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. Author manuscript; available in PMC: 2014 Dec 15.
Published in final edited form as: Cell Rep. 2014 Oct 2;9(1):378–390. doi: 10.1016/j.celrep.2014.08.067

Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits

Jeremy R Haag 1,2, Brent Brower-Toland 3, Elysia K Krieger 3, Lyudmila Sidorenko 4, Carrie D Nicora 5, Angela D Norbeck 5, Andre Irsigler 6,#, Huachun LaRue 3, Jan Brzeski 4, Karen McGinnis 6, Sergey Ivashuta 3, Ljiljana Pasa-Tolic 5, Vicki L Chandler 4, Craig S Pikaard 1,2,*
PMCID: PMC4196699  NIHMSID: NIHMS627471  PMID: 25284785

Summary

Unlike nuclear multisubunit RNA polymerases I, II and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA Polymerases IV and V are non-essential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their twelve subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits, but differ from each other only in their largest subunits. Use of alternative catalytic second-subunits, which are non-redundant for development and paramutation, yields at least two subtypes of Pol IV, and three subtypes of Pol V in maize. Pol IV/V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

Keywords: transcription, gene silencing, epigenetic regulation, DNA-dependent RNA polymerase, RNA-directed DNA methylation, chromatin modification

Introduction

Plants are unique in having evolved multisubunit RNA polymerases IV and V in addition to Pols I, II and III, the three ubiquitous nuclear DNA-dependent RNA polymerases of eukaryotes. Pols IV and V synthesize noncoding RNAs for transcriptional silencing of transposons, repetitive elements and a subset of genes (Haag and Pikaard, 2011; Herr et al., 2005; Kanno et al., 2005b; Onodera et al., 2005; Pontier et al., 2005; Ream et al., 2014). Both are 12-subunit enzymes (Ream et al., 2009) that evolved as specialized forms of Pol II, as shown by mass spectrometry (Huang et al., 2009; Law et al., 2011a; Ream et al., 2009) and phylogenetic analyses (Luo and Hall, 2007; Onodera et al., 2005; Tucker et al., 2011).

Pols IV and V play distinct roles in RNA-directed DNA methylation (RdDM) in Arabidopsis (Law and Jacobsen, 2010; Matzke et al., 2009; Pikaard et al., 2013; Wierzbicki, 2012; Zhang and Zhu, 2011). Pol IV acts early in the process, generating transcripts that serve as templates for RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) (Xie et al., 2004), which physically associates with Pol IV (Haag et al., 2012; Law et al., 2011a). Other Arabidopsis Pol IV-associated proteins include CLSY (a sub-family of SWI2/SNF2-like putative ATP-dependent DNA translocases) (Smith and Baulcombe, 2007) and SHH1 (a.k.a. DTF1), a BAH domain protein that binds histone H3 that is dimethylated on lysine 9 (H3K9me2) and unmethylated on lysine 4, thereby recruiting Pol IV to heterochromatin (Law et al., 2013; Law et al., 2011a; Zhang et al., 2013). Double-stranded RNAs generated by Pol IV and RDR2 are diced by DCL3 into 24 nt small interfering RNAs (siRNAs) that are bound by ARGONAUTE 4 (AGO4), AGO6 or AGO9 (Havecker et al., 2010; Mallory and Vaucheret, 2010). The Argonaute-siRNA complexes bind to Pol V transcripts, whose synthesis requires a multi-protein complex that includes a SWI2/SNF2-family ATP-dependent DNA translocase (DRD1), a protein related to the hinge domains of cohesins and condensins (DMS3), and a single-stranded DNA binding protein (RDM1) (Gao et al., 2010; Kanno et al., 2005a; Kanno et al., 2008; Law et al., 2010; Wierzbicki et al., 2008; Wierzbicki et al., 2009b). AGO4 can also interact with the C-terminal domain of the Pol V largest subunit, NRPE1 (El-Shami et al., 2007). Pol V-dependent recruitment of AGO-siRNA complexes enables subsequent recruitment of de novo DNA methylation, histone modification and chromatin remodeling machineries, yielding chromatin states refractive to Pol I, II or III transcription (Wierzbicki, 2012; Wierzbicki et al., 2008; Wierzbicki et al., 2009b).

Paramutation is an epigenetic phenomenon in which a functional allele can be inactivated upon exposure to a silenced allele of the same gene (Arteaga-Vazquez and Chandler, 2010; Erhard and Hollick, 2011). How alleles communicate in paramutation is unclear, but maize orthologs of Arabidopsis RdDM pathway proteins are required. These include MOP1 (MEDIATOR OF PARAMUTATION1), the ortholog of Arabidopsis RDR2 (Alleman et al., 2006), and RMR6 (REQUIRED TO MAINTAIN REPRESSION6), which corresponds to the Pol IV largest subunit, NRPD1 (Erhard et al., 2009). Likewise, RMR1 (REQUIRED TO MAINTAIN REPRESSION1) encodes a paralog of Arabidopsis CLSY or DRD1 proteins, suggesting possible roles in Pol IV or Pol V transcription (Hale et al., 2009).

The second-largest subunits of Pols IV and V in Arabidopsis (ecotype Col-0) are encoded by a single gene, NRP(D/E)2 (also known as NRP(D/E)2a) (Herr et al., 2005; Kanno et al., 2005b; Onodera et al., 2005; Pontier et al., 2005). A closely-linked paralog, NRP(D/E)2b is non-functional. NRP(D/E)2 is most similar to NRPB2, the Arabidopsis gene uniquely encoding the second subunit of Pol II (Onodera et al., 2005). Interestingly, maize has three NRP(D/E)2-like genes (designated with the suffixes a, b and c) and two NRPB2-like genes. Independent studies have shown that the paramutation mutants mop2 and rmr7 correspond to mutant alleles of NRP(D/E)2-likea (Sidorenko et al., 2009; Stonaker et al., 2009), but the affected polymerase(s) are unclear. To address this question, we affinity-purified maize Pol II or epitope-tagged Pol IV, Pol V or MOP1 and identified their subunits and associated proteins using mass spectrometry. Surprisingly, the fundamental difference between maize Pols IV and V is their use of distinct largest subunits. Moreover, we show that Pols IV and V can assemble using two, or all three, respectively, of the NRP(D/E)2 subunit variants (a, b, and c). Alternative forms of Pol II likewise assemble using two NRPB2 subunit variants. Maize Pol IV associates with the RNA-dependent RNA polymerase, MOP1, paralleling Pol IV-RDR2association in Arabidopsis, and with SWI2/SNF2 family proteins RMR1 and CHR167, suggesting that the latter are functionally analogous to Arabidopsis CLSY proteins. Maize Pol V likewise associates with paralogs of Arabidopsis RdDM pathway proteins, including ARGONAUTE121 (related to Arabidopsis AGO6), CHR127 (related to Arabidopsis DRD1) and DMS3. Two closely related proteins, SHH2a and SHH2b associate with both Pols IV and V in maize, suggesting that both enzymes are targeted to heterochromatic regions in the same way, analogous to Arabidopsis Pol IV targeting by SHH1/DTF1 (Law et al., 2013; Zhang et al., 2013). Collectively, our proteomic analyses provide evidence for ongoing Pol IV/V functional diversification as well as protein partnerships important for paramutation.

RESULTS

Subunit compositions of maize RNA polymerases II, IV and V

In the maize genome, single copy genes encode the largest subunits of RNA polymerases II, IV and V (Fig. 1A; accession numbers for genes used to generate the phylogenetic tree are provided in Table S1). The encoded proteins (NRPB1, NRPD1 and NRPE1, respectively) have magnesium ion binding motifs (Metal A site) and conserved domains A-H typical of multisubunit RNA polymerase largest subunits (Fig. 1B; see also Fig. S1a). The C-terminal domain (CTD) of the Pol II largest subunit, NRPB1 consists of repeating seven amino acid (heptad) motifs, as in all eukaryotes (Egloff and Murphy, 2008; Hsin and Manley, 2012), that are absent in NRPD1 and NRPE1 of Pols IV and V, respectively (Haag and Pikaard, 2011). Instead, maize NRPD1, as in Arabidopsis, has a short CTD with a so-called DeCL domain, named for its similarity to the DEFECTIVE CHLORLOPLASTS AND LEAVES protein implicated in chloroplast ribosomal RNA processing (Bellaoui and Gruissem, 2004). The CTD of the maize Pol V largest subunit, NRPE1 also has a DeCL domain, plus two imperfect repeats of 27-amino acids. Aside from the conserved DeCL domains, maize and Arabidopsis Pol V CTDs display little, if any, primary sequence conservation. Whereas the A. thaliana (ecotype Col-0) Pol V CTD has ten imperfect repeats of 16-amino acid sequence, and a glutamine/serine (QS)-rich domain at the extreme C-terminus of the protein, these features are absent in the maize Pol V CTD. However, maize and Arabidopsis Pol V CTDs have in common the occurrence of numerous WG or GW amino acid pairs (11 in maize; 18 in Arabidopsis). In Arabidopsis, these WG/GW “AGO-hook” motifs facilitate Argonaute protein interactions (El-Shami et al., 2007).

