NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation

Abstract

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.[Keywords: DNA methylation; transcriptional gene silencing; siRNAs; Pol IV; Pol V] Supplemental material is available at http://www.genesdev.org.

A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development

“…Therefore, the rdm4 mutation releases the silencing of RD29A-LUC but not 35S-NPTII. This effect is very similar to that of nrpd1, nrpe1, nrpd2, drd1, and ago6 mutations (Zheng et al 2007;He et al 2009b). Because the TGS of the 35S-NPTII transgene is mediated by an siRNA-independent pathway (He et al 2009b), these results suggest that, like NRPD1, NRPE1, NRPD2, DRD1, and AGO6, RDM4 functions specifically in the siRNA-dependent pathway of TGS.…”

“…A T-DNA mutagenized population in the ros1-1 background was screened to identify new components in the RdDM pathway, based on the luminescence phenotype of RD29A-LUC. This screen recovered not only mutants in known RdDM pathway components-such as NRPD1, NRPE1, NRPD2/NRPE2, AGO4, HEN1, DRD1, and HDA6 (He et al 2009b)-but also several new components. One of the new components is designated as RDM4.…”

“…A T-DNA mutagenized ros1-1 population was generated as described previously (He et al 2009b). A 3-d cold stratification was used to promote the uniformity of seed germination.…”

“…We recently found another subunit, RDM2/NRPD4, that is shared by Pol IV and Pol V, and that is also necessary for the RdDM pathway (He et al 2009b). Like Pol II (Cramer 2002), Pol IV and Pol V are multisubunit complexes (Huang et al 2009;Ream et al 2009).…”

“…The 24-nt siRNAs are generated in a pathway involving the putative DNA-directed RNA polymerase IV (Pol IV), RDR2 (RNA-dependent RNA polymerase 2), and DCL3 (Dicer-like RNA Pol IV and Pol V have distinct largest subunits-NRPD1 and NRPE1, respectively-but share some common subunits such as NRPD/E2 and NRPD/E4 (He et al 2009b). Some subunits of Pol IV and Pol V are also shared with Pol II (Huang et al 2009;Ream et al 2009).…”