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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

基本信息

批准号：
7220652
负责人：
CRAIG Stuart PIKAARD
金额：
$ 25.58万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7220652
关键词：
Active Sites Address Amino Acids Arabidopsis 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 Disruption Double-Stranded RNA Embryo Escherichia coli Eukaryota Eukaryotic Cell Event Fission Yeast Fragile X Syndrome Genes Health Heterochromatin Histones Human Lead Light Link Maintenance Malignant Neoplasms Mediating Methylation Methyltransferase Mitochondria Modification Mouse-ear Cress Nematoda Nuclear 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 Thinking Transcript Trisomy Viral Work day ds-DNA fly gene therapy genetic resource human DICER1 protein insight mutant novel pol genes programs tool

项目摘要

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）：在人类、果蝇、线虫、裂殖酵母和植物等多种生物体中，小干扰 RNA (siRNA) 调节重要的过程，包括病毒防御、反转录转座子沉默、特定 mRNA 失活和着丝粒维护。 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相当清楚。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位点的机制尚不清楚。新发现的拟南芥异染色质 siRNA 途径所需的活性是核 RNA 聚合酶 IV (pol IV)。 pol IV 的两个最大亚基与大肠杆菌 DNA 依赖性 RNA 聚合酶的催化亚基以及真核细胞核 RNA 聚合酶 I、II 和 III 的相应亚基相似。 pol IV 亚基的破坏导致异染色质分散，同时重复基因和逆转录转座子的胞嘧啶甲基化和 siRNA 产生损失。最简单的假设是 pol IV 为 Dicer 生成的 siRNA 生成前体转录本，从而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 最大的亚基在假定的活性位点处也具有非规范氨基酸，这表明 pol IV 具有新型聚合酶活性的假设。 pol IV 依赖性 siRNA 生产还需要 RNA 依赖性 RNA 聚合酶 2 (RDR2) 和胞嘧啶甲基转移酶活性（DRM 或 MET1）这一发现表明，双链 RNA 或甲基化 DNA 可能是 pol IV 转录的专门模板。本研究的目的是： 1) 确定 pol IV 的模板、产物和亚基； 2) 推论 pol IV 在异染色质 siRNA 途径中发挥作用的步骤，以及 3) 检验以下假设：两种不同形式的 pol IV 在促进整体异染色质组织中非冗余地发挥作用。染色质修饰越来越多地与 siRNA 相关，并与多种癌症以及 Rett、ICF、Prader-Willi、Beckwith-Wiedemann 和脆性 X 综合征有关。染色体不稳定也与着丝粒中 siRNA 的参与有关，可导致三体性或胚胎致死。此外，RNA 干扰有望成为对抗疾病的基因治疗工具。因此，我们的研究利用拟南芥独特的遗传资源，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。