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 recruited, resulting in de novo cytosine methylation and establishment of repressive histone modifications ().

Eukaryotes decode their genomes using three essential nuclear DNA-dependent RNA polymerases, RNA Polymerases I, II, and III (abbreviated as Pol I, Pol II, and Pol III) (). In plants, two additional multisubunit RNA polymerases, Pol IV and Pol V, are not strictly required for viability but are important for development, transposon taming, antiviral and antibacterial defense, and interallelic communications mediating paramutation ().

Pol V transcripts can be detected at specific intergenic loci such as IGN5 (), therefore we examined whether 9b-1 mutants are impaired for Pol V transcription. IGN5 transcripts are readily detected in wild-type plants or Pol IV mutants (nrpd1) but are substantially reduced in nrpe1 mutants ( Figure 4 B). IGN5 transcript abundance is not affected in the 9b-1 mutant ( Figure 4 B), suggesting that Pol V's ability to synthesize RNA is not impaired and that a step downstream of RNA synthesis might be impaired, instead.

MRD1 is representative of a small set of Arabidopsis loci at which RdDM and silencing requires Pol IV but not Pol V. Thus, MRD1 is expressed at low levels in Pol V mutants (nrpe1-11), as in wild-type plants, but is substantially derepressed in Pol IV mutants (nrpd1-3), unlike AtSN1 or soloLTR elements that require both Pol IV and Pol V for silencing ( Figure 4 A ). In 9a-1 or 9b-1 mutants, MRD1 is not derepressed, suggesting that Pol IV function is not impaired in either single mutant ( Figure 4 A, lanes 4 and 5).

(D) Locations of the eight amino acids that are polymorphic in the Arabidopsis 9a and 9b subunits, mapped (in red) onto a space-filling rendering of yeast Rpb9 (green) within a Pol II elongation complex (PDB:1Y1W). Rpb1 is colored gray, Rpb2 is blue, Rpb5 is gold, and the DNA is pink. The image on the right is rotated clockwise relative to the image on the left to show the position of the DNA duplex. Rpb9 amino acids colored red correspond to the amino acids that align with the polymorphic amino acids of the Arabidopsis 9a and 9b subunits, and are numbered based on the Arabidopsis sequences.

(C) Amino acid sequence alignment of yeast Rpb9 with Arabidopsis NRP(B/D/E)9a and NRP(B/D/E)9b. Amino acids that differ in the 9a and 9b subunits are highlighted. Numbers correspond to amino acid positions of the Arabidopsis 9a and 9b proteins, and not (necessarily) to yeast Rpb9.

(A) Pol IV-dependent, but Pol V-independent, silencing of locus MRD1. RNA isolated from wild-type (Col-0), nrpd1-3, nrpe1-11, 9a-1, or 9b-1 mutants was subjected to RT-PCR using primers specific for MRD1, soloLTR, or AtSN1.

Pol IV is required for the biogenesis of ∼95% of all 24 nt siRNAs (). For instance, 24 nt siRNAs corresponding to 5S rRNA genes and soloLTR retrotransposons are severely depleted in nrpd1-3 mutants ( Figure 3 D) (). In 9a-1 or 9b-1 mutants, siRNAs are detected at wild-type levels, suggesting that Pol IV activity is not impaired. Interestingly, reductions in siRNA levels observed in nrpe1 mutants are also not apparent in 9a-1 or 9b-1 mutants, suggesting that RNA synthesis by Pol V is also unimpaired.

A complete NRP(B/D/E)9b transgene, transcribed from its native promoter and containing all its introns, rescues all 9b-1 mutant phenotypes, restoring wild-type leaf morphologies ( Figure S3 A ), NRP(B/D/E)9b mRNA production ( Figure S3 B), AtSN1 and soloLTR silencing ( Figure S3 B), and cytosine methylation ( Figure S3 C). We conclude that 9b-1 mutant phenotypes are due solely to mutation of the NRP(B/D/E)9b gene.

(A) A transgene containing a genomic clone of NRP(B/D/E)9b, under control of its native promoter and containing all introns and exons, was engineered to contain a FLAG epitope at the protein's C terminus. This transgene was transformed into 9b-1 mutants to test for restoration of wild-type morphological and molecular phenotypes. Multiple independent transformants are shown, all displaying wild-type morphological phenotypes.

