Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing and sliced target retention

Abstract

RNA-directed DNA methylation (RdDM) in plants is guided by 24 nt siRNAs generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs function as passenger strands during 24 nt siRNA incorporation into AGO4. The 23 nt RNAs are then sliced into 11 and 12 nt fragments, with 12 nt fragments remaining associated with AGO4. Slicing recapitulated with recombinant AGO4 and synthetic RNAs reveals that siRNAs of 21-24 nt, and having any 5-prime terminal nucleotide, can guide slicing, with sliced RNAs then retained by AGO4. RdDM target locus RNAs that copurify with AGO4 in vivo also display a sequence signature of slicing. Comparing plants expressing slicing-competent versus slicing-defective AGO4 shows that slicing elevates cytosine methylation levels at virtually all RdDM loci. We propose that siRNA passenger strand elimination and AGO4 tethering to sliced target RNAs are distinct modes by which AGO4 slicing enhances RdDM.