Atypical RNA polymerase subunits required for RNA-directed DNA methylation

Abstract

RNA-directed DNA methylation, one of several RNA interference–mediated pathways in the nucleus1, has been documented in plants2,3 and in human cells4,5. Despite progress in identifying the DNA methyltransferases, histone-modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation1, the mechanism remains incompletely understood. We screened for mutants defective in RNA-directed DNA methylation and silencing of a transgene promoter in Arabidopsis thaliana and identified three drd complementation groups6. DRD1 is a SNF2-like protein6 required for RNA-directed de novo methylation. We report here that DRD2 and DRD3 correspond to the second-largest subunit and largest subunit, respectively, of a fourth class of DNA-dependent RNA polymerase (polymerase IV) that is unique to plants. DRD3 is a functionally diversified homolog of NRPD1a or SDE4, identified in a separate screen for mutants defective in post-transcriptional gene silencing7,8. The identical DNA methylation patterns observed in all three drd mutants suggest that DRD proteins cooperate to create a substrate for RNA-directed de novo methylation.

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Figure 1: Non-CG methylation is lost in drd mutants.
Figure 2: α′ promoter dsRNA and siRNAs accumulate in drd mutants.
Figure 3: Structure of the gene NRPD1b and positions of point mutations.
Figure 4: Transposon reactivation in drd mutants.
Figure 5: DNA methylation and siRNA accumulation from 5S rDNA repeats.
Figure 6: Distinct Pol IV complexes act in RNA-directed DNA methylation.

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Acknowledgements

We thank C. Pikaard, A. Herr and D. Baulcombe for discussions and preprints and the Monsanto Company for marker information. This work was supported by funds from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.

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Correspondence to Marjori Matzke.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Bisulfite sequencing. (PDF 109 kb)

Supplementary Fig. 2

Sequence comparisons. (PDF 735 kb)

Supplementary Fig. 3

DRD protein-dependent RNA-directed de novo methylation. (PDF 11 kb)

Supplementary Fig. 4

AtSN1 methylation analysis. (PDF 3419 kb)

Supplementary Table 1

Oligonucleotides and small RNA probes. (PDF 6 kb)

Supplementary Methods (PDF 23 kb)

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Kanno, T., Huettel, B., Mette, M. et al. Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet 37, 761–765 (2005). https://doi.org/10.1038/ng1580

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