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 small interfering RNA (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 base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3′ terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt 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 strand discrimination. [Display omitted] •RNA polymerase IV, RDR2, and DCL3 are sufficient for siRNA synthesis in vitro•Nontemplate-strand-induced Pol IV termination triggers RDR2 synthesis of dsRNA•RDR2 adds an untemplated terminal nucleotide to its transcripts’ 3′ ends•DCL3 generates 24- and 23-nt siRNAs; 23-nt siRNAs often have untemplated termini siRNA-directed DNA methylation is important for genome defense against transposons and viruses. Singh et al. recapitulate siRNA biogenesis in vitro using NUCLEAR RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE 2, and DICER-LIKE 3, providing mechanistic insights into Pol IV termination, RDR2 activation, siRNA precursor strand specification, and DCL3 dicing.