PolV(PolIVb) function in RNA-directed DNA methylation requires the conserved active site and an additional plant-specific subunit

Two forms of a plant-specific RNA polymerase (Pol), PolIV(PolIVa) and PolV(PolIVb), currently defined by their respective largest subunits [NRPD1(NRPD1a) and NRPE1(NRPD1b)], have been implicated in the production and activity of 24-nt small RNAs (sRNAs) in RNA-directed DNA methylation (RdDM). Prevailing models support the view that PolIV(PolIVa) plays an upstream role in RdDM by producing the 24-nt sRNAs, whereas PolV(PolIVb) would act downstream at a structural rather than an enzymatic level to reinforce sRNA production by PolIV(PolIVa) and mediate DNA methylation. However, the composition and mechanism of action of PolIV(PolIVa)/PolV(PolIVb) remain unclear. In this work, we have identified a plant-specific PolV(PolIVb) subunit, NRPE5a, homologous to NRPB5a, a common subunit shared by PolI-III and shown here to be present in PolIV(PolIVa). Our results confirm the combinatorial diversity of PolIV(PolIVa)/PolV(PolIVb) subunit composition and indicate that these plant-specific Pols are eukaryotic-type polymerases. Moreover, we show that nrpe5a-1 mutation differentially impacts sRNAs accumulation at various PolIV(PolIVa)/PolV(PolIVb)-dependent loci, indicating a target-specific requirement for NRPE5a in the process of PolV(PolIVb)-dependent gene silencing. We then describe that the triad aspartate motif present in the catalytic center of PolV(PolIVb) is required for recapitulation of all activities associated with this Pol complex in RdDM, suggesting that RNA polymerization is important for PolV(PolIVb) to perform its regulatory functions.