Figure 1.

Figure 1

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Maize Pol II, IV and V largest subunits. (A) Neighbor-joining tree, with bootstrap values, generated by MUSCLE alignment of full-length RNAP largest subunits. Maize sequences are in red. (B) Domain features of the maize NRPB1, NRPD1 and NRPE1 proteins, including conserved domains A-H, Metal A catalytic centers (invariant aspartates are colored red), NRPB1 C-terminal domain heptad repeats (open arrowheads), DeCL domains, of NRPD1 and NRPE1 (green), NRPE1 WG/GW-rich regions (yellow) and 27aa repeats (closed arrowheads). (C) Immunoblots of immunoprecipitated NRPB1, or FLAG epitope-tagged NRPD1 or NRPE1 using antibodies recognizing native NRPB1, NRPD1 or NRPE1. (D) Affinity-purified Pols II, IV and V are functional for transcription in vitro. A 32 nt DNA template annealed to a 16 nt RNA oligonucleotide, yielding an 8 bp DNA-RNA hybrid region (diagrammed at left), was incubated with affinity purified Pols II, IV and V and nucleotide triphosphates, including alpha32P-CTP, in the presence or absence of 5 ug/ml alpha-amanitin. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis, transferred to filter paper, dried under vacuum, and visualized by phosphorimaging. See also Figure S1a and Table S1.

Transgenic maize cell cultures expressing full-length NRPD1 or NRPE1 fused at their C-termini to tandem FLAG and HA epitope tags were used for affinity capture of Pol IV or Pol V via anti-FLAG immunoprecipitation (IP). Maize Pol II was IP’ed using an antibody recognizing the CTD heptad repeats of NRPB1. Immunoblot analyses using antibodies specific for Pol II, IV or V largest subunits (NRPB1, NRPD1 or NRPE1, respectively) revealed that each polymerase had been isolated free of cross-contamination (Fig. 1C). Affinity-purified Pol II, IV and V complexes are functional for transcription, as shown by their ability to synthesize radioactively labeled RNA transcripts in vitro (Fig. 1D). As in Arabidopsis (Haag et al., 2012), transcription by maize Pols IV and V is insensitive to the fungal toxin, alpha-amanitin, whereas Pol II activity is inhibited (Fig. 1D).

Tryptic peptides of affinity-purified maize Pol II, IV and V were analyzed by liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). Peptides of NRPB1, NRPD1 or NRPE1 were detected only in Pol II, Pol IV or Pol V samples, respectively (summarized in Table 1; for additional details see Tables S2-S4), consistent with the immunoblotting results (Fig. 1C). Eleven of the expected twelve subunits were identified for each polymerase, but no peptides corresponding to any of three twelfth-subunit paralogs were detected (Table 1). The twelfth subunit is one of five subunits common to RNA polymerases I, II and III in eukaryotes, and is also common to Arabidopsis Pols IV and V (Ream et al., 2009). Our failure to detect 12th subunit peptides may be a consequence of the small size of the proteins (~52 amino acids), thus limiting the number of potential tryptic fragments amenable to ionization and detection.

Table 1.

Proteins identified by mass spectrometry in affinity-purified Pols II, IV or V

Proteins associated with affinity-purified Pols II, IV or V. Numerical values are the percentage of the full-length protein represented by unique peptides, excluding peptides that could match related paralogs. A “*” symbol denotes that peptides matching the protein were detected, but also match one or more paralogs. A “#” symbol denotes that proteins are identical, and indistinguishable. For additional details, see Tables S2-S4.

Yeast
Protein		 Maize homologs Affinity Purified Protein Names/Synonyms
NRPB1
(Pol II)		 NRPD1
(Pol IV)		 NRPE1
(Pol V)
DNA-dependent RNA polymerase subunits Rpb1 GRMZM2G044306 26% NRPB1
GRMZM2G007681 50% 2% NRPD1/ RMR6
GRMZM2G153797 59% NRPE1
Rpb2 GRMZM2G084891 6% NRPB2a
GRMZM2G113928 2% NRPB2b
GRMZM2G054225 14% 28% NRP(D/E)2a/ MOP2/ RMR7
GRMZM2G427031 3% 16% NRP(D/E)2b
GRMZM2G133512 * 4% NRPE2c
Rpb3 GRMZM2G402295 58% 50% 63% NRP(B/D/E)3
Rpb4 GRMZM2G119393 60% NRPB4
GRMZM2G453424 49% 51% NRP(D/E)4
Rpb5 GRMZM2G476009 47% NRPB5a
GRMZM2G099183 38% NRPB5b
GRMZM2G469969 44% 48% NRP(D/E)5
Rpb6 GRMZM2G013600 * * * NRP(B/D/E)6a
GRMZM2G086904 * * * NRP(B/D/E)6b
Rpb7 GRMZM2G179346 23% NRPB7
GRMZM2G040702 26% 42% NRP(D/E)7
Rpb8 GRMZM2G034326 61% 78% 77% NRP(B/D/E)8
GRMZM2G347789 NRPB8-like
Rpb9 GRMZM2G046061 37% NRPB9a
GRMZM2G023028 37% NRPB9b
GRMZM5G898768 18% 33% NRP(D/E)9
Rpb10 NP_001152395 35% 42% 59% NRP(B/D/E)10a
GRMZM5G803992 * * 42% # NRP(D/E)10b
GRMZM5G834335 * * 42% # NRP(D/E)10c
Rpb11 GRMZM2G130207 32% 48% 48% NRP(B/D/E)11
GRMZM2G043461 * * * NRPB11-like
Rpb12 GRMZM2G146331 NRPB12a
GRMZM2G322661 NRPB12b
GRMZM2G540834 NRPB12c
Polymerase-associated proteins RNA-dep
RNAP		 GRMZM2G042443 29% M0P1/RDR101
GRMZM2G145201 * RDR102
Swi2/Snf2
family		 GRMZM2G154946 4% RMR1
GRMZM2G178435 15% CHR167 RMR1-like
GRMZM5G574858 8% CHR127/ Zm_DRD1a
GRMZM2G393742 * CHR156/ Zm_DRD1b
- NP_001132336 5% Zm_DMS3
- GRMZM2G111204 5% Zm_SHH2a
GRMZM2G126170 2% Zm_SHH2b
Iwr1 GRMZM2G098603 9% 25% 8% IWR1/Zm_DMS4/Zm_RDM4
Ago1 GRMZM2G589579 * AG0105
GRMZM2G089743 * AGO119
GRMZM2G347402 4% AGO121
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*

= One or more identified peptides also matched this protein, but are not unique to this protein

Multiple subtypes of Pols II, IV and V assemble using alternative second subunits

Maize has five genes encoding proteins similar to the yeast Pol II second-subunit, Rpb2. Two group with the Arabidopsis Pol II second-subunit gene, NRPB2 (Fig. 2A, Table 1; see also Fig. S1b). Both of these proteins, NRPB2a and NRPB2b, are detected in affinity-purified Pol II (Table 1, Fig. 2A; see also Table S2), indicating that either can assemble into Pol II. The remaining three maize Rpb2 paralogs are similar to the Arabidopsis NRP(D/E)2 gene that encodes the second subunits of Pols IV and V, but segregate into two monocot-specific clades (Fig. 2A). The protein encoded by NRP(D/E)2-likea, the gene disrupted in mop2 and rmr7 mutants, is present in both Pol IV and Pol V, as is its closest paralog, NRP(D/E)2-likeb (Table 1 and Fig. 2A; see also Tables S3, S4). We have thus renamed the proteins NRP(D/E)2a and NRP(D/E)2b to reflect their confirmed associations with both Pol IV and Pol V. Peptides unique to NRP(D/E)2-likec were identified only in Pol V; the protein is thus renamed NRPE2c (Table 1 and Fig. 2A; see also Table S4).

