Roles of RNA Polymerases IV and V in siRNA-mediated gene silencing

RNA 聚合酶 IV 和 V 在 siRNA 介导的基因沉默中的作用

• 批准号: 8041251
• 负责人: CRAIG Stuart PIKAARD
• 金额: $ 31.96万
• 依托单位: TRUSTEES OF INDIANA 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8041251
• 关键词: Accounting; Address; Affinity Chromatography; Agriculture; Alleles; Arabidopsis; Area; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biotechnology; C-terminal; Cells; Centromere; Chromatin; Chromatin Structure; Complex; Consensus Sequence; Coupled; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Polymerase II; DNA Polymerase III; DNA Transposable Elements; DNA-Directed RNA Polymerase; DRD1 gene; Development; Eukaryota; Fission Yeast; Fragile X Syndrome; Functional RNA; Funding; Gene Silencing; Genes; Genetic; Genetic Transcription; Genome; Genome Stability; Genomics; Goals; Grant; Hereditary Disease; Heterochromatin; Histones; Human; Human Genetics; In Vitro; Maintenance; Malignant Neoplasms; Mediating; Medical; Modeling; Parents; Pathway interactions; Plants; Play; Polymerase; Process; Production; Protein Family; Proteins; RNA; RNA Interference; RNA Polymerase I; RNA Polymerase II; RNA-Directed RNA Polymerase; Recruitment Activity; Retrotransposon; Ribonuclease III; Role; SWI2/SNF2; Signal Transduction; Site; Small Interfering RNA; Small RNA; Specific qualifier value; Technology; Tertiary Protein Structure; Testing; Transcript; Untranslated RNA; Virus; Work; X Inactivation; base; chromatin modification; chromatin remodeling; cohesin; condensin; design; dosage; fly; human DICER1 protein; human FBXW7 protein; human disease; imprint; maternal imprint; member; novel; promoter; transcription factor

DESCRIPTION (provided by applicant): All eukaryotes use three essential DNA-dependent RNA polymerases to decode the genetic information in chromosomal DNA, namely RNA Polymerases I, II and III. Remarkably, plants have evolved two additional RNA polymerases, abbreviated as Pol IV and Pol V. These novel RNA polymerases play non- redundant roles in RNA-directed DNA methylation, silencing of transposable elements, large-scale heterochromatin organization, long-distance spreading of silencing signals, and proper temporal and morphological development. Affinity purification and mass spec analyses revealed that Pol IV and Pol V are specialized forms of RNA Polymerase II, with half of their twelve subunits encoded by the same genes. Three subunits differ between Pol IV and Pol V and presumably account for their unique functions. Pol IV and Pol V are best understood with respect to their roles in the Arabidopsis siRNA-directed DNA methylation pathway. Pol IV acts early in the pathway, generating transcripts that are required for the biogenesis of 24 nt short interfering RNAs (siRNAs) that are loaded into ARGONAUTE 4 (AGO4). Independent of siRNA biogenesis, Pol V generates noncoding transcripts at target loci. siRNA-AGO4 complexes bind to these Pol V transcripts, facilitating recruitment of AGO4 to the adjacent chromatin. In subsequent steps that are not understood, the de novo DNA methyltransferase, DRM2 and histone modifying activities are recruited to target loci, generating heterochromatin that is refractive to transcription by conventional polymerases such as Pol II and Pol III. Major questions in need of answers include: what are the templates used by Pol IV and Pol V?; how are Pol IV and Pol V recruited to these templates?; are Pol IV and Pol V transcription units specified by conventional promoters or by chromatin structures?; how are Pol IV and Pol V coordinated with other proteins of the gene silencing machinery; and how do the unique subunits of Pol II, Pol IV and Pol V confer the unique functions of these novel polymerases? Using genetics and genomics as well as cell biological and biochemical approaches, our specific aims are designed to find answers to these questions. In diverse eukaryotes, including humans, flies, worms and fission yeast, siRNAs and noncoding RNAs essential processes through chromatin modifications. Examples include transposon silencing, centromere maintenance, X-chromosome inactivation and imprinting of maternal or paternal alleles. DNA methylation and chromatin modifications are also implicated in Rett, ICF, Prader-Willi, Beckwith-Wiedemann and Fragile X syndromes, as well as numerous forms of cancer. By understanding how noncoding RNAs and siRNAs specify sites of DNA methylation and gene silencing, our study will contribute to the long-term goal of understanding these processes with respect to human disease. PUBLIC HEALTH RELEVANCE: Large non-coding RNAs and small RNAs are critical for X-chromosome inactivation and dosage control, imprinting of genes expressed from only one parent, defense against retrotransposons and viruses, heterochromatin formation at centromeres and genome stability. Alterations in DNA methylation and heterochromatin are involved in multiple human diseases and genetic disorders, including cancer, and targeted gene silencing using small RNA interference (RNAi) technologies are being used in medical and agricultural biotechnology applications. Our studies of RNA polymerases IV and V are significant with respect to all of these areas, especially the roles of intergenic noncoding RNAs and siRNAs in chromatin-mediated gene silencing.

