In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.DOI: http://dx.doi.org/10.7554/eLife.09591.001

Highlights d RNA polymerase IV, RDR2, and DCL3 are sufficient for siRNA synthesis in vitro d Nontemplate-strand-induced Pol IV termination triggers RDR2 synthesis of dsRNA d RDR2 adds an untemplated terminal nucleotide to its transcripts' 3 0 ends d DCL3 generates 24-and 23-nt siRNAs; 23-nt siRNAs often have untemplated termini

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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. KeywordsNuclear RNA Polymerase IV, noncoding RNA, RNA silencing, RNA-directed DNA methylation, transcription termination, dicing, ncRNA processing mismatched nucleotides and the short size of Pol IV transcripts (Zhai et al., 2015). However, RDR2 has terminal transferase activity that can add untemplated nucleotides to RNA 3' ends, suggesting an alternative hypothesis for the mismatched nucleotides (Blevins et al., 2015).Whether RDR2's terminal transferase activity might act on Pol IV transcripts, RDR2 transcripts, or both is unknown.Pol IV transcribes single-stranded (ss) DNA but lacks significant activity using sheared double-stranded (ds) DNA in vitro Onodera et al., 2005). Our current study provides an explanation, showing that when Pol IV is engaged in transcription of a ssDNA strand it terminates within 12-18 nt after encountering dsDNA. Importantly, Pol IV termination induced in this manner is key to channeling the transcript to RDR2, which converts the Pol IV transcript into dsRNA. We show that single-stranded M13 bacteriophage DNA can template siRNA biogenesis in vitro, with Pol IV synthesizing first strand transcripts, RDR2 synthesizing the second strands and DCL3 dicing the duplexes into both 24 bp and 23bp siRNAs, as in vivo.DNA-mismatched nucleotides are present at precursor and siRNA 3' ends, as in vivo, with sequencing showing these to be hallmarks of RDR2 transcripts, not Pol IV transcripts.Collectively, the reactions of Pol IV, RDR2 and DCL3 are necessary and sufficient for siRNA biogenesis and can account for the short length of P4R2 RNAs, the origin of untemplated 3' nucleotides, the mechanism of Pol IV-RDR2 coupling and the channeling of RNAs from DNA template transcription to siRNA strand discrimination.