RNA-directed DNA methylation, one of several RNA interference-mediated pathways in the nucleus, has been documented in plants and in human cells. Despite progress in identifying the DNA methyltransferases, histone-modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation, 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 groups. DRD1 is a SNF2-like protein 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 silencing. 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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