Figure 2.

Figure 2

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Second-subunit diversity, and positions of Pol II, IV and V common versus distinct subunits. (A) Neighbor-joining tree, with bootstrap values, generated from MUSCLE alignment of full-length RNAP second subunits. Maize subunits are highlighted in red. (B). NRPD1 and NRPE1 co-immunoprecipitate with epitope-tagged NRP(D/E)2a-FLAG-HA. Proteins immunoprecipitated from the indicated lines using anti-FLAG resin were subjected to immunoblotting using antibodies recognizing native NRPD1, native NRPE1 or the HA epitope. (C) Based on yeast Pol II (PDB: 2VUM), positions of subunits common to Pols II, IV and V (in color) are displayed in views of the leading face (left) or backside (right) of the enzyme. The DNA helix (green) and nascent RNA (red) are visible at the center of the leading face image. (D) Positions of subunits that differ in Pols IV and V compared to Pol II are displayed in views of the leading face (left) or from above, looking down onto the template DNA within the enzyme. Pol IV/V-specific subunits occupy the leading face of the enzyme; common subunits cluster on the trailing face. See also Figures S1b-l and Table S1.

As an independent test of NRP(D/E)2a associations with Pols IV and V, a cell-free extract of transgenic maize expressing NRP(D/E)2a fused to tandem, C-terminal FLAG and HA epitope tags was immunoprecipitated using anti-FLAG resin. The affinity-purified proteins were then analyzed by immunoblotting, using antibodies recognizing the largest subunits of native Pol IV (NRPD1) or Pol V (NRPE1) or the HA tag on the recombinant NRP(D/E)2a-FLAG-HA protein (Fig. 2B), as well as by mass spectrometry. NRPD1 and NRPE1 both co-purify with NRP(D/E)2a, as shown by immunoblotting (Fig. 2B) and by mass spectrometry (Table 2; Table S5). Interestingly, a different engineered form of the protein, FLAG-NRP(D/E)2a, which has the FLAG epitope fused to the amino terminus of the protein, co-purified only with Pol V (Table 2; Table S6), suggesting that the N-terminal epitope tag interferes with Pol IV assembly.

Table 2.

Proteins co-purifying with N- or C-terminally tagged maize NRP(D/E)2a.

Proteins co-purifying with maize NRP(D/E)2a epitope-tagged at the amino or carboxyl terminus . Annotation is the same as for Table 1. For additional details, see Tables S5 and S6.

Yeast
Protein		 Maize homologs Recombinant form of NRP(D/E)2a analyzed
N-terminal
FLAG tag		 C-terminal
FLAG-HA tag		 Names/Synonyms
DNA-dependent RNA polymerase subunits Rpb1 GRMZM2G044306 NRPB1
GRMZM2G007681 2% NRPD1/ RMR6
GRMZM2G153797 16% 9% NRPE1
Rpb2 GRMZM2G084891 NRPB2a
GRMZM2G113928 NRPB2b
GRMZM2G054225 14% 18% NRP(D/E)2a /MOP2/RMR7
GRMZM2G427031 * * NRP(D/E)2b
GRMZM2G133512 * * NRPE2c
Rpb3 GRMZM2G402295 47% 54% NRP(B/D/E)3
Rpb4 GRMZM2G119393 NRPB4
GRMZM2G453424 30% 8% NRP(D/E)4
Rpb5 GRMZM2G476009 NRPB5a
GRMZM2G099183 NRPB5b
GRMZM2G469969 36% 23% NRP(D/)E5
Rpb6 GRMZM2G013600 * NRP(B/D/E)6a
GRMZM2G086904 * NRP(B/D/E)6b
Rpb7 GRMZM2G179346 NRPB7
GRMZM2G040702 7% NRP(D/E)7
Rpb8 GRMZM2G034326 52% 26% NRP(B/D/E)8
GRMZM2G347789 NRPB8-like
Rpb9 GRMZM2G046061 NRPB9a
GRMZM2G023028 NRPB9b
GRMZM5G898768 12% NRP(D/E)9
Rpb10 NP_001152395 * 42% NRP(B/D/E)10a
GRMZM5G803992 35% # * NRP(B/D/E)10b
GRMZM5G834335 35% # * NRP(B/D/E)10c
Rpb11 GRMZM2G130207 33% 41% NRP(B/D/E)11
GRMZM2G043461 * * NRPB11-like
Rpb12 GRMZM2G146331
GRMZM2G322661
GRMZM2G540834
Polymerase-associated proteins RdRP GRMZM2G042443 3% M0P1/RDR101
GRMZM2G145201 RDR102
Swi2/Snf2 GRMZM2G154946 RMR1
GRMZM2G178435 CHR167/RMR1-like
GRMZM5G574858 CHR127/Zm_DRD1a
GRMZM2G393742 CHR156/Zm_DRD1b
- GRMZM2G309152 Zm_DMS3
- GRMZM2G111204 10% 5% Zm_SHH2a
GRMZM2G126170 15% 5% Zm_SHH2b
Iwr1 GRMZM2G098603 IWR1/Zm_DMS4/Zm_RDM4
Ago1 GRMZM2G589579 AG0105
GRMZM2G089743 AGO119
GRMZM2G347402 AGO121
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*

= One or more identified peptides also matched this protein, but are not unique to this protein

Subunits indicative of Pol II/IV/V divergence

Multiple subunits of maize Pols II, IV and V are encoded by the same genes, namely the third, sixth, eighth, tenth, and eleventh subunits (Table 1; Fig. 2C; see also Fig. S1c, f, h, i, j, k). The 12th subunit is also expected to be common to all three polymerases (as well as to Pols I and III), but was undetected, as discussed previously. The third and eleventh subunits of Pols II, IV and V interact to form a sub-complex (Ulmasov et al., 1996) and are homologs of the two alpha subunits of bacterial RNA polymerase, which mediate activator-dependent transcription initiation (Ebright and Busby, 1995). Subunits six, eight, ten and twelve are common to all eukaryotic RNA polymerases examined thus far and are thought to be important for RNA polymerase assembly (Werner and Grohmann, 2011).

Six subunits define the evolutionary split between Pol II and Pols IV/V in maize: subunits one, two, four, five, seven and nine (See Table 1; Fig. 2D), revealing similarities and important differences compared to Arabidopsis. As in Arabidopsis, the maize Pol IV and Pol V fourth subunits are encoded by the same gene, NRP(D/E)4, which is distinct from the Pol II fourth subunit gene, NRPB4 (Table 1; see also Fig. S1d). In Arabidopsis, the fifth subunits of Pols I, II, III and IV are encoded by the same gene, NRP(A/B/C/D)5, but the corresponding subunit of Pol V, NRPE5, is encoded by a distinct gene (Lahmy et al., 2009; Ream et al., 2009). Interestingly, maize has a clear ortholog of Arabidopsis NRPE5 (GRMZM2G469969) which surprising encodes the fifth subunit of both Pols IV and V (Table 1, Fig. 3A; Tables S3, S4; Fig. S1e). We thus designate this gene NRP(D/E)5. The seventh subunits of Pols IV and V are encoded by different genes in Arabidopsis, yet the same gene encodes the seventh subunits of Pols IV and V in maize (Table 1; Fig. 3B; Tables S3, S4; Fig. S1g). Whereas the same two ninth subunit variants are used alternatively by Pols II, IV and V in Arabidopsis, maize Pols IV and V make use of a unique ninth subunit, NRP(D/E)9 that is distinct from two NRPB9 variants used by Pol II (Table 1; Fig. S1i).

Figure 3.

Figure 3

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Phylogenetic analyses of proteins homologous to yeast Rpb5, yeast Rpb7 or Arabidopsis SHH1. (A) Neighbor-joining tree, with bootstrap values, for Rpb5 homologs; maize NRP(D/E)5, NRPB5a and NRPB5b proteins are highlighted in red. (B) Neighbor-joining tree for Rpb7 homologs, with NRP(D/E)7, NRPB7 proteins highlighted in red. (C) Neighbor-joining tree for SHH1 homologs in Arabidopsis, rice (O. sativa) and maize. See also Figures S1e, g, q and Table S1.

