A Pre-mRNA-Splicing Factor Is Required for RNA-Directed DNA Methylation in Arabidopsis

Wei Chen,Hao-Ran Zhou,Daisuke Miki,Lan Yang,Ze-Yang Ma,Xin-Jian He,Jian-Kang Zhu,Heng Zhang,Zhimin Zheng,Chao-Feng Huang,Kai Tang,Renyi Liu

doi:10.1371/journal.pgen.1003779

Abstract

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.

Highlights

Cytosine methylation in eukaryotic cells is an epigenetic mark that plays important roles in diverse biological processes, such as the silencing of genes and transposons [1,2], X inactivation [3], paramutation [4], and imprinting [5]
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
Plants have evolved a pathway for active DNA demethylation that is initiated by the ROS1 subfamily of 5-methylcytosine DNA glycosylases, to counteract the RNA-directed DNA methylation (RdDM) pathway to prevent undesirable silencing

Summary

Introduction

Cytosine methylation in eukaryotic cells is an epigenetic mark that plays important roles in diverse biological processes, such as the silencing of genes and transposons [1,2], X inactivation [3], paramutation [4], and imprinting [5]. Cytosine methylation can occur in all three sequence contexts: CG, CHG and CHH (H = A, T, or C). De novo cytosine methylation in all three sequence contexts can be catalyzed by DRM2 [10] in a pathway known as RNA-directed DNA methylation (RdDM) [11,12]. In this pathway, a plant-specific RNA polymerase IV is recruited to transcribe transposons and some endogenous repeat loci and the transcripts are copied into double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASE2 (RDR2) [13,14,15,16].

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