Tandemly repeated 5S ribosomal RNA genes are also subject to RdDM. Southern blots of genomic DNA digested with HaeIII and hybridized to a 5S rRNA gene probe show a ladder of bands in wild-type Col-0 plants ( Figure 3 C), with larger bands reflecting increased methylation among adjacent genes. HaeIII methylation is reduced in nrpd1-3, nrpe1-1, and 9b-1 mutants, but not in 9a-1 mutants.

Retrotransposon silencing correlates with Pol IV and Pol V-dependent RNA-directed DNA methylation (RdDM), rendering methylation-sensitive AluI sites within soloLTR elements and HaeIII sites within AtSN1 elements resistant to AluI or HaeIII digestion. Consequently, PCR using primers that flank the restriction sites amplifies the interval in wild-type (Col-0) plants ( Figure 3 B). However, in nrpd1-3 or nrpe1-11 mutants, loss of RdDM allows AluI and HaeIII to cleave the DNA, and PCR fails ( Figure 3 B). Methylation is similarly lost in 9b-1 mutants, but not in 9a-1 mutants ( Figure 3 B).

Loci silenced by Pol IV and Pol V include soloLTR and AtSN1 retroelements, whose expression is undetectable in wild-type plants (Col-0) but prevalent in nrpd1-3 (Pol IV largest subunit) or nrpe1-11 (Pol V largest subunit) mutants ( Figure 3 A ). In 9a-1 mutants, soloLTR and AtSN1 silencing is unaffected. However, these retroelements are derepressed in 9b-1 mutants ( Figure 3 A).

(D) RNA blot hybridization analysis of inflorescence small RNAs using 5S rRNA gene (siR1003), soloLTR, and microRNA (miR160) probes. The same blot was stripped and rehybridized sequentially. miR160 serves as an RNA loading control.

(B) Cytosine methylation assay involving PCR amplification of soloLTR and AtSN1 retrotransposons following incubation of genomic DNA with the methylation sensitive restriction endonucleases, HaeIII or AluI. The control locus, At2g19920, lacks HaeIII sites. Diagrams show positions of restriction endonuclease recognition sites within the amplicon.

(A) RT-PCR analysis of soloLTR and AtSN1 retroelement expression, comparing Pol IV and Pol V largest subunit null mutants, nrpd1-3 and nrpe1-11, respectively, with 9a-1, 9b-1, and wild-type (Col-0). The loss of silencing in the 9b-1 mutant is restored by a full-length 9b transgene (see Figure S3 ).

Among the progeny of heterozygotes carrying a recessive allele of an essential gene, heterozygotes should outnumber homozygotes 2:1. However, we observed a nearly 1:1 ratio of heterozygotes to homozygous wild-type plants ( Figure 2 E), suggesting a defect in male or female (or both) transmission of the mutant alleles. Reciprocal crosses showed reduced transmission of 9a-1 or 9b-1 alleles through both the male and female gametophytes ( Figure S2 ). This allele transmission behavior differs from null mutations eliminating catalytic subunits of Pols I, II, or III, which show zero transmission via the egg donor ().

We showed previously that null mutant alleles for catalytic subunits of Pols I, II and III are not transmitted maternally due to 100% lethality in haploid female gametophytes (). By contrast, female or male gametophytes that are 9a-1 9b-1 double mutants are able to transmit the mutant alleles to the next generation, indicating that the double mutant does not cause obligate gametophyte lethality. However, the mutant alleles are transmitted at slightly reduced frequency (32%–41%; the expected frequency is 50% for nondeleterious alleles), suggesting some decrease in gametophyte fitness, especially for male gametophytes (pollen).

The top table shows the results for an experiment in which plants homozygous for the 9a-1 allele and hemizygous for the 9b-1 allele were crossed, as the female or male parent, to wild-type (+/+) plants; their progeny were then scored for the presence of each mutant allele. The bottom table shows the results of an equivalent experiment in which plants hemizygous for 9a-1 and homozygous for 9b-1 were crossed to wild-type plants.