Pol IV/V-associated proteins include chromatin binding, chromatin remodeling, paramutation, and Argonaute proteins

A number of proteins implicated in paramutation or RdDM were detected in association with Pols IV and/or V (Table 1). These include two proteins similar to Arabidopsis SHH2 (Law et al., 2011b) associated with both Pol IV and Pol V (Table 1, Fig. 3C; Fig. S1q). The related Arabidopsis protein, SHH1 (a.k.a. DTF1) helps target Pol IV to heterochromatin by binding to histone H3 dimethylated on lysine 9 and unmethylated on lysine 4 (Law et al., 2013; Zhang et al., 2013). Association of maize SHH2 with Pol IV and Pol V suggests similar modes of chromatin recruitment for both enzymes.

A known paramutation protein detected in association with Pol IV is MEDIATOR OF PARAMUTATION1 (MOP1), the maize ortholog of Arabidopsis RDR2 (Alleman et al., 2006). Pol IV-MOP1 association was confirmed in reciprocal co-immunoprecipitation experiments in which epitope-tagged versions of the proteins were isolated from cell extracts using antibodies specific for the epitope tags and then detected on immunoblots using antibodies recognizing native NRPD1 or MOP1 (Fig. 4A-B).

Figure 4.

Figure 4

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Proteins associated with Pols IV and/or Pol V in maize. (A) MOP1 associates with NRPD1 (Pol IV). Immunoprecipitated Pols II, IV or V, and controls, were subjected to immunoblot analysis using anti-MOP1 antibody. (B) NRPD1 detection in association with MOP1. FLAG-MOP1 was immunoprecipitated and subjected to immunoblot analysis using anti-FLAG (to verify IP) or anti-NRPD1 antibodies. (C) Neighbor-joining tree, with bootstrap values, generated from MUSCLE alignment of Arabidopsis, rice and maize proteins most similar to maize RMR1, CHR167, CHR127 and CHR166 (in red). DDM1 serves as an outgroup. (D) Neighbor-joining tree, with bootstrap values, generated from MUSCLE alignment of full-length Arabidopsis, rice and maize Argonaute proteins most similar to Arabidopsis AGO4. Fission yeast (S. pombe) Ago1 serves as an outgroup. See also Figures S1o, p and Table S1.

Pol IV-MOP1 association was also confirmed by LC-MS/MS analyses of affinity-purified FLAG-MOP1 complexes (Table 3; see also Table S7). MOP1-associated proteins include the Pol IV largest subunit, NRPD1 and the Pol IV-associated protein, RMR1 (see later section), but not the Pol V largest subunit, NRPE1, or Pol V-associated proteins. Pol IV associated with MOP1 included the NRP(D/E)10b or NRP(D/E)10c variant forms of the tenth subunit, whose alternative use was unclear upon analysis of NRPD1-associated proteins (compare Tables 1 and 3). Moreover, MOP1 appears to preferentially associate with the Pol IV subtype containing NRP(D/E)2a; no peptides for NRP(D/E)2b were detected.

Table 3.

Proteins co-purifying with FLAG-tagged MOP1. Annotation is the same as for Table 1. For additional details, see Table S7.

Yeast
Protein		 Maize homologs Coverage by
peptides unique to
indicated protein		 Protein Names & Synonyms
DNA-dependent RNA polymerase subunits Rpbl GRMZM2G044306 NRPB1
GRMZM2G007681 18% NRPD1/ RMR6
GRMZM2G153797 NRPE1
Rpb2 GRMZM2G084891 NRPB2a
GRMZM2G113928 NRPB2b
GRMZM2G054225 14% NRP(D/E)2a/MOP2/RMR7
GRMZM2G427031 * NRP(D/E)2b
GRMZM2G133512 * NRPE2c
Rpb3 GRMZM2G402295 43% NRP(B/D/E)3
Rpb4 GRMZM2G119393 NRPB4
GRMZM2G453424 44% NRP(D/E)4
Rpb5 GRMZM2G476009 NRPB5a
GRMZM2G099183 NRPB5b
GRMZM2G469969 32% NRP(D/E)5
Rpb6 GRMZM2G013600 * NRP(B/D/E)6a
GRMZM2G086904 * NRP(B/D/E)6b
Rpb7 GRMZM2G179346 NRPB7
GRMZM2G040702 16% NRP(D/E)7
Rpb8 GRMZM2G034326 47% NRP(B/D/E)8
GRMZM2G347789 NRPB8-like
Rpb9 GRMZM2G046061 NRPB9a
GRMZM2G023028 NRPB9b
GRMZM5G898768 12% NRP(D/E)9
Rpb10 NP_001152395 * NRP(B/D/E)10a
GRMZM5G803992 35% # NRP(D/E)10b
GRMZM5G834335 35% # NRP(D/E)10c
Rpb11 GRMZM2G130207 33% NRP(B/D/E)11
GRMZM2G043461 * NRPB11-like
Rpb12 GRMZM2G146331 NRPB12a
GRMZM2G322661 NRPB12b
GRMZM2G540834 NRPB12c
Polymerase-associated proteins RdRP GRMZM2G042443 55% M0P1/RDR101
GRMZM2G145201 * RDR102
Swi2/Snf2 GRMZM2G154946 RMR1
GRMZM2G178435 CHR167/RMR1-like
GRMZM5G574858 CHR127/Zm_DRD1a
GRMZM2G393742 CHR156/Zm_DRD1b
- GRMZM2G309152 Zm_DMS3
- GRMZM2G111204 3% Zm_SHH2a
GRMZM2G126170 13% Zm_SHH2b
Iwr1 GRMZM2G098603 4% IWR1/Zm_DMS4/Zm_RDM4
Ago1 GRMZM2G589579 AGO105
GRMZM2G089743 AGO119
GRMZM2G347402 AGO121
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*

= One or more identified peptides also matched this protein, but are not unique to this protein

REQUIRED TO MAINTAIN REPRESSION1 (RMR1) is a known paramutation protein (Hale et al, 2007) that we found in association with Pol IV (NRPD1), along with its paralog, CHR167 (Table 1, Fig. 4C). These are SWI2/SNF2 family proteins, and thus putative ATP-dependent DNA translocases. Both proteins are related to Arabidopsis CLSY proteins (Fig. 4C; Fig. S1p), which associate with Pol IV and are involved in Pol IV and RDR2-dependent siRNA biogenesis (Law et al., 2011a; Smith et al., 2007), and to DRD1 (Fig. 4C), which facilitates Pol V transcription in Arabidopsis. However, a more closely related DRD1-like protein, CHR127 was detected in association with maize Pol V (Table 1). A maize ortholog of Arabidopsis DMS3 (Kanno et al., 2008), a protein that associates with DRD1 and is required for Pol V transcription (Law et al., 2010; Wierzbicki et al., 2008; Wierzbicki et al., 2009a), was also identified in association with Pol V (Table 1; Fig. S1m).

AGO121, a maize protein similar to Arabidopsis AGO6, associates with maize Pol V (Table 1, Fig. 4D; Fig. S1o). AGO4 and AGO6 are the principal 24 nt siRNA-binding Argonaute proteins involved in RNA-directed DNA methylation in vegetative tissues of Arabidopsis, suggesting that AGO121 plays an analogous role in 24 nt siRNA-mediated silencing in maize.

The maize ortholog of the yeast Iwr1 protein was detected in association with Pols II, IV and V (Table 1; Fig. S1n). This is consistent with the involvement of yeast Iwr1 in the nuclear import of RNA polymerases assembled in the cytoplasm, and with the previous identification of IWR1 as a protein involved in RdDM that can associate with Arabidopsis Pol V (Czeko et al., 2011; He et al., 2009; Kanno et al., 2010; Law et al., 2011b). The presence of IWR1 in affinity-purified MOP1 complexes (Table S7) suggests that MOP1 and Pol IV associate prior to IWR1 dissociation from Pol IV.

