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

描述（由申请人提供）：所有真核生物都使用三种必需的DNA依赖性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，植物已经进化出两种另外的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、转座因子的沉默、大规模异染色质组织、沉默信号的长距离传播以及适当的时间和形态发育中发挥非冗余作用。亲和纯化和质谱分析表明，Pol IV和Pol V是RNA聚合酶II的特殊形式，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，可能是它们独特功能的原因。 Pol IV和Pol V在拟南芥siRNA指导的DNA甲基化途径中的作用是最好理解的。Pol IV在途径的早期起作用，产生加载到ARGONAUTE 4（AGO 4）中的24 nt短干扰RNA（siRNA）的生物发生所需的转录物。独立于siRNA生物发生，Pol V在靶基因座产生非编码转录物。siRNA-AGO 4复合物与这些Pol V转录物结合，促进AGO 4募集到相邻的染色质。在不被理解的后续步骤中，从头DNA甲基转移酶、DRM 2和组蛋白修饰活性被募集到靶基因座，产生对常规聚合酶如Pol II和Pol III的转录具有折射性的异染色质。 需要回答的主要问题包括：PolIV和PolV使用的模板是什么？Pol IV和Pol V是如何被招募到这些模板中的？Pol IV和Pol V转录单位是由常规启动子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的独特功能？利用遗传学和基因组学以及细胞生物学和生物化学方法，我们的具体目标旨在找到这些问题的答案。 在多种真核生物中，包括人类、苍蝇、蠕虫和裂殖酵母，siRNA和非编码RNA通过染色质修饰进行必需的过程。实例包括转座子沉默、着丝粒维持、X染色体失活和母本或父本等位基因的印记。DNA甲基化和染色质修饰也与Rett、ICF、Prader-Willi、Beckwith-Wiedemann和脆性X综合征以及许多形式的癌症有关。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点，我们的研究将有助于了解这些过程与人类疾病的长期目标。 公共卫生相关性：大的非编码RNA和小RNA对于X染色体失活和剂量控制、仅从一个亲本表达的基因的印记、对逆转录转座子和病毒的防御、着丝粒处的异染色质形成和基因组稳定性至关重要。DNA甲基化和异染色质的改变涉及多种人类疾病和遗传性疾病，包括癌症，并且使用小RNA干扰（RNAi）技术的靶向基因沉默正在用于医学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有重要意义，特别是基因间非编码RNA和siRNA在染色质介导的基因沉默中的作用。

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.