To test 9a and 9b redundancy, homozygous 9a-1 and 9b-1 mutants were crossed, resulting F1 plants were selfed, and their progeny genotyped. In siliques of F2 plants homozygous for 9a-1 and heterozygous for 9b-1, in which 25% of the F3 seeds are expected to be homozygous 9a-1 9b-1 double mutants, 30% (55/181 analyzed) of the seeds arrested in development and 70% developed normally ( Figure 2 D). Similar results were observed for the progeny of plants homozygous for 9b-1 but heterozygous for 9a-1. In arrested seeds, which are translucent, embryos failed to develop past the globular stage ( Figure 2 D). Among plants germinated from seeds collected from siliques of plants that were homozygous for either 9a-1 or 9b-1 and heterozygous for the other mutation, no 9a-1 9b-1 double mutants were identified ( Figure 2 E). Because lethality is a consequence of lost Pol II function (), but not disrupted Pol IV or Pol V function (), we conclude that the 9a and 9b subunits are mostly redundant with respect to Pol II functions required for viability and development, such that only the double mutant is embryo lethal.

Wild-type (ecotype Col-0) and 9a-1 mutant plants are indistinguishable, but leaves of 9b-1 mutants are more ovate, have shorter petioles and display less downward edge curling ( Figure 2 C). Other 9b-1 phenotypes include smaller trichomes on the first true leaves, more prominent leaf midveins, changes in the cuticular wax coating on the leaves, and shorter siliques. These morphological differences presumably result from altered Pol II-dependent gene expression given that null mutations eliminating Pol IV or Pol V largest subunits (nrpd1-3 or nrpe1-11, respectively) do not induce similar phenotypes. Moreover, 9b-1 phenotypes are neither suppressed nor enhanced in 9b-1 nrpd1-3 or 9b-1 nrpe1-11 double mutants.

Arabidopsis NRP(B/D/E)9a (At3g16980) and NRP(B/D/E)9b (At4g16265) genes have similar intron/exon structures ( Figure 2 A ). T-DNA insertion alleles, designated nrp(b/d/e)9a-1 (Salk_032670) and nrp(b/d/e)9b-1 (Salk_031043), are disrupted within introns 2 or 1, respectively ( Figure 2 A). Transcripts of the 9a and 9b (abbreviated for brevity) genes are readily detected in wild-type plants ( Figure 2 B) but not in 9a-1 or 9b-1 mutants ( Figure 2 C).

(E) Homozygous 9a-1 9b-1 double mutants are not recovered among seedling progeny of plants homozygous for 9a-1 or 9b-1 and heterozygous for the other allele. Heterozygote under-representation results from reduced transmission of mutant alleles via both the male and female gametophytes (see Figure S2 ).

(D) Ovules within a silique of a plant homozygous for 9a-1 and heterozygous for 9b-1 (upper image). Opaque ovules (blue arrows) contain fully developed embryos whose primary roots and (folded-over) cotyledons can be observed by differential interference microscopy (image at lower left). By contrast, translucent ovules (red arrows), occurring at the expected frequency of 9a-1 9b-1 double mutants, lack mature embryos (bottom center and bottom right images). The image at bottom right shows a blow-up of the region circled in the bottom center image, revealing an embryo arrested at the globular stage of development.

(B) RT-PCR amplification of NRP(B/D/E)9a, NRP(B/D/E)9b or actin mRNAs in wild-type (Col-0), Pol V largest subunit null mutant (nrpe1-11), or 9a-1 or 9b-1 mutant plants. Actin RT-PCR reactions in which reverse transcriptase was omitted were also conducted (bottom row) to assess potential DNA contamination of the RNA samples.

(A) Positions of T-DNA insertions within the nrp(b/d/e)9a-1 and nrp(b/d/e)9b-1 alleles are indicated by triangles. Filled boxes represent exons; lines within boxes represent introns. Genes At3g16980 and At4g16265 encode the 9a and 9b protein sequences whose accession numbers (shown in Figure 1 and Figure S1 ) are NP_188323 and NP_567490, respectively.