Discussion

Functionally distinct polymerase subtypes use alternative catalytic subunits

Catalytic centers of multisubunit RNA polymerases are formed by the largest and second-largest subunits (Cramer et al., 2008) such that mutants defective for either subunit are expected to have the same phenotypes. The fact that maize nrpd1 (rmr6) and nrp(d/e)2a (mop2/rmr7) loss-of-function mutants are both impaired for paramutation fits this expectation. However, developmental abnormalities observed in nrpd1 mutants are not typical of nrp(d/e)2a mutants (Sidorenko et al., 2009; Stonaker et al., 2009), an observation made even more surprising by our finding that NRP(D/E)2a is the preferred second-subunit for both Pol IV and Pol V (based on peptide abundance), indicating that nrp(d/e)2a mutants should be impaired for both activities. The fact that Pol IV can also assemble using NRP(D/E)2b, and Pol V can make alternative use of NRP(D/E)2b or NRP(D/E)2c provides a solution to this apparent paradox, allowing the deduction that Pol IV and/or Pol V subtypes assembled using alternative second-subunits are functionally distinct. The evidence suggests that Pol IVNRP(D/E)2a and Pol IVNRP(D/E)2b subtypes are redundant with respect to functions important for development, such that a nrp(d/e)2a nrp(d/e)2b double mutant would be necessary to recapitulate nrpd1 developmental phenotypes. By contrast, the Pol IVNRP(D/E)2b subtype must not be redundant with Pol IVNRP(D/E)2a with respect to paramutation, given that nrp(d/e)2a single mutants are impaired for paramutation, like nrpd1 mutants.

One plausible explanation for polymerase subtype non-redundancy might be different interactions with partner proteins. In this regard, it is noteworthy that Pol IV associated with MOP1 contains the NRP(D/E)2a subunit, but no NRP(D/E)2b peptides were detected. Different Pol IV and Pol V subtypes might also target different subsets of loci, or may have different enzymatic properties. Alternatively, the different polymerase subtypes may be enriched in different cell types, tissues or organs, consistent with observation that NRPE2c is highly expressed only in tassels and pollen (Sidorenko et al, 2009), perhaps explaining why the protein was detected only in trace amounts in Pol V isolated from callus cells in our study. Variation in non-catalytic subunits, such as the sixth and tenth subunits, may contribute additional functional diversity to maize Pols IV and V subtypes, as well as to Pol II subtypes.

New insights into the Pol II - Pol IV/V evolutionary split and Pol IV/V diversification

The fundamental difference between Pols IV and V in maize is their use of different largest subunits. This is surprising in light of our prior results in Arabidopsis, in which we showed that Pols IV and V differ in three subunits: their largest, fifth-largest and seventh-largest subunits (Ream et al., 2009). Arabidopsis Pol V makes use of a fifth subunit encoded by a gene (NRPE5) that is distinct from the single-copy gene that encodes the corresponding subunits of Pols I, II, III and IV. Importantly, the maize ortholog of Arabidopsis NRPE5 (Fig. 3A) encodes the 5th subunit of both Pols IV and V (see Table 1). This argues against the Arabidopsis-centric hypothesis that emergence of a Pol V-specific fifth subunit was a critical event in the functional diversification of Pols IV and V.

Maize Pols IV and V also make use of seventh subunits encoded by the same gene, unlike Arabidopsis. This is less surprising given that phylogenetic analyses indicate that the gene duplication giving rise to thet NRPD7 and NRPE7 genes in Arabidopsis is not deeply rooted, even among dicots (Fig. 3B; see also (Tucker et al., 2011)). The seventh subunit, in partnership with the fourth subunit, is expected to form a stalk-like sub-complex located adjacent to the RNA exit channel, as in archaeal RNA polymerases or eukaryotic Pols I, II and III (Cramer et al., 2008; Cramer et al., 2001; Werner and Grohmann, 2011). In the context of Pol II, the subunit 4/7 subcomplex is important for multiple aspects of RNA processing and, in yeast, can even dissociate from the core polymerase and traffic with RNA within the cell (Harel-Sharvit et al., 2010; Mitsuzawa et al., 2003; Sampath and Sadhale, 2005). Importantly, the fourth and seventh subunits of maize Pols IV and V are the same, yet distinct from the corresponding subunits of Pols I, II and III. This suggests that the 4/7 subcomplex is likely important for functions common to both Pols IV and V, yet different from those of other polymerases.

Another maize -Arabidopsis difference concerns the ninth subunits of Pols II, IV and V. In the context of Pol II, the ninth subunit is important for RNA cleavage activity, stimulated by TFIIS, for the correction of misincorporated nucleotides, backtracking to overcome polymerase stalling, or transcription termination (Hemming et al., 2000; Koyama et al., 2007; Nesser et al., 2006; Walmacq et al., 2009). Arabidopsis expresses two alternative ninth subunits that are 92% identical and are detected in similar abundance in Pols II, IV or V (Law et al., 2011a; Ream et al., 2009). Genetic experiments in Arabidopsis showed that these alternative subunits are redundant for Pol II functions required for viability, but not for RNA-directed DNA methylation (Tan et al., 2012). Instead, only the NRP(B/D/E)9b variant is required for RdDM; the 9a variant is dispensable (Tan et al., 2012). In light of these studies, it is intriguing that maize Pols IV and V make use of a ninth subunit distinct from the two alternative ninth subunits used by Pol II (see Table 1), suggesting important Pol IV/V-specific ninth-subunit functions that await definition.

Maize Pols IV and V differ only in their largest subunits and the most obvious difference between their NRPD1 and NRPE1 proteins is the presence of a longer C-terminal domain in NRPE1. Pol V interaction with AGO proteins is attributable to the CTD of NRPE1 in Arabidopsis (El-Shami et al., 2007), suggesting that maize Pol V interaction with AGO121 may similarly involve the WG and GW-rich region of the CTD. Pol V-specific interactions with CHR127/Zm-DRD1 and ZmDMS3, and Pol IV-specific interactions with MOP1 and RMR1 must also be attributable, directly or indirectly, to the different largest subunits of Pols IV and V. By contrast, interactions with SHH2a and SHH2b, by both Pols IV and V, are presumably mediated by subunits common to Pols IV and V. Guided by these insights, identification of Pol IV or Pol V sequences that interact with partner proteins to coordinate RNA-directed DNA methylation and/or paramutation is a priority for future studies.

Materials & Methods

Identification and Cloning of RNA Polymerase Subunits Gene models for maize NRPD1, NRPE1 and NRP(D/E)2a were deduced based on sequence similarity to Arabidopsis and rice orthologs. Sequences corresponding to GRMZM2G007681, GRMZM2G153797 and GRMZM2G054225 were targeted for cloning and overexpression. Full-length cDNAs were obtained by RT-PCR amplification of isolated total RNA.

C-terminal FLAG-HA tagged NRPD1, NRPE1 and NRP(D/E)2a were generated by cDNA amplification using KOD DNA polymerase (Novagen) and cloning into pCR-Blunt IITOPO (Invitrogen). Plant expression vectors pMON124573 (NRPD1-C tag), pMON124574 (NRPDE2a-C tag), pMON124576 (NRPE1-C tag)] were used for Agrobacterium-mediated plant transformation (Armstrong, 2003). Transgenic (R0) plants were selected on medium containing glyphosate and verified by PCR and Southern blot hybridization.

To generate N-terminal FLAG-tagged NRP(D/E)2a (NCBI GQ453405), cDNA from 5 day old seedlings (Brzeska et al., 2010) was amplified with primers vc2817F and vc2817R. MOP1 (NCBI JQ248126) was amplified using primers KM510F and KM511R and RNA from B73 embryos dissected from mid-maturation kernels using the Agilent Plant RNA Isolation Mini Kit (Santa Clara, CA). Primers carried attB sites for GATEWAY LR cloning (Invitrogen) into the plant expression vector, pEarleyGate402 (http://sites.bio.indiana.edu/~pikaardlab/Vectors%20homepage.html). Resulting constructs were transformed into HiII embryos using A. tumefaciens at the Iowa State Plant Transformation Facility (Ames, IA).

Transgenic plant production, growth and genotyping

FLAG::MOP1 plants, grown in a greenhouse with 16 hours of light and 8 hours of dark were screened for transgene expression by RT-PCR using FLAG tag primer KM666F and MOP1 coding region primer, KM668R. Tissues flash frozen in liquid nitrogen, and stored at −80°C. FLAG::NRP(D/E)2a callus was screened for transgene expression by RT-PCR using a FLAG primer (vc4856F) and NRP(D/E)2a-specific primer, vc4856R3. The transgene was detected by PCR using either the Ubiquitin1 promoter primer vc6139F and NRP(D/E)2a coding region primer vc6139R, or the NRP(D/E)2a coding region primer vc2987F and OCS3′R1 (NCBI MCG524A) primer vc2556AR.

