A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis.
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.
Shanghai Center for Plant Stress Biology and Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China ; State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, China.
Chao-Feng
CF
Huang
Daisuke
D
Miki
Kai
K
Tang
Hao-Ran
HR
Zhou
Zhimin
Z
Zheng
Wei
W
Chen
Ze-Yang
ZY
Ma
Lan
L
Yang
Heng
H
Zhang
Renyi
R
Liu
Xin-Jian
XJ
He
Jian-Kang
JK
Zhu
D017398:Alternative Splicing / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics*; D056466:Histone Demethylases / Q000235:genetics; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000235:genetics*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism
D029681:Arabidopsis Proteins; C548147:DMS4 protein, Arabidopsis; D009687:Nuclear Proteins; C587158:RDM16 protein, Arabidopsis; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; C471271:ROS1 protein, Arabidopsis; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D056466:Histone Demethylases