Poiv(poiivb) Function In Rna-directed Dna Methylation Requires The Conserved Active Site And An Additional Plant-specific Subunit

摘要

Two forms of a plant-specific RNA polymerase (Pol), PoIIV(PoIIVa) and PoIV(PoIIVb), 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 PoIIV(PoIIVa) plays an upstream role in RdDM by producing the 24-nt sRNAs, whereas PoIV(PoIIVb) would act downstream at a structural rather than an enzymatic level to reinforce sRNA production by PoIIV(PoIIVa) and mediate DNA methylation. However, the composition and mechanism of action of PoIIV-(PoIIVa)/PoIV(PoIIVb) remain unclear. In this work, we have identified a plant-specific PoIV(PoIIVb) subunit, NRPE5a, homologous to NRPB5a, a common subunit shared by Poll-Ill and shown here to be present in PoIIV(PoIIVa). Our results confirm the combinatorial diversity of PoIIV(PoIIVa)/PoIV(PoIIVb) subunit composition and indicate that these plant-specific Pols are eukaryotic-type poly-merases. Moreover, we show that nrpe5a-1 mutation differentially impacts sRNAs accumulation at various PoIIV(PoIIVa)/PoIV(PoIIVb)-dependent loci, indicating a target-specific requirement for NRPE5a in the process of PoIV(PoIIVb)-dependent gene silencing. We then describe that the triad aspartate motif present in the catalytic center of PoIV(PoIIVb) is required for recapitulation of all activities associated with this Pol complex in RdDM, suggesting that RNA polymerization is important for PoIV(PoIIVb) to perform its regulatory functions.