Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA ( rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b ( a subunit of the RNA Polymerase IV complex, RNA PoIIV), NRPD2 ( a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent-disruption of one class does not affect the other-suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.