Type II corn callus AT824 was initiated (Gordon-Kamm et al., 1990) and cryopreserved and reinitiated as described in (Aves K, 1992). AT824 callus suspension was maintained on N6 media at 28 °C as described (Aves K, 1992; Gordon-Kamm et al., 1990). NRPD1-C, NRP(D/E)2a-C and NRPE1-C transgenic corn cell lines were created by microprojectile bombardment (Biolistic particle delivery system PDS-1000/He) at 1350 psi. Post bombardment, cells were cultured for 7 days, then spread on selection plates containing 25 mg/L paramomycin. Resistant colonies were identified 5 weeks post-selection, confirmed by PCR and immunoblot analyses and maintained as individual cell lines on medium containing 25 mg/L paramomycin.

Sample preparation for mass spectrometry

Frozen cells (70 g) from callus suspension cultures (non-transgenic, NRPD1-FLAG-HA, NRPE1-FLAG-HA or NRP(D/E)2a-FLAG-HA genotypes), or from 100g combined husk and ear tissues harvested 3-5 days post-silk emergence (non-transgenic, FLAG-NRP(D/E)2a and FLAG-MOP1) was homogenized at 4 °C in a blender with extraction buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 5 mM DTT, 1mM PMSF and 1:300 plant protease inhibitor cocktail (Sigma)], filtered through two layers of Miracloth (Calbiochem), and centrifuged twice at 10,000 × g, 20 min, 4°C. Supernatants were incubated with anti-FLAG-M2 resin (Sigma) for 3 hr in 15 mL conical tubes using 25 μL of resin per 14 mL of extract. Resin was pelleted at 200 × g for 2 min, was washed five times with 10 mL extraction buffer, and resuspended in an equal volume Gentle Ag/Ab Elution Buffer (Pierce). After 2 min, resin was pelleted and proteins in the supernatant were concentrated in an Ultracel-10k centrifugal filter (Millipore), with three serial buffer exchanges of 25 mM ammonium bicarbonate, to a final volume of ~100 μL.

NRPB1 was purified from 34 g of non-transgenic husk and ear tissues as above. The cell-free extract was pre-cleared with Protein G agarose (Pierce) for 30 min at 4°C before the supernatant was split for control [mouse IgG serum (Calbiochem)] or anti-NRPB1 [clone 8WG16 (Millipore)] immune-affinity purifications. Samples were incubated with antibodies 2 hr, 50 μL Protein G agarose (Pierce) was added and incubated an additional 2 hrs, and resin was washed, eluted and concentrated, as above.

Mass spectrometry of affinity purified protein complexes

Samples (100 μl) were mixed with 400 μl of 0°C methanol, 100 μl of 0°C chloroform and 300 μl of 0°C water, incubated 2 min on ice and subjected to centrifugation at 12,000 × g, 2 min, 0°C. The supernatant was removed, mixed by vortexing with 300 μl methanol and subjected to centrifugation at 12,000 × g, 5 min. The supernatant was removed and the protein pellet dried briefly at room temperature prior to addition of 50 μl of 50 mM ammonium bicarbonate, pH 8.5. Protein concentration was estimated using a Coomassie dye-binding assay (Pierce, Rockford, IL). 2,2,2-Trifluoroethanol (TFE) (Sigma, St. Louis, MO) was added to a final concentration of 50% (v/v). The sample was sonicated in an ice-water bath for 1 minute and incubated at 60°C for 2 hr with shaking at 300 rpm. Samples were reduced with 2 mM dithiothreitol (DTT) (Sigma, St. Louis, MO) for 1hr, 37°C with shaking at 300 rpm, then diluted 5-fold with 50 mM ammonium bicarbonate. 1 mM CaCl2 and sequencing-grade modified porcine trypsin (Promega, Madison, WI) were added at a 1:50 (w/w) trypsin-to-protein ratio and incubated 3 hr at 37°C. Samples were concentrated in a Speed Vac to a volume of ~30 μl, centrifuged at 12,000 × g, and supernatants subjected to LC-MS analysis using LTQ-Orbitrap and LTQ-Orbitrap Velos mass spectrometry (Thermo Scientific, Waltham, MA) as previously described (Ream et al., 2009). Acquired tandem mass spectra were searched against the annotated Z. mays genome (www.maizesequence.org, release 5b.60) supplemented with additional RNA polymerase subunit and chromatin modifier sequences identified in The Chromatin Database (http://www.chromdb.org/), Qian et al. (Qian et al., 2011), and NCBI GenBank by tblastn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using SEQUEST (Eng et al., 1994), allowing for partial tryptic cleavage and methionine oxidation. Non-modified peptides or those only containing an oxidized methionine were filtered to a 2% false discovery rate (FDR), based on MSGF scores (http://proteomics.ucsd.edu/Software/MSGeneratingFunction.html).

Antibody production

Antibodies against unique NRPD1 and NRPE1 sequences were raised in rabbits by immunization with multiple peptide antigens (KLH-linked). NRPD1 antibiodies were raised against peptides ELHREPPEAILNAIKFDC and KVRNFEKNHLDTRRQSTE. NRPE1 sera were raised against peptides CDGTGLLGKAPQADWGPRFDAD and SQRNNPGRPPRRPDER.

Anti-MOP1 antibodies were raised against a peptide comprising amino acids 1028-1127 and affinity purified from crude sera using bacterially expressed 6xHis-MOP1(aa1028-1127) immobilized on PVDF membrane, as previously described (Haag et al, 2012).

Immunoprecipitation and immunoblotting

Frozen cell pellets of maize callus (1-2 g), or husk and ear tissues (4 g), were ground in liquid nitrogen and suspended in 14 mL extraction buffer (see above), filtered through Miracloth, and subjected to centrifugation at 16,000 × g for 15 min, 4 °C. Supernatants were incubated 2-3 hrs at 4 °C with 50 μL anti-FLAG-M2 slurry or pre-cleared with mouse IgG serum for 30 min at 4 °C prior to incubation with mouse IgG serum or anti-NRPB1 (clone 8WG16) for 2 hrs followed by incubation with 50 μL Protein G agarose slurry for 1-2 hrs, 4 °C. Resin was washed three times in extraction buffer and eluted in two bed volumes of 2x SDS sample buffer by boiling. Proteins were subjected to SDS-PAGE on 7.5% Tris-glycine gels and transferred to nitrocellulose membranes. Blots were incubated with antibodies in TBST + 5% (w/v) nonfat dried milk. Antibody dilutions were: 1:3,000 anti-FLAG-HRP (Sigma), 1:3000 anti-HA, clone 9E10 (Sigma), 1:500 anti-NRPD1, 1:500 anti-NRPE1, 1:250 anti-MOP1, 1:2,000 anti-NRPB1, clone 8WG16 (Millipore), and 1:5,000 to 1:10,000 donkey anti-mouse-HRP (Santa Cruz Biotech). ECL and ECL Plus reagents (GE Healthcare) were used for chemiluminescent detection on film.

Phylogenetic analyses

Aligned proteins were identified by tblastn searches using A. thaliana and S. cerevisiae protein sequences as queries against NCBI (http://blast.ncbi.nlm.nih.gov/), maizesequence (www.maizesequence.org), The Chromatin Database (http://www.chromdb.org/), Joint Genome Institute (http://www.phytozome.net/), and Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml) databases. Sequences were aligned using ClustalW2 or MUSCLE and conserved sequences highlighted using BOXSHADE v3.31. Phylogenetic analysis was by the neighbor-joining method, with 1000 bootstrap replications, using Geneious software.

Supplementary Material

1
2
3

Highlights.

  • Alternative second-subunits yield two Pol IV and three Pol V subtypes in maize.

  • Pol IV/V subtypes are non-redundant for development and paramutation.

  • Only the largest subunit discriminates Pol IV from Pol V in maize.

  • Pols IV and V associate with common, as well as different, protein partners.

Acknowledgments

J.R.H. and C.S.P. designed the study and wrote the paper. Monsanto generated transgenic callus lines and NRPD1 and NRPE1 antibodies. FLAG-NRP(D/E)2a and FLAG-MOP1 plants were generated by L.S. and A.I., respectively. J.B. generated the MOP1 antibody. LC-MS/MS was performed by C.D.N., A.D.N., and L.P.-T. Maize polymerase subunit genes were identified and annotated by J.R.H., L.S., and B.B.-T. All other experiments and analyses were performed by J.R.H. We thank John Lemon (IU) and Benjamin Echalier (UA) and their staffs for helping with plant care; Hong Liu and Jiyan Ma for technical assistance; and Jason Osborne (Monsanto Immunoassay) and Todd Ziegler (Monsanto Custom Expression) for reagents. Portions of this research were supported by the NIH National Center for Research Resources (RR18522) and the W.R. Wiley Environmental Molecular Science Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy, located at PNNL and operated by Battelle Memorial Institute under DOE contract DE-AC05-76RL01830. Pikaard laboratory studies were supported by National Institutes of Health grant GM077590 and C.S.P.’s support as an Investigator of the Howard Hughes Medical Institute and the Gordon & Betty Moore Foundation. Opinions are those of the authors and do not necessarily reflect the views of our sponsors.

