A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis.
A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis.
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DOI：
10.1371/journal.pgen.1003779
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Zhu JK
中科院分区：
文献类型：
--
作者：
Huang CF;Miki D;Tang K;Zhou HR;Zheng Z;Chen W;Ma ZY;Yang L;Zhang H;Liu R;He XJ;Zhu JK
胞嘧啶DNA甲基化是一种稳定的表观遗传标记，经常与基因和转座元件(TES)的沉默有关。在拟南芥中，DNA甲基化的建立是通过RNA指导的DNA甲基化(RdDM)途径。在这里，我们报告了RDM16的鉴定和特性，RDM16是RdDM途径中的一个新因子。RDM16的突变降低了DNA甲基化水平，并部分释放了报告基因的沉默以及DNA去甲基酶ROS1-1突变背景中的一些内源基因组位点。Rdm16突变体具有形态缺陷，对盐胁迫和脱落酸(ABA)敏感。基于图谱的克隆和互补实验鉴定了RDM16，它编码一个mRNA剪接前因子3，是U4/U6 SnRNP的一个组成部分。RNA-seq分析表明，rdm16发生了308个内含子保留事件，证实了rdm16参与了植物的前mRNA剪接。RNA-seq和mRNA表达分析也显示RDM16突变不影响已知RdDM基因的前mRNA剪接，提示RDM16可能直接参与RdDM。在显示RDM16依赖的DNA甲基化的座位上的小RNA表达分析表明，与先前报道的假定剪接因子突变体不同，rdm16不影响小RNA水平；相反，rdm16突变导致Pol V转录本水平下降。芯片分析显示，RDM16在某些Pol V靶点上有浓集。我们的结果表明，RDM16通过影响Pol V转录水平来调节DNA甲基化。最后，我们的全基因组DNA甲基化分析表明，RDM16调控TES和基因周围区域的整体甲基化，并优先针对依赖于POL IV的DNA甲基化位点和ROS1靶点。因此，我们的工作有助于理解RdDM及其与活性DNA去甲基化的相互作用。植物和动物都利用胞嘧啶DNA甲基化作为重要的表观遗传标记来抑制转座元件(TES)、重复序列和基因，这对基因组的完整性和发育至关重要。在植物中，DNA的从头甲基化可以通过RNA引导的DNA甲基化(RdDM)途径来介导。植物还进化出一条活性DNA去甲基化的途径，该途径由5-甲基胞嘧啶DNA糖基酶的ROS1亚家族启动，以抵消RdDM途径，以防止不必要的沉默。在本研究中，我们发现了RdDM途径中的一个新因子RDM16。我们证明RDM16是一种前mRNA剪接因子，它在调节DNA甲基化和基因沉默方面的作用不是通过影响siRNA水平或编码已知RdDM组分的基因的表达或剪接，而可能是通过影响Pol V转录本。我们还发现RDM16优先影响ROS1靶基因座。总之，我们的发现有助于理解RdDM及其与ROS1介导的DNA去甲基化的相互作用。
Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation. Both plants and animals utilize cytosine DNA methylation as an important epigenetic mark to suppress transposable elements (TEs), repeat sequences and genes, which is crucial for the genome integrity and development. In plants, de novo DNA methylation can be mediated by the RNA-directed DNA methylation (RdDM) pathway. Plants have also evolved a pathway for active DNA demethylation that is initiated by the ROS1 subfamily of 5-methylcytosine DNA glycosylases, to counteract the RdDM pathway to prevent undesirable silencing. In this study, we identified RDM16, a new factor in the RdDM pathway. We show that RDM16 is a pre-mRNA splicing factor and its function in the regulation of DNA methylation and gene silencing is not through influencing siRNA levels or the expression or splicing of genes encoding known RdDM components, but likely through affecting Pol V transcripts. We also show that RDM16 preferentially affects ROS1 target loci. Together, our findings contribute to the understanding of RdDM and its interactions with ROS1-mediated DNA demethylation.
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影响因子：
4.5
作者：
Henderson IR;Deleris A;Wong W;Zhong X;Chin HG;Horwitz GA;Kelly KA;Pradhan S;Jacobsen SE
通讯作者：
Jacobsen SE
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通讯作者：
Fischer RL
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作者：
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通讯作者：
Bhattacharya, SS
影响因子：
4
作者：
Grewal, Shiv I. S.
通讯作者：
Grewal, Shiv I. S.