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，植物进化出了另外两种RNA聚合酶，缩写为Pol IV和Pol v。这些新的RNA聚合酶在RNA定向的DNA甲基化、转座元件的沉默、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，它们的12个亚基中有一半是由相同的基因编码的。Pol IV和Pol V之间的三个亚基不同，这可能解释了它们独特的功能。Pol IV和Pol V在拟南芥sirna导向的DNA甲基化途径中的作用是人们了解得最好的。Pol IV在该通路的早期起作用，产生装载到ARGONAUTE 4 （AGO4）中的24nt短干扰rna （sirna）的生物发生所需的转录本。独立于siRNA生物发生，Pol V在目标位点产生非编码转录物。siRNA-AGO4复合物与这些Pol V转录本结合，促进AGO4招募到邻近的染色质上。在随后的未知步骤中，从头开始的DNA甲基转移酶、DRM2和组蛋白修饰活性被招募到目标位点，产生异染色质，这种异染色质被传统的聚合酶（如Pol II和Pol III）转录。需要回答的主要问题包括：Pol IV和Pol V使用的模板是什么？Pol IV和Pol V如何被招募到这些模板中？Pol IV和Pol V转录单位是否由常规启动子或染色质结构指定？Pol IV和Pol V如何与基因沉默机制的其他蛋白质协调；以及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，我们的具体目标是找到这些问题的答案。在多种真核生物中，包括人类、苍蝇、蠕虫和裂变酵母，sirna和非编码rna通过染色质修饰是必不可少的过程。例子包括转座子沉默、着丝粒维持、x染色体失活和母亲或父亲等位基因的印记。DNA甲基化和染色质修饰也与Rett、ICF、Prader-Willi、beckwithi - 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理解这些过程与人类疾病的长期目标。

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.