植物小分子RNA参与许多重要的生物学过程，精细胞是保障植物成功受精的前提之一，但小分子RNA在精细胞形成中的功能研究鲜少。申请人长期致力于小分子RNA的作用机制及其在植物精细胞形成中的调控机理。主要成绩: 率先发现植物的Pol II为siRNA途径所必需，阐明了多种RNA聚合酶互作的分子机制；发现蛋白酶体APC通过调节miRNA的产生促进精细胞形成，解析了蛋白降解与miRNA协同调控精细胞形成的作用机制；发现与siRNA介导的DNA甲基化有关的Cajal body穿梭于营养核与生殖核，提示了miRNA与siRNA可能协同调控植物精细胞形成的分子特征。近五年以第一或通讯作者在Gene Dev和Plant Cell等发表论文4篇。花粉的营养核和精细胞核呈现出不均衡的表观遗传修饰，但其调控机制尚不清楚。申请人拟研究siRNA依赖且穿梭于营养核与生殖核的ARID1的作用机制，丰富对父本遗传信息重编程机制的认识。

Small RNAs are 20-24 nucleotides RNAs that function as sequence-specific regulators of gene expression at both the transcriptional and posttranscriptional levels in eukaryotes. Small RNA-directed gene silencing is important for many developmental processes and understanding the mechanisms has becoming increasingly important. Recent studies from the applicant mainly focused on the molecular mechanisms of small RNA-directed gene silencing and the regulation by small RNAs during sperm cell formation. Her major discoveries include: (1) showed the necessity of DNA-dependent RNA polymerase II (Pol II) in siRNA-directed gene silencing (Zheng et al., 2009), and demonstrated Pol II is required for Pol IV and Pol V recruitment to siRNA loci. Thus this study took a leading role in investigating the interplays among three polymerases; (2) found that APC (Anaphase Promoting Complex) is required for miRNA biogenesis by facilitating Pol II recruitment, and demonstrated APC-mediated miRNA biogenesis promotes sperm cell formation by limiting the expression of the substrate in a dual manner (Zheng et al., 2011); (3) showed Cajal body, a processing center for siRNA-directed DNA methylation, is developmentally regulated during sperm cell formation, and uncovered the phenomena of miRNA and siRNA coordinately regulating sperm cell formation (the co-corresponding paper: Scarpina et al., 2013). Recent studies showed the expression of TEs (transposable elements) is activated in the vegetative nuclei, but highly repressed in sperm nuclei, However, the mechanism of unequal epigenetic modifications between the vegetative cell and sperm cells remains unclear. Therefore, in this proposed study, the applicant aims to answer the question: how pollen maintains the opposite pattern of epigenetic modifications in the vegetative nucleus and sperm nuclei? Specifically, we will focus on the molecular mechanism of ARID1, a transcription factor identified due to small RNAs enriched in the promoter region, regulated by siRNA. We will use genetic, cell biological, biochemical, and genomic methods to characterizing the dependence of ARID1 subcellular localization and stability on siRNAs, and isolating genes involved in the regulation of ARID1 stability by siRNA. We hope that our study will broaden the knowledge about molecular mechanism of reprogramming of paternal genetic information.

本项目主要集中研究植物体内的小分子RNA的产生及其在生殖发育过程中的作用。众所周知，植物的小RNA包括miRNA和siRNA，目前关于miRNA的产生途径已经非常清楚，大致框架如下：Pol II将miRNA基因转录出具有颈环结构的primary miRNA (pri-miRNA)，pri-miRNA经过核酸内切酶DCL1复合物在颈环的底部切割成发卡结构的precursor miRNA (pre-miRNA)，pre-miRNA继续被DCL1复合物从顶部切割成成熟的21 nt为主的miRNA。DCL1复合物中已经鉴定到多种辅助蛋白，HYL1是其中最核心也是功能研究相对清楚的一个。HYL1是一个双链RNA结合蛋白，其主要通过结合pri-miRNA和pre-miRNA帮助DCL1剪切。而且HYL1被发现是一个短寿命且受到磷酸化调控的蛋白。尽管miRNA产生途径已经了解的很清楚，但是miRNA产生的核心复合物本身的调控，尤其是转录后水平的调控依然相当不清楚。此外，花粉中小RNA如何参与其表观遗传信息的重编程以及配子细胞中富集的小RNA到底有什么生物学功能也不清楚。项目主持人在本人才项目的支持下，主要围绕模式植物拟南芥中miRNA产生的调控机制和小分子RNA介导的表观遗传调控在植物生殖发育过程中的功能展开研究。通过遗传学、生物化学、细胞生物学和基因组学的手段，项目执行期间，我们取得主要研究成绩有三方面:1)首次鉴定了pre-mRNA拼接过程中产生的套索RNA通过与pre-miRNA竞争结合miRNA加工复合物,从而干扰miRNA的加工；2）证明动植物中高度保守的蛋白磷酸酶复合物PP4/SMEK1通过与MAPK信号通路拮抗调控miRNA加工复合物的重要组分HYL1的蛋白稳态，实现对miRNA产生的精细调控；3）在实验室前期鉴定的转录因子ARID1通过与miR159互作调控精细胞成熟的研究基础上，发现ARID1与MET1在生殖细胞中相互抑制，介导了大孢子母细胞细胞命运的决定。后续正在围绕套索RNA与小分子RNA互作维持基因组稳定、小分子RNA参与调控花粉中表观遗传信息重编程的分子机制及其在早期种子发育中的作用等方面展开研究。这些工作为深入阐明植物体内miRNA的加工以及植物生殖发育过程中小分子RNA介导的表观遗传调控的功能提供了重要的依据。