描述(申请人提供)：所有真核生物都使用三种基本的依赖DNA的RNA聚合酶来解码染色体DNA中的遗传信息，即RNA聚合酶I、II和III。值得注意的是，植物还进化了另外两种RNA聚合酶，缩写为POL IV和POL V。这些新型RNA聚合酶在RNA指导的DNA甲基化、转座元件沉默、大规模异染色质组织、沉默信号的远距离传播以及适当的时间和形态发育中发挥着非多余的作用。亲和纯化和质谱分析表明，Pol IV和Pol V是RNA聚合酶II的特化形式，其12个亚基中有一半由相同的基因编码。Pol IV和Pol V之间有三个亚基不同，推测这三个亚基具有独特的功能。POL IV和POL V在拟南芥siRNA指导的DNA甲基化途径中的作用被最好地理解。POL IV在该途径中起早期作用，产生24个核苷酸短干扰RNA(SiRNAs)生物发生所需的转录本，这些siRNAs被装载到ArgAerte 4(AGO4)中。与siRNA生物发生无关，Pol V在目标位点产生非编码转录本。SiRNA-AGO4复合体与这些Pol V转录本结合，促进AGO4向相邻染色质的募集。在未知的后续步骤中，从头开始的DNA甲基转移酶、DRM2和组蛋白修饰活性被招募到靶点，产生异染色质，这些异染色质是由Pol II和Pol III等传统聚合酶折射转录的。需要回答的主要问题包括：Pol IV和Pol V使用什么模板？Pol IV和Pol V如何被招募到这些模板？Pol IV和Pol V转录单位是由传统启动子指定的还是由染色质结构指定的？Pol IV和Pol V如何与基因沉默机制中的其他蛋白质协调；Pol II、Pol IV和Pol V的独特亚基如何赋予这些新型聚合酶独特的功能？利用遗传学和基因组学以及细胞生物学和生化方法，我们的具体目标是找到这些问题的答案。在各种真核生物中，包括人、苍蝇、蠕虫和分裂酵母，siRNAs和非编码RNAs通过染色质修饰进行必要的过程。例如，转座子沉默、着丝粒维持、X染色体失活和母系或父系等位基因的印记。DNA甲基化和染色质修饰也与Rett、ICF、Prader-Willi、Beckwith-Wiedemann和Fragile X综合征以及多种形式的癌症有关。通过了解非编码RNA和siRNA如何指定DNA甲基化和基因沉默的位置，我们的研究将有助于理解这些与人类疾病有关的过程的长期目标。 与公共卫生相关：大的非编码RNA和小的RNA对于X染色体的失活和剂量控制、只有一个亲本表达的基因的印记、对反转录转座子和病毒的防御、着丝粒异染色质的形成和基因组的稳定性是至关重要的。DNA甲基化和异染色质的改变涉及多种人类疾病和遗传疾病，包括癌症，使用小RNA干扰(RNAi)技术的靶向基因沉默正被用于医疗和农业生物技术应用。我们对RNA聚合酶IV和V的研究对于所有这些领域都具有重要意义，特别是基因间非编码RNAs和siRNAs在染色质介导的基因沉默中的作用。