植物、动物和真菌的转录组研究发现转录组中含有大量非编码RNA，但其功能尚不清楚。本项目申请人实验室在拟南芥中鉴定了RNA聚合酶Pol IV的转录物，Pol IV和 Pol V的转录物或作为siRNA的前体或作为支架募集siRNA到其同源染色质区域，共同参与RNA指导的DNA甲基化（RdDM）。RdDM能沉默转座子和重复序列以及调控蛋白质基因的表达。迄今为止，非编码RNA的转录、加工和降解机制几乎一无所知，非编码RNA如何被核内RNA加工复合体识别，并被区别于蛋白质编码RNA对待，也尚不清楚。本项目旨在系统分析Pol IV和 Pol V转录产物的序列特征；研究非编码RNA的生物合成和加工机制。研究结果将有助于阐明表观基因组的构成，也有助于我们了解不仅植物中，而且动物中基因组稳定性的调控，因为动物中的piRNAs和植物中的siRNAs在调控基因组稳定性方面有着相似的功能。

Transcriptome studies in plants, animals, and fungi have revealed pervasive transcription in genomes and the presence of multitudes of noncoding transcripts, although the functions of the noncoding transcripts are largely unknown. Recent studies in Arabidopsis from my group and that of Dr. Craig Pikaard have identified nuclear, long noncoding transcripts generated by RNA polymerase IV (Pol IV) and Pol V, respectively. These transcripts serve as precursors to small interfering RNAs (siRNAs) or scaffolds to recruit siRNAs to chromatin and are integral components in RNA-directed DNA methylation, a mechanism to silence transposable elements and repeats and to regulate the expression of protein-coding genes. Although these and other studies point to an emerging role of nuclear noncoding RNAs in regulating gene expression or genome stability, the mechanisms underlying the transcription, processing, and degradation of the noncoding transcripts are almost completely unknown. It is also unknown how these noncoding transcripts are recognized and treated differently from protein-coding transcripts by nuclear RNA processing machinery. This project aims to systematically identify Pol IV- and Pol V-dependent noncoding RNAs and their features by high-throughput sequencing, and to study the mechanisms governing the biogenesis and processing of the noncoding transcripts. As these transcripts are critical to the epigenetic landscape of plant genomes, the mechanisms that produce, process, and degrade these transcripts are expected to help shape the epigenome. Our efforts to understand the transcription and metabolism of these long noncoding transcripts will contribute to knowledge of genome stability not only in plants but also in animals, as piRNA-mediated epigenome specification in animal germlines invokes similar mechanisms as siRNA-mediated epigenome specification in plants.

本项目旨在通过研究参与RNA指导的DNA甲基化(RdDM)过程中长链非编码RNA的代谢来进一步揭示RdDM的分子机制。RdDM是一种基因防御机制它能通过DNA甲基化沉默基因组中转座子、重复序列等被认为是“外源”的序列元件。但是目前人们对这些序列元件如何被识别还知之甚少。RdDM的第一步就是植物所特有的RNA polymerase IV (Pol IV)识别并转录这些元件。Pol IV的转录产物作物作为前体随后被加工成小干扰RNAs (siRNAs)，这些siRNAs能指导DNA甲基转移酶甲基化其同源序列。因此 Pol IV如何识别其靶序列对于理解RdDM的机理至关重要。本项目我们采用一个创新的实验设计方法，第一次成功在基因组上鉴定到大量的依赖Pol IV的转录产物并且描述其特征。依赖Pol IV的转录产物的发现有利于我们研究Pol IV在基因组上的转录区域以及这些区域的遗传特征，为理解Pol IV的作用机制奠定了基础。本项目通过遗传筛选鉴定了两个在DNA甲基化的下游调控基因表达的因子，这些因子能抑制DNA甲基化引起的基因沉默促进基因表达，但并不影响DNA甲基化。我们发现RdDM存在负调控机制，这些研究开辟了研究表观遗传调控基因表达的新领域。