Footnotes

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The authors declare that no competing interests exist.

References

  1. Alleman M, Sidorenko L, McGinnis K, Seshadri V, Dorweiler JE, White J, Sikkink K, Chandler VL. An RNA-dependent RNA polymerase is required for paramutation in maize. Nature. 2006;442:295–298. doi: 10.1038/nature04884. [DOI] [PubMed] [Google Scholar]
  2. Armstrong CR, J. Agrobacterium-mediated plant transformation method. 2003.
  3. Arteaga-Vazquez MA, Chandler VL. Paramutation in maize: RNA mediated trans-generational gene silencing. Curr Opin Genet Dev. 2010;20:156–163. doi: 10.1016/j.gde.2010.01.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aves K GD, Willets N, Zachweija S, Mann M, Spencer T, Flick C, Gordon-Kamm W. Transformation of an elite maize inbred line through microprojectile bombardment & regenerable, embryogenic callus. In vitro. 1992;28:124A. [Google Scholar]
  5. Bellaoui M, Gruissem W. Altered expression of the Arabidopsis ortholog of DCL affects normal plant development. Planta. 2004;219:819–826. doi: 10.1007/s00425-004-1295-5. [DOI] [PubMed] [Google Scholar]
  6. Brzeska K, Brzeski J, Smith J, Chandler VL. Transgenic expression of CBBP, a CXC domain protein, establishes paramutation in maize. Proc Natl Acad Sci U S A. 2010;107:5516–5521. doi: 10.1073/pnas.1001576107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cramer P, Armache KJ, Baumli S, Benkert S, Brueckner F, Buchen C, Damsma GE, Dengl S, Geiger SR, Jasiak AJ, et al. Structure of eukaryotic RNA polymerases. Annual review of biophysics. 2008;37:337–352. doi: 10.1146/annurev.biophys.37.032807.130008. [DOI] [PubMed] [Google Scholar]
  8. Cramer P, Bushnell DA, Kornberg RD. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science. 2001;292:1863–1876. doi: 10.1126/science.1059493. [DOI] [PubMed] [Google Scholar]
  9. Czeko E, Seizl M, Augsberger C, Mielke T, Cramer P. Iwr1 directs RNA polymerase II nuclear import. Mol Cell. 2011;42:261–266. doi: 10.1016/j.molcel.2011.02.033. [DOI] [PubMed] [Google Scholar]
  10. Ebright RH, Busby S. The Escherichia coli RNA polymerase alpha subunit: structure and function. Curr Opin Genet Dev. 1995;5:197–203. doi: 10.1016/0959-437x(95)80008-5. [DOI] [PubMed] [Google Scholar]
  11. Egloff S, Murphy S. Cracking the RNA polymerase II CTD code. Trends Genet. 2008;24:280–288. doi: 10.1016/j.tig.2008.03.008. [DOI] [PubMed] [Google Scholar]
  12. El-Shami M, Pontier D, Lahmy S, Braun L, Picart C, Vega D, Hakimi MA, Jacobsen SE, Cooke R, Lagrange T. Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev. 2007;21:2539–2544. doi: 10.1101/gad.451207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eng JK, McCormack AL, Yates JR., III An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry. 1994;5:976–989. doi: 10.1016/1044-0305(94)80016-2. [DOI] [PubMed] [Google Scholar]
  14. Erhard KF, Jr., Hollick JB. Paramutation: a process for acquiring trans generational regulatory states. Curr Opin Plant Biol. 2011;14:210–216. doi: 10.1016/j.pbi.2011.02.005. [DOI] [PubMed] [Google Scholar]
  15. Erhard KF, Jr., Stonaker JL, Parkinson SE, Lim JP, Hale CJ, Hollick JB. RNA polymerase IV functions in paramutation in Zea mays. Science. 2009;323:1201–1205. doi: 10.1126/science.1164508. [DOI] [PubMed] [Google Scholar]
  16. Gao Z, Liu HL, Daxinger L, Pontes O, He X, Qian W, Lin H, Xie M, Lorkovic ZJ, Zhang S, et al. An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature. 2010;465:106–109. doi: 10.1038/nature09025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O’Brien JV, Chambers SA, Adams WR, Jr., Willetts NG, et al. Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants. Plant Cell. 1990;2:603–618. doi: 10.1105/tpc.2.7.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haag JR, Pikaard CS. Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nature Reviews Molecular Cell Biology. 2011;12:483–492. doi: 10.1038/nrm3152. [DOI] [PubMed] [Google Scholar]
  19. Haag JR, Ream TS, Marasco M, Nicora CD, Norbeck AD, Pasa-Tolic L, Pikaard CS. In Vitro Transcription Activities of Pol IV, Pol V, and RDR2 Reveal Coupling of Pol IV and RDR2 for dsRNA Synthesis in Plant RNA Silencing. Mol Cell. 2012;48:811–818. doi: 10.1016/j.molcel.2012.09.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hale CJ, Erhard KF, Jr., Lisch D, Hollick JB. Production and processing of siRNA precursor transcripts from the highly repetitive maize genome. PLoS Genet. 2009;5:e1000598. doi: 10.1371/journal.pgen.1000598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harel-Sharvit L, Eldad N, Haimovich G, Barkai O, Duek L, Choder M. RNA Polymerase II Subunits Link Transcription and mRNA Decay to Translation. Cell. 2010;143:552–563. doi: 10.1016/j.cell.2010.10.033. [DOI] [PubMed] [Google Scholar]
  22. Havecker ER, Wallbridge LM, Hardcastle TJ, Bush MS, Kelly KA, Dunn RM, Schwach F, Doonan JH, Baulcombe DC. The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell. 2010;22:321–334. doi: 10.1105/tpc.109.072199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. He XJ, Hsu YF, Zhu S, Liu HL, Pontes O, Zhu J, Cui X, Wang CS, Zhu JK. A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development. Genes Dev. 2009;23:2717–2722. doi: 10.1101/gad.1851809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hemming SA, Jansma DB, Macgregor PF, Goryachev A, Friesen JD, Edwards AM. RNA polymerase II subunit Rpb9 regulates transcription elongation in vivo. The Journal of biological chemistry. 2000;275:35506–35511. doi: 10.1074/jbc.M004721200. [DOI] [PubMed] [Google Scholar]
  25. Herr AJ, Jensen MB, Dalmay T, Baulcombe DC. RNA polymerase IV directs silencing of endogenous DNA. Science. 2005;308:118–120. doi: 10.1126/science.1106910. [DOI] [PubMed] [Google Scholar]
  26. Hsin JP, Manley JL. The RNA polymerase II CTD coordinates transcription and RNA processing. Genes Dev. 2012;26:2119–2137. doi: 10.1101/gad.200303.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Huang L, Jones AM, Searle I, Patel K, Vogler H, Hubner NC, Baulcombe DC. An atypical RNA polymerase involved in RNA silencing shares small subunits with RNA polymerase II. Nat Struct Mol Biol. 2009;16:91–93. doi: 10.1038/nsmb.1539. [DOI] [PubMed] [Google Scholar]
  28. Kanno T, Aufsatz W, Jaligot E, Mette MF, Matzke M, Matzke AJ. A SNF2-like protein facilitates dynamic control of DNA methylation. EMBO Rep. 2005a;6:649–655. doi: 10.1038/sj.embor.7400446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kanno T, Bucher E, Daxinger L, Huettel B, Bohmdorfer G, Gregor W, Kreil DP, Matzke M, Matzke AJ. A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation. Nat Genet. 2008;40:670–675. doi: 10.1038/ng.119. [DOI] [PubMed] [Google Scholar]
  30. Kanno T, Bucher E, Daxinger L, Huettel B, Kreil DP, Breinig F, Lind M, Schmitt MJ, Simon SA, Gurazada SG, et al. RNA-directed DNA methylation and plant development require an IWR1-type transcription factor. EMBO Rep. 2010;11:65–71. doi: 10.1038/embor.2009.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, Daxinger L, Kreil DP, Matzke M, Matzke AJ. Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet. 2005b;37:761–765. doi: 10.1038/ng1580. [DOI] [PubMed] [Google Scholar]
  32. Koyama H, Ito T, Nakanishi T, Sekimizu K. Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeast. Genes to cells : devoted to molecular & cellular mechanisms. 2007;12:547–559. doi: 10.1111/j.1365-2443.2007.01072.x. [DOI] [PubMed] [Google Scholar]
