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An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation

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Abstract

DNA methylation is an important epigenetic mark in many eukaryotes1,2,3,4,5. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4–6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

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Figure 1: Effects of rdm1 on RD29A-LUC and 35S-NPTII silencing, DNA methylation and small RNAs.
Figure 2: Effects of rdm1-1 on siRNA and transcript levels and DNA methylation at endogenous RdDM target loci.
Figure 3: Immunoblot analysis of RDM1 and its interaction with AGO4, DRM2 and NRPB1.
Figure 4: Sub-nuclear localization of RDM1 and its co-localization with other components of the RdDM pathway.

Change history

  • 06 May 2010

    Reference 23 was updated to reflect a change in the title.

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Acknowledgements

This work was supported by National Institutes of Health grants (J.-K.Z.), Austrian Fonds zur Förderung der wissenschaftlichen Forschung (M.M. and Z.J.L.), National Science Foundation Career Award (H.J.), Edward Mallinckrodt Foundation Award (O.P.), and Agence Nationale de la Recherche (T.L.). We thank E. Richards for his gift of methylated DNA oligonucleotides, B. Stevenson for technical assistance and T. Kanno for discussions.

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Z.G., H.-L.L, X.H., W.Q., H.L., M.X., S.Z., D.M., and X.Z. contributed Figs 1, 2, 3b–d, Supplementary Figs 1–6 and 9–12, and Supplementary Table 2. L.D, Z.J.L., A.J.M. and M.M. contributed the rdm1-4 allele (Supplementary Fig. 5) and data on its characterization (Supplementary Fig. 7). O.P. and C.S.P contributed Fig. 4, Supplementary Fig. 8 and Supplementary Table 1. D.P and T.L. contributed Fig. 3a. J.-K.Z designed the experiments and wrote the paper together with Z.G., H.J., O.P., C.S.P. and M.M.

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Correspondence to Jian-Kang Zhu.

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Gao, Z., Liu, HL., Daxinger, L. et al. An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106–109 (2010). https://doi.org/10.1038/nature09025

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