GSE201234
AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [WGBS]
In plants, transcriptional silencing by RNA-directed DNA methylation (RdDM) is mediated by ARGONAUTE 4 (AGO4) and 24 nt short-interfering RNAs (siRNAs) that are generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs serve as the passenger strands of 23/24 nt duplexes loaded into AGO4. The 24 nt siRNAs then guide AGO4 slicing of the passenger strands, generating 11 and 12 nt cleavage products. Unexpectedly, we find that the 12 nt products remain associated with the guide strand-AGO4 complexes. Long noncoding RNAs generated at RdDM loci are similarly sliced and retained by AGO4.  These results suggest a model in which RNA POLYMERASE V transcripts at target loci are sliced repeatedly as transcription elongation proceeds, sequentially releasing AGO4-siRNA-scaffold RNA complexes that independently recruit the RdDM machinery. Consistent with this hypothesis, plant lines expressing wild-type versus slicing-defective AGO4 show quantitative variation in cytosine methylation and siRNA levels within RdDM loci. Overall design: Whole-genome bisulfite sequencing of wild-type Arabidopsis thaliana ecotype/strain Wassilewskija, ago4-4 null-mutant plants, and ago4-4 plants that stably express transgenes expressing either wild-type or slicing-defective AGO4.