DNA-dependent RNA Polymerases IV and V: Orchestrators of RNA-mediated Transcriptional Silencing in Plants

Plants are remarkable in having five nuclear DNA-dependent RNA polymerases. In addition to RNA Polymerases I, II and III, which are ubiquitous among eukaryotes and essential for viability, plants have evolved two additional multisubunit plant-specific RNA polymerases, abbreviated as Pol IV and Pol V.  Pols IV and V evolved as specialized forms of RNA Polymerase II and play non-redundant roles in siRNA-mediated DNA methylation and gene silencing. In vitro transcription studies show that Pol IV and Pol V are alpha-amanitin insensitive DNA-dependent RNA polymerases. Pol IV transcripts serve as templates for RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), generating double-stranded RNAs that are diced by DICER-LIKE3. Resulting 24 nt siRNAs are loaded primarily into AGO4. At target loci, Pol V generates noncoding RNAs that are required for DNA methylation of the loci. AGO4-siRNA effector complexes bind to these Pol V transcripts in order to be recruited to their target sites. Our efforts are focused on a biochemical understanding of Pol IV and Pol V functions and the channeling of RNA intermediates in the siRNA-directed DNA methylation pathway.