维持。SiRNAs靶向同源RNAs在DICER和RISC介导的RNA干扰(RNAi)途径中降解的机制已经相当清楚。相比之下，siRNAs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还知之甚少。新发现的拟南芥异染色质siRNA途径所需的一个活性是核RNA聚合酶IV(PolIV)。PolIV的两个最大的亚基类似于大肠杆菌DNA依赖的RNA聚合酶的催化亚基以及真核RNA聚合酶I、II和III的相应亚基。PolIV亚基的破坏导致异染色质的分散，与重复基因和反转座子上胞嘧啶甲基化和siRNA产生的损失一致。最简单的假设是，PolIV为迪格尔产生的siRNAs生成前体转录产物，将异染色质修饰定向到同源序列。有趣的是，PolIV似乎缺乏以双链DNA为模板的传统RNA聚合酶活性。POL IV最大的亚基在推测的活性部位也有非规范的氨基酸，这表明POL IV具有新的聚合酶活性的假设。PolIV依赖的siRNA的产生还需要RNA依赖的RNA聚合酶2(RDR2)和胞嘧啶甲基转移酶活性(DRM或MET1)，这表明双链RNA或甲基化的DNA可能是PolIV转录的专门模板。拟议的研究目的是：1)确定PolIV的模板、产物和亚基；2)推断PolIV在异染色质siRNA途径中的作用步骤；3)验证两种不同形式的PolIV在全球异染色质组织中非冗余作用的假设。染色质的改变越来越多地与siRNA的参与有关，并与许多癌症以及Rett、ICF、Prader-Willi、Beckwith-Wiedemann和Fragile X综合征有关。染色体不稳定，也与着丝粒的siRNA参与有关，可导致三体或胚胎死亡。此外，RNA干扰显示出作为对抗疾病的基因治疗工具的前景。我们的研究利用了拟南芥独特的遗传资源，因此可能有助于揭示与人类健康相关的siRNA产生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.