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

所有真核生物都使用三种基本的依赖DNA的RNA聚合酶来解码遗传信息 储存在DNA中，即RNA聚合酶I、II和III。值得注意的是，植物多了两种RNA聚合酶， 缩写为Pol IV和Pol V，从Pol II进化到专门合成引导基因的RNA 沉默，在所有生物体中控制反转录转座子、病毒或基因的重要过程 对发展很重要。Pols IV和V是复杂的RNA指导的DNA甲基化(RdDM)的关键 路径。该途径由Pol IV启动，与RNA-4结成伙伴关系并进行物理结合- 依赖于RNA聚合酶RDR2将DNA转录成短双链RNA(DsRNAs)。这些 然后，dsRNAs被DICER内切酶DCL3从两端修剪，产生24个核苷酸的短干扰 RNA(SiRNAs)。装载到ArgAerte4(AGO4)中的siRNAs引导siRNA-AGO复合体到位点 在Pol V转录中，它们与Pol V转录本以及Pol V的C-末端结构域结合 最大的亚基。DNA甲基转移酶DRM2(人类DNMT3的同源基因)被招募，甲基化 Pol V转录的DNA中的胞嘧啶。由此形成的异染色质对转录是折射的。 POLS I、II或III。然而，POLS IV和V在这个染色质环境中不被抑制。相反，帮助者 识别胞嘧啶甲基化或抑制性组蛋白修饰的蛋白质被认为能招募Pol IV V，取代转录因子，不需要常规的启动子元件。 关于RdDM过程，我们有很多不了解的地方。是什么解开了波尔斯四号和 为了获得模板链？Pol IV和RDR2活性是如何偶联合成dsRNA的？ Pol V转录本在哪里开始和结束，有多少siRNA-AGO复合体可以结合它们？你要做什么 推测的POL IV和POL V辅助蛋白真的起作用吗？通过设计新的生化分析组合 通过对基因、基因组和结构的研究，我们的目标是详细回答这些问题。 在各种真核生物中，如人、苍蝇、蠕虫和分裂酵母，非编码RNA引导染色质 对着丝粒功能、转座子沉默、X染色体失活或印记重要的修饰 父亲或母亲的等位基因。与我们的研究特别相关的是piRNA途径，它引导 沉默人类生殖系中的转座子，从而服务于相同的目的，并使用相同的DNA 甲基化机制，作为植物的RdDM途径。受控的DNA甲基化是至关重要的，因此 异常的DNA甲基化和染色质修饰参与了Rett，ICF，Prader-Willi， Beckwith-Wiedemann综合征和脆性X综合征，以及大多数形式的癌症。通过了解生物发生学 以及非编码RNA在DNA甲基化和基因沉默中的靶向机制，我们的研究将 对对人类发展和疾病至关重要的基本过程有新的理解。

All eukaryotes use three essential DNA-dependent RNA polymerases to decode genetic information stored in DNA, namely RNA Polymerases I, II and III. Remarkably, plants have two more RNA polymerases, abbreviated as Pol IV and Pol V, that evolved from Pol II to specialize in the synthesis of RNAs that guide gene silencing, an important process in all living organisms for controlling retrotransposons, viruses or genes important for development. Pols IV and V are key to a complicated RNA-directed DNA methylation (RdDM) pathway. The pathway is initiated by Pol IV, acting in partnership and physical association with a RNA- dependent RNA polymerase, RDR2 to transcribe DNA into short double-stranded RNAs (dsRNAs). These dsRNAs are then trimmed, from either end, by the DICER endonuclease, DCL3, yielding 24 nt short interfering RNAs (siRNAs). The siRNAs, loaded into ARGONAUTE 4 (AGO4), guide the siRNA-AGO complexes to sites of Pol V transcription, where they bind to Pol V transcripts as well as to the C-terminal domain of the Pol V largest subunit. The DNA methyltransferase, DRM2 (the ortholog of human DNMT3) is recruited, methylating cytosines within the Pol V-transcribed DNA. Resulting heterochromatin formation is refractive to transcription by Pols I, II or III. However, Pols IV and V are not repressed in this chromatin environment. Instead, helper proteins that recognize cytosine methylation or repressive histone modifications are thought to recruit Pols IV and V, taking the place of transcription factors and dispensing with conventional promoter elements. There is much that we do not understand about the RdDM process. What unwinds DNA for Pols IV and V to gain access to template strands? How are Pol IV and RDR2 activities coupled for dsRNA synthesis? Where do Pol V transcripts begin and end, and how many siRNA-AGO complexes can bind them ? What do the presumed Pol IV and Pol V helper proteins actually do? By devising new biochemical assays combined with genetic, genomic and structural studies, our goal is to answer these questions in mechanistic detail. In eukaryotes as diverse as humans, flies, worms and fission yeast, noncoding RNAs guide chromatin modifications important for centromere function, transposon silencing, X-chromosome inactivation or imprinting of paternal or maternal alleles. Of special relevance to our studies is the piRNA pathway that directs the silencing of transposons in the human germline, thereby serving the same purpose, and using the same DNA methylation machinery, as the RdDM pathway of plants. Controlled DNA methylation is critical, such that aberrant DNA methylation and chromatin modification is implicated in the pathology of Rett, ICF, Prader-Willi, Beckwith-Wiedemann and Fragile X syndromes, and in most forms of cancer. By understanding the biogenesis and targeting mechanisms of noncoding RNAs in DNA methylation and gene silencing, our studies will contribute new understanding of fundamental processes important for human development and disease.