  33. Lahmy S, Pontier D, Cavel E, Vega D, El-Shami M, Kanno T, Lagrange T. PolV(PolIVb) function in RNA-directed DNA methylation requires the conserved active site and an additional plant-specific subunit. Proc Natl Acad Sci U S A. 2009;106:941–946. doi: 10.1073/pnas.0810310106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Law JA, Ausin I, Johnson LM, Vashisht AA, Zhu JK, Wohlschlegel JA, Jacobsen SE. A protein complex required for polymerase V transcripts and RNA- directed DNA methylation in Arabidopsis. Curr Biol. 2010;20:951–956. doi: 10.1016/j.cub.2010.03.062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Law JA, Du J, Hale CJ, Feng S, Krajewski K, Palanca AM, Strahl BD, Patel DJ, Jacobsen SE. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature. 2013 doi: 10.1038/nature12178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet. 2010;11:204–220. doi: 10.1038/nrg2719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Law JA, Vashisht AA, Wohlschlegel JA, Jacobsen SE. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV. PLoS genetics. 2011a;7:e1002195. doi: 10.1371/journal.pgen.1002195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Law JA, Vashisht AA, Wohlschlegel JA, Jacobsen SE. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV. PLoS Genet. 2011b;7:e1002195. doi: 10.1371/journal.pgen.1002195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Luo J, Hall BD. A multistep process gave rise to RNA polymerase IV of land plants. J Mol Evol. 2007;64:101–112. doi: 10.1007/s00239-006-0093-z. [DOI] [PubMed] [Google Scholar]
  40. Mallory A, Vaucheret H. Form, function, and regulation of ARGONAUTE proteins. Plant Cell. 2010;22:3879–3889. doi: 10.1105/tpc.110.080671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJ. RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol. 2009;21:367–376. doi: 10.1016/j.ceb.2009.01.025. [DOI] [PubMed] [Google Scholar]
  42. Mitsuzawa H, Kanda E, Ishihama A. Rpb7 subunit of RNA polymerase II interacts with an RNA-binding protein involved in processing of transcripts. Nucleic Acids Res. 2003;31:4696–4701. doi: 10.1093/nar/gkg688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nesser NK, Peterson DO, Hawley DK. RNA polymerase II subunit Rpb9 is important for transcriptional fidelity in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:3268–3273. doi: 10.1073/pnas.0511330103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Onodera Y, Haag JR, Ream T, Nunes PC, Pontes O, Pikaard CS. Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell. 2005;120:613–622. doi: 10.1016/j.cell.2005.02.007. [DOI] [PubMed] [Google Scholar]
  45. Pikaard CS, Haag JR, Pontes OM, Blevins T, Cocklin R. A Transcription Fork Model for Pol IV and Pol V-dependent RNA-Directed DNA Methylation. Cold Spring Harb Symp Quant Biol. 2013 doi: 10.1101/sqb.2013.77.014803. [DOI] [PubMed] [Google Scholar]
  46. Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, Hakimi MA, Lerbs-Mache S, Colot V, Lagrange T. Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev. 2005;19:2030–2040. doi: 10.1101/gad.348405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Qian Y, Cheng Y, Cheng X, Jiang H, Zhu S, Cheng B. Identification and characterization of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in maize. Plant Cell Rep. 2011;30:1347–1363. doi: 10.1007/s00299-011-1046-6. [DOI] [PubMed] [Google Scholar]
  48. Ream T, Haag J, Pikaard CS. Plant multisubunit RNA polymerases IV and V. In: Murakami K, Trakselis M, editors. Nucleic Acid Polymerases. Springer-Verlag; Berlin Heidelberg: 2014. pp. 289–308. [Google Scholar]
  49. Ream TS, Haag JR, Wierzbicki AT, Nicora CD, Norbeck AD, Zhu JK, Hagen G, Guilfoyle TJ, Pasa-Tolic L, Pikaard CS. Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II. Mol Cell. 2009;33:192–203. doi: 10.1016/j.molcel.2008.12.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sampath V, Sadhale P. Rpb4 and Rpb7: a sub-complex integral to multi-subunit RNA polymerases performs a multitude of functions. IUBMB Life. 2005;57:93–102. doi: 10.1080/15216540500078905. [DOI] [PubMed] [Google Scholar]
  51. Sidorenko L, Dorweiler JE, Cigan AM, Arteaga-Vazquez M, Vyas M, Kermicle J, Jurcin D, Brzeski J, Cai Y, Chandler VL. A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes. PLoS Genet. 2009;5:e1000725. doi: 10.1371/journal.pgen.1000725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Smith LM, Baulcombe DC. Dissection of silencing signal movement in Arabidopsis. Plant Signal Behav. 2007;2:501–502. doi: 10.4161/psb.2.6.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Smith LM, Pontes O, Searle I, Yelina N, Yousafzai FK, Herr AJ, Pikaard CS, Baulcombe DC. An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell. 2007;19:1507–1521. doi: 10.1105/tpc.107.051540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stonaker JL, Lim JP, Erhard KF, Jr., Hollick JB. Diversity of Pol IV function is defined by mutations at the maize rmr7 locus. PLoS Genet. 2009;5:e1000706. doi: 10.1371/journal.pgen.1000706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tan EH, Blevins T, Ream T, Pikaard C. Functional consequences of subunit diversity in RNA polymerases II and V. Cell Reports. 2012 doi: 10.1016/j.celrep.2012.01.004. doi:10.1016/j.celrep.2012.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tucker SL, Reece J, Ream TS, Pikaard CS. Evolutionary History of Plant Multisubunit RNA Polymerases IV and V: Subunit Origins via Genome-Wide and Segmental Gene Duplications, Retrotransposition, and Lineage-Specific Subfunctionalization. Cold Spring Harbor symposia on quantitative biology. 2011 doi: 10.1101/sqb.2010.75.037. [DOI] [PubMed] [Google Scholar]
  57. Ulmasov T, Larkin RM, Guilfoyle TJ. Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II. J Biol Chem. 1996;271:5085–5094. doi: 10.1074/jbc.271.9.5085. [DOI] [PubMed] [Google Scholar]
  58. Walmacq C, Kireeva ML, Irvin J, Nedialkov Y, Lubkowska L, Malagon F, Strathern JN, Kashlev M. Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II. The Journal of biological chemistry. 2009;284:19601–19612. doi: 10.1074/jbc.M109.006908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Werner F, Grohmann D. Evolution of multisubunit RNA polymerases in the three domains of life. Nat Rev Microbiol. 2011;9:85–98. doi: 10.1038/nrmicro2507. [DOI] [PubMed] [Google Scholar]
  60. Wierzbicki AT. The role of long non-coding RNA in transcriptional gene silencing. Curr Opin Plant Biol. 2012;15:517–522. doi: 10.1016/j.pbi.2012.08.008. [DOI] [PubMed] [Google Scholar]
  61. Wierzbicki AT, Haag JR, Pikaard CS. Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell. 2008;135:635–648. doi: 10.1016/j.cell.2008.09.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wierzbicki AT, Ream TS, Haag JR, Pikaard CS. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat Genet. 2009a;41:630–634. doi: 10.1038/ng.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wierzbicki AT, Ream TS, Haag JR, Pikaard CS. RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nature genetics. 2009b;41:630–634. doi: 10.1038/ng.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2004;2:642–652. doi: 10.1371/journal.pbio.0020104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhang H, Ma ZY, Zeng L, Tanaka K, Zhang CJ, Ma J, Bai G, Wang P, Zhang SW, Liu ZW, et al. DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc Natl Acad Sci U S A. 2013;110:8290–8295. doi: 10.1073/pnas.1300585110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhang H, Zhu JK. RNA-directed DNA methylation. Curr Opin Plant Biol. 2011;14:142–147. doi: 10.1016/j.pbi.2011.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

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NIHMS627471-supplement-1.pdf (14MB, pdf)
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NIHMS627471-supplement-2.docx (109.8KB, docx)
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