MINIYO and QUATRE-QUART 2 Are Required for the Assembly of RNA Polymerase V in Arabidopsis

RNA polymerases IV and V (Pol IV and Pol V) are plant-specific polymerases required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Their subunit compositions largely resemble that of Pol II. However, the mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we performed a forward genetic screen and identified novel mutant alleles of MINIYO (IYO), QUATRE-QUART 2 (QQT2) and NUCLEAR RNA POLYMERASE B/D/E 11 (NRPB/D/E11) that are defective in RdDM. We found that Pol IV-dependent small interfering RNAs (siRNAs) and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, dissociated from other Pol V subunits in the iyo and qqt2 mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. Furthermore, we found that IYO and QQT2 were mutually dependent for their binding to the NRPE3 subassembly to facilitate the assembly of Pol V holoenzyme. Our findings reveal IYO and QQT2 as cofactors for Pol V assembly and provide mechanistic insights into how RNA polymerases are assembled in Arabidopsis. To investigate the effects of iyo-4, qqt2-2 and nrpb/d/e11-1 mutations on Arabidopsis DNA methylome, we carried out whole genome bisulfite sequencing. The destabilization of AGO4 in the iyo-4, qqt2-2 and nrpb/d/e11-1 mutants suggests that IYO, QQT2 and NRPB/D/E11 could be required for the production of 24-nt siRNAs. To test this, we performed small RNA deep sequencing analysis.