Pol IV and RDR2 associate in vivo
We rescued an nrpd1-3
null mutant lacking the Pol IV largest subunit with a FLAG epitope-tagged NRPD1 transgene (NRPD1-FLAG), allowing Pol IV affinity purification using anti-FLAG resin. Trypsin digestion and LC-MS/MS mass spectrometry identified peptides of Pol IV’s twelve core subunits (Ream et al., 2009
) as well as ten peptides corresponding to RDR2 (), confirming a recent report (Law et al., 2011
As an independent test of Pol IV- RDR2 interaction, we rescued an rdr2-1
null mutant with a RDR2
transgene (see Figures S1A–C
) that includes the RDR2
promoter, all exons and introns, and a C-terminal HA epitope tag. Following anti-HA immunoprecipitation (IP) and immunoblotting, RDR2-HA is readily detected using anti-RDR2 antisera (, lane 2, row 2), as are the catalytic subunits of Pol IV, NRPD1 and NRPD2 (, lane 2, rows 3 and 5). LC-MS/MS analysis of affinity-purified RDR2-HA identified nine of the twelve Pol IV subunits, including major (3a), and alternative (3b) forms of the third subunit (Tables S1 and S2
). No Pol I, II, III or V-specific subunits were detected.
Consistent with the RDR2-HA IP and mass spectrometry results, RDR2 co-IPs with FLAG-tagged NRPD1 (, lane 3) but not with Pol V (NRPE1-FLAG, lane 4), Pol II (NRPB2-FLAG; lane 7), or Pols I or III (lane 2, row 8). NRPD1 does not co-IP with RNA-DEPENDENT RNA POLYMERASE 6 (, lane 6 and lane 3), involved in 21 nt siRNA biogenesis (Figure S1D
), indicative of Pol IV’s specificity for RDR2. No association between RDR2 and DCL3 was detected by immunoblot (, lanes 2 and 5) or LC-MS/MS analyses.
To test if Pol IV and RDR2 might associate via RNA, we made use of Pol IV rendered catalytically inactive (Haag et al., 2009
) by changing to alanines the three invariant aspartates of the NRPD1 Metal A site (). Whereas nrpd1-3
null mutants are rescued by a wild-type NRPD1-FLAG
bearing the active site mutations (ASM) fails to restore siRNA biogenesis, RNA-directed DNA methylation or transposon silencing (Haag et al., 2009
). For example, a soloLTR element silenced in wild-type cells (, lane 1), but derepressed in nrpd1-3
(Pol IV) or nrpe1–11
(Pol V) null mutants (lanes 3 and 4), is re-silenced by NRPD1-FLAG
transgenes (lanes 5 and 6), but not by NRPD1(ASM)-FLAG
(active site mutant) or NRPE1(ASM)-FLAG
transgenes (, lanes 7 and 8).
NRPD1-FLAG or NRPD1(ASM)-FLAG associate with equivalent amounts of NRPD2 (the Pol IV second subunit) and RDR2 (, compare lanes 4 and 5), suggesting that active site mutations do not disrupt Pol IV assembly or RDR2 association. Pol IV-RDR2 association is also unaffected by RNase A (, lane 2). Collectively, these results suggest that Pol IV-RDR2 interaction does not require Pol IV transcripts or other RNAs.
