Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V.

Unlike yeast and metazoans, Arabidopsis thaliana and Populus trichocarpa (poplar) each have two genes orthologous to RBP9, and maize and rice have three ( Figure 1 and Figure S1 ). Orthologs of yeast Rpa12 and Rpc11, the Rpb9-like subunits of Pols I and III, respectively, form separate clades ( Figure 1 ). Both Arabidopsis Rpb9 orthologs copurify with affinity-purified RNA polymerases II, IV, or V (), such that their comprehensive names are NRP(B/D/E)9a and NRP(B/D/E)9b. Despite the fact that the two proteins differ at only 8 of their 114 amino acids, we show here that these ninth subunit variants are incompletely redundant for Pol II and nonredundant for Pol V functions.

Human, Drosophila, zebrafish, Chlamydomonas, Arabidopsis, poplar, maize, or rice proteins homologous to yeast (S. cerevisiae) Rpb9 (Pol II), or its Pol I or Pol III paralogs, were identified by searching the NCBI Reference Sequence (RefSeq) or maizeGBD (in the case of Zea mays GRMZM2G009673) databases using BLASTp. Multiple alignment of the sequences was conducted using MUSCLE (see Figure S1 ) in order to generate the phylogenetic tree. Bootstrap values are indicated for each branchpoint.

In yeast, the 12 Pol II subunits are each encoded by unique genes, ten of which are essential. One of the nonessential genes encodes the ninth-largest subunit, Rpb9. rpb9 deletion strains are viable, but temperature-sensitive (). Rpb9 is implicated in multiple aspects of polymerase II function, including initiation (), processivity (), transcriptional fidelity, proofreading (), and transcription-coupled DNA repair ().

Arabidopsis thaliana Pols II, IV, and V each have 12 core subunits (). Pol II, IV, and V largest subunits are encoded by unique genes: NRPB1, NRPD1, and NRPE1, respectively ("NRP" denotes "Nuclear RNA Polymerase"; "B, D, and E," as the second, fourth, and fifth letters of the alphabet, denote Pols II, IV, or V; the numeral 1 indicates the largest subunit). Pol IV and V second-largest subunits are encoded by the same gene, NRP(D/E)2, which is distinct from the corresponding Pol II subunit gene, NRPB2 (). The two largest subunits interact to form the catalytic center for RNA synthesis, with noncatalytic subunits playing structural and regulatory roles for initiation, elongation, termination, or RNA processing (). Most of the noncatalytic subunits of Pols II, IV, and V are encoded by the same genes ().

Pol IV and Pol V functions are best understood with respect to RNA-directed DNA methylation (), a process in which 24 nt short interfering RNAs (siRNAs) direct the cytosine methylation, and silencing, of complementary DNA sequences. Pol IV acts early in the pathway, working in partnership with RNA-DEPENDENT RNA POLYMERASE 2 to produce double-stranded RNAs that are diced into siRNAs and loaded (primarily) into ARGONAUTE 4 (AGO4) (). Independent of siRNA biogenesis, Pol V generates RNA transcripts at loci that undergo RdDM () and AGO4 binds these Pol V transcripts () as well as Pol V itself (). Chromatin modifying activities are subsequently