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ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis

Abstract

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms1,2,3,4,5,6. Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation7,8,9,10. In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway8,10,11,12,13. These demethylases have critical roles in erasing DNA methylation and preventing TGS of target genes7,8,10. However, it is not known how the demethylases are targeted to specific sequences. Here we report the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutants and ros1 ros3 double mutants suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts shows that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins co-localize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.

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Figure 1: The ros3 mutation causes transcriptional gene silencing.
Figure 2: DNA hypermethylation in ros3 and suppression of ros3 by nrpd1a.
Figure 3: ROS3 binds small RNAs.
Figure 4: Co-localization of ROS3 with ROS1 in the nucleus of Arabidopsis mesophyll cells and assay of ROS1 and ROS3 mRNA levels.

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Acknowledgements

This work was supported by National Institutes of Health grants R01GM070795 and R01GM059138 (J.-K.Z.), R01GM077590 and 1R01GM060380 (C.S.P.), Edward Mallinckrodt Foundation (O.P.) and China Scholarship Council scholarship 2007104542 (F.Z.).

Author Contributions X.Z. did the cloning, mutant analysis, RNA binding and other experiments. J.Z. and A.K. contributed to mutant analysis. D.M., F.Z. and K.I. contributed to DNA methylation analysis. W.-X.L. contributed to small RNA results. O.P. and C.S.P. contributed immunostaining data. J.-K.Z. designed the project and wrote the paper.

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Correspondence to Jian-Kang Zhu.

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Zheng, X., Pontes, O., Zhu, J. et al. ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis. Nature 455, 1259–1262 (2008). https://doi.org/10.1038/nature07305

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