Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

In eukaryotes with multiple small RNA pathways the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the siRNA biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV) whose atypical termination mechanism, induced by nontemplate DNA basepairing, channels transcripts to the associated RNA-dependent RNA polymerase, RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs then typically adds an extra untemplated 3’ terminal nucleotide to the second strands. The dicer endonuclease, DCL3 cuts resulting duplexes to generate 24 and 23nt siRNAs. The 23nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling and RNA channeling from template DNA transcription to siRNA guide strand/passenger strand discrimination.