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

  • ️Tue Jan 01 2008

. 2008 Oct 30;455(7217):1259-62.

doi: 10.1038/nature07305. Epub 2008 Sep 24.

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

Xianwu Zheng et al. Nature. 2008.

Abstract

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms. Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation. 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 pathway. These demethylases have critical roles in erasing DNA methylation and preventing TGS of target genes. 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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Figures

Figure 1
Figure 1. The ros3 mutation causes transcriptional gene silencing

a, Stress-induced expression of the RD29A-LUC transgene in wild type (WT) and ros3 mutant plants after treatment with 300 mM NaCl for 5 hr. b, Like ros1, the ros3 mutant plants are sensitive to kanamycin. c, Northern blots showing that ros3 reduces the transcript levels of LUC, NPTII and endogenous RD29A, but not of the control, COR15A. Plants were either untreated (Control) or treated with cold (4°C) for 24 hr, 100 µM ABA for 3 hr, or 300 mM NaCl for 5 hr. d, Nuclear run-on assay showing the pre-mRNA levels of LUC, NPTII and RD29A genes in wild-type and ros3. COR15A and CBF3 were used as controls.

Figure 2
Figure 2. DNA hypermethylation in ros3 and suppression of ros3 by nrpd1a

a and b Bisulfite sequencing analysis of promoter methylation status of the endogenous RD29A (a) and RD29A-LUC transgene (b) in WT, ros1, ros3, ros1ros3, ros1 complemented with wild type ROS1 transgene, and ros3 complemented with wild type ROS3 transgene. c, Suppression of ros3 by the nrpd1a-1 mutation. Seedlings of WT, ros3 and nrpd1aros3 double mutant grown in MS medium for three weeks were transferred to a filter paper soaked with 300 mM NaCl for 5 hr. Left, picture of the seedlings; right, luminescence image. d, Quantification of luminescence in (c). Error bars represent standard deviation (n=20). e, DNA methylation status at the transgene RD29A promoter in ros3 and nrpd1aros3 plants.

Figure 3
Figure 3. ROS3 binds small RNAs

a, Diagram of ROS3 and its truncated mutant forms. b, Coomassie stained SDS-PAGE gel showing the recombinant proteins used for RNA binding assays. c, ROS3 but not ROS3ΔNC binds 21-,24- and 26-nt single stranded RNA of sequence b. Lanes 1, 3, 5, 7, 9, 11 and 13, ROS3; lanes 2, 4, 6, 8, 10, 12 and 14, ROS3ΔNC. d, ROS3ΔC but not ROS3ΔN or ROS3ΔNC binds 24-nt single stranded RNA of sequence b. Lane 1, ROS3; lane 2, ROS3ΔN; lane 3, ROS3ΔC; lane 4, ROS3ΔNC. e, Protein concentration-dependent binding to 24-nt single stranded RNA (b) and competition by unlabeled small RNA. Lanes 1–4, increasing binding to 24-nt (b) small RNA by increasing ROS3 protein concentration; lanes 5–9, competition by increasing amount of cold 24-nt RNA (b). f, ROS3 binds 25-nt RNA (c) and RNA (d) in vitro. RNAs of sequences b and a are used as controls. Lanes 1, 3, 5 and 7, ROS3; lanes 2, 4, 6 and 8, ROS3ΔN.

Figure 4
Figure 4. Co-localization of ROS3 with ROS1 in the nucleus of Arabidopsis mesophyll cells and assay of ROS1 and ROS3 mRNA levels

a, Localization of ROS3 and ROS1 by their respective antibodies. b, Dual immunolocalization of ROS3 and ROS1 with use of anti-ROS3 and anti-Myc. c, ROS1 localization in ros3 mutant and ROS3 localization in ros1 showing their inter-dependence for appropriate localization. Size bars correspond to 5 µm. d, Quantitative RT-PCR assay of relative ROS1 and ROS3 transcript levels in the various genotypes.

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