Role of RNA polymerase IV in the heterochromatic siRNA pathway

RNA聚合酶IV在异染色质siRNA途径中的作用

• 批准号: 7599659
• 负责人: CRAIG Stuart PIKAARD
• 金额: $ 5.05万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7599659
• 关键词: Active Sites; Address; Amino Acids; Arabidopsis; Biochemical; Biochemical Genetics; Biological Assay; Catalytic Domain; Cell Nucleus; Centromere; Chloroplasts; Chromatin; Chromosomal Instability; Cytosine; DNA; DNA Methylation; DNA Polymerase I; DNA-Directed RNA Polymerase; DRD1 gene; Data; Defect; Development; Disease; Double-Stranded RNA; Embryo; Escherichia coli; Eukaryota; Event; Fission Yeast; Fragile X Syndrome; Genes; Genetic; Health; Heterochromatin; Human; Lead; Light; Link; Maintenance; Malignant Neoplasms; Mediating; Methylation; Methyltransferase; Mitochondria; Modification; Mouse-ear Cress; Nematoda; Nuclear; Nuclear RNA; Organism; Paper; Pathway interactions; Physical condensation; Plants; Polymerase; Printing; Process; Production; Proteins; Publishing; RNA Degradation; RNA Interference; RNA Interference Pathway; RNA Polymerase I; RNA chemical synthesis; RNA-Directed RNA Polymerase; Reporting; Research; Research Personnel; Retroelements; Retrotransposon; Rice; Role; Sequence Homologs; Small Interfering RNA; Specific qualifier value; Testing; Transcript; Trisomy; Viral; Work; chromatin modification; combat; ds-DNA; fly; gene therapy; genetic resource; histone modification; human DICER1 protein; insight; mutant; novel; programs; tool

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.

描述（由申请人提供）：在像人类，苍蝇，线虫，裂变酵母和植物等多样的生物中，小型干扰RNA（siRNAS）调节了包括病毒防御的基本过程，包括病毒防御，沉默逆转转身，特定mRNA和特定mRNA和Centromere维护。 siRNA靶向同源RNA在dicer和RISC介导的RNA干扰（RNAI）途径中降解的机制已得到充分了解。相比之下，对siRNA的直接组蛋白修饰和/或胞嘧啶甲基化事件的机制却众所周知。拟南芥的异质siRNA途径中需要的新发现的活性是核RNA聚合酶IV（POL IV）。 POL IV的两个最大亚基类似于大肠杆菌DNA依赖性RNA聚合酶的催化亚基以及真核核RNA聚合酶I，II和III的相应亚基。 pol IV亚基的破坏会导致异染色质分散与重复基因和逆转座子的胞嘧啶甲基化和siRNA产生的损失一致。最简单的假设是，Pol IV生成了将异染色质修饰的dicer生成的siRNA的前体转录本。有趣的是，使用双链DNA作为模板，Pol IV似乎缺乏常规的RNA聚合酶活性。 Pol IV最大的亚基在假定活性位点也具有非典型的氨基酸，这表明POL IV具有新型聚合酶活性的假设。 POL IV依赖性siRNA的产生还需要RNA依赖性RNA聚合酶2（RDR2）和胞嘧啶甲基转移酶活性（DRM或MET1）的发现表明，POL IV转录的双链RNA或甲基化DNA可以专业化。拟议研究的目的是：1）确定POL IV的模板，产品和亚基； 2）推断POL IV在异型siRNA途径中起作用的步骤和3）检验以下假设：两种不同形式的Pol IV的两种形式在为全球异染色质组织贡献的情况下非重新起作用。染色质的修饰越来越多地与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。染色体不稳定性也与centromeres的siRNA受累有关，可能导致trisomies或胚胎致死性。此外，RNA干扰显示了与疾病打击的基因治疗工具的希望。我们的研究利用了拟南芥独特的遗传资源，因此可以阐明与人类健康有关的siRNA生产和染色质修饰的机制。