Discussion

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Cramer P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. In mutants lacking the 9b subunit, Pol IV-dependent siRNA biogenesis is not impaired, nor is silencing of MRD1, a locus whose RdDM and repression is dependent on Pol IV but not Pol V. However, loci that require both Pol IV and Pol V for silencing are derepressed in 9b-1 mutants. Based on these observations, we deduce that loss of silencing in 9b-1 mutants is due to a defect in Pol V function. Interestingly, Pol V transcription does not appear to be impaired in 9b-1 mutants, based on IGN5 transcript production and siRNA abundance at loci where siRNA levels depend, in part, on Pol V activity. Therefore, we reason that the impairment of Pol V function in 9b mutants is not due to a decreased ability of Pol V to synthesize RNA, but an impairment of a regulatory function, possibly mediated by interactions with other proteins. Consistent with this hypothesis, the eight amino acids that are different in the 9a and 9b subunits are predicted to be exposed on the surface of the proteins, based on their homology to yeast Rpb9 ( Figure 4 C), whose structure is known. Figure 4 D shows a space filling model in which the predicted positions of the polymorphic amino acids of 9a and 9b are mapped onto the corresponding amino acid positions of Rpb9 within a yeast Pol II elongation complex (PDB:1Y1W) ().

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Cramer P. Evolution of two modes of intrinsic RNA polymerase transcript cleavage. Rpb9 has two zinc finger domains, Zn1 and Zn2, which are located near the N and C termini of the protein. The Zn2 domain shares homology with the elongation/transcript cleavage factor, TFIIS and is thought to catalyze transcript cleavage events in partnership with TFIIS. Transcript cleavage is important for RNA 3′-end processing, transcription termination, and polymerase backtracking that allows for correction of misincorporated nucleotides, escape from an arrested state, or DNA repair at damaged sites (). The Rpb9-paralogous subunits of Pols I and III, Rpa12, and Rpc11, also possess transcript cleavage activity and are stronger endonucleases than Rpb9, suggesting that Rpb9 has evolved to be regulated by TFIIS or other factors (). Several of the amino acid differences between NRP(B/D/E)9a and NRP(B/D/E)9b occur within the Zn2 domain (amino acids 77, 82, and 109). Arabidopsis has multiple genes encoding TFIIS-like proteins, leading us to speculate that the eight amino acids that differ between the 9a or 9b proteins might specify interactions with different TFIIS-like proteins, or with proteins that mediate chromatin modifications at Pol V-transcribed loci.

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Young R.A. Yeast RNA polymerase II subunit RPB9 is essential for growth at temperature extremes. The single multisubunit RNA polymerases used by archaea closely resemble eukaryotic RNA Polymerase II except that they lack an Rpb9-like subunit (); likewise, yeast strains deleted for the RPB9 gene are viable (). These observations have suggested that Rpb9 is an important, but nonessential, regulatory subunit in eukaryotes. However, our results show that NRPB9 function is essential in Arabidopsis. Either NRPB9a or NRPB9b (the subunits named in the context of Pol II) is sufficient for viability, but embryogenesis cannot be completed in nrpb9a nrpb9b double mutants.

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Pikaard C.S. Sex-biased lethality or transmission of defective transcription machinery in Arabidopsis. It is noteworthy that nrpb9a nrpb9b mutants develop further than do null mutants for catalytic subunits of Pols I, II, and III, in which female gametophytes fail to develop and are never fertilized, such that no embryogenesis takes place (). Therefore, NRPB9-mediated functions may be partially dispensable in plants, as in yeast and archaea, specifically at the haploid gametophytic stage of the plant life cycle. However, NRPB9 function is essential during the diploid sporophyte stage of the plant life cycle, at one or more steps required for embryo development, beginning at the globular embryo stage. NRPB9 functions must also affect later vegetative development in order to explain the distinct phenotypes of nrpb9b mutants.

Our data suggest that Pol IV functions are not impaired in 9a-1 or 9b-1 mutants. One possibility is that the ninth subunit is dispensable for Pol IV function. Alternatively, the 9a and 9b subunits might be redundant in the context of Pol IV, as they are for most Pol II functions. Unfortunately, the lethality of the 9a-1 or 9b-1 double mutant precludes an easy test of whether a functional ninth subunit is required for Pol IV activity.