In Vitro Transcription Activities of Pol IV, Pol V, and RDR2 Reveal Coupling of Pol IV and RDR2 for dsRNA Synthesis in Plant RNA Silencing

SummaryIn Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases Pol IV and Pol V and the putative RNA-dependent RNA polymerase RDR2. Here we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to α-amanitin, and differ in their ability to displace the nontemplate DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is nonfunctional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis.

► RNA polymerase activity is demonstrated for Pol IV, Pol V, and RDR2
► Unlike Pol II, in vitro transcription by Pol IV and Pol V requires an RNA primer
► Pol IV and Pol V are resistant to α-amanitin, a potent inhibitor of Pol II
► Pol V transcription of template DNA is impeded by downstream dsDNA