Affinity-purified Pol IV-RDR2 complexes generate transcripts in vitro
In the search for templates for Pols IV or V (e.g. see Fig. S2
), we found that Pol IV, like Pol II, will transcribe a tripartite oligonucleotide template that mimics a transcription bubble (). Features of this template include an 8 bp RNA-DNA hybrid, single stranded DNA and RNA upstream of the hybrid region and double-stranded DNA downstream of the DNA-RNA hybrid (). Pols I and II transcribe such tripartite templates, extending the RNA in a DNA-templated manner (Kuhn et al., 2007
; Lehmann et al., 2007
Pol IV displays DNA-dependent RNA polymerase activity
Using the tripartite template, Arabidopsis Pol II and Pol IV-RDR2 complexes catalyze alpha 32
P-CTP incorporation into RNA extension products that can be resolved on sequencing gels and visualized by autoradiography (). The RNA of the tripartite template is 16 nt; its DNA-templated extension can yield a full-length product of 32 nt. Consistent with previous studies using yeast Pol II, Arabidopsis Pol II catalyzes the synthesis of RNA products up to 32 nt (lane 5) and is inhibited by 5 μg/ml α-amanitin (lane 6). The Pol IV-RDR2 complex generates abundant 12–16 nt transcripts and longer transcripts up to 32 nt (lane 2). Pol IV-RDR2 transcripts are insensitive to α-amanitin (, lane 3), consistent with the divergence in Pols IV and V of the α-amanitin binding pocket of Pol II (Figure S3
) and the expected α-amanitin insensitivity of RNA-dependent RNA polymerases, such as RDR2. Cloning and sequencing of RNA-primed extension products confirmed that all Pol II, IV and V transcripts are DNA-templated (Figure S4
Catalytically crippled (ASM) and wild-type Pol IV both associate with RDR2 (). Affinity purified Pol IV(ASM)-RDR2 generates 12–16 nt RNA products (, lane 4) as efficiently as wild-type Pol IV-RDR2 (lanes 1 and 2) but most long transcription products are absent. Long RNAs dependent on the Pol IV active site are interpreted to be DNA-templated Pol IV transcripts. Transcripts unaffected by mutating the Pol IV active site are presumably generated by RDR2; these are mostly smaller than the 16 nt RNA oligonucleotide in the reactions (a 5′ end-labeled aliquot of this RNA is present in lane 8), consistent with RDR2 transcribing the 16 nt RNA. A transcript of ~26 nt generated by the Pol IV(ASM)-RDR2 complex (lane 4) is also RDR2-dependent based on subsequent experiments using Pol IV isolated from an rdr2 null mutant background (see below).
We next deconstructed the tripartite template, testing its component oligonucleotides as templates (). Pol IV-RDR2 or Pol II transcription reactions performed using a bipartite template, consisting of the 16 nt RNA hybridized to the 31 nt DNA template, yielded products similar to those obtained using the tripartite template (, lanes 6–8; compare to lanes 2–4), indicating that non-template DNA downstream of the DNA-RNA hybrid is dispensable. In fact, transcription was more robust without the need to displace the non-template DNA oligonucleotide, allowing more full-length transcription by Pols II and IV.
Using only the 31 nt DNA oligonucleotide as the template, a ladder of transcription products were generated by both Pol IV-RDR2 and Pol II (, lanes 9–12). Many of these products were less abundant using the Pol IV active site mutant form of the Pol IV-RDR2 complex, indicating that Pol IV (like Pol II) is able to transcribe single-stranded DNA to some extent. The 12–16 nt RNA products obtained using the tripartite or bipartite templates are absent in reactions containing only the 31 nt DNA template (lanes 9–12). Conversely, transcription reactions using the 16 nt RNA oligonucleotide alone support 12–16 nt RNA production in Pol IV-RDR2 and Pol IV(ASM)-RDR2 reactions (, lanes 14 and 15), but not in Pol II reactions (lane 16), consistent with these being RDR2 transcripts templated by the 16 nt RNA oligonucleotide.
Genetic and biochemical testing of Pol IV-RDR2 interdependence
Because Pol IV and RDR2 copurify, we disentangled them by introgressing NRPD1-FLAG or NRPD1(ASM)-FLAG transgenes into an rdr2-1 null mutant background and by introgressing an RDR2-HA transgene into a Pol IV null mutant (nrpd1-3). In the rdr2-1 mutant background, affinity purified NRPD1 or NRPD1(ASM) lack associated RDR2, as expected (, lanes 4 and 5). Likewise, RDR2-HA normally associates with Pol IV ( lane 2), but not in an nrpd1-3 null mutant background (, lane 3).
Pol IV transcription is independent of RDR2, but RDR2 requires Pol IV
As shown previously, Pol IV-RDR2 generates both long (>16 nt) and short (<16 nt) transcripts using the bipartite template (, lane 2), with most long transcripts dependent on the Pol IV active site (lane 3). Importantly, 12–16 nt transcripts are no longer produced in reactions utilizing Pol IV isolated from an rdr2 mutant (, lane 4), consistent with their synthesis by RDR2. Products of ~16 and 31 nt observed in anti-FLAG and anti-HA IP controls from non-transgenic plants (, lanes 1 and 6, respectively) are due to end-labeling activities that are neither Pol IV nor RDR2-dependent.
Pol IV-RDR2 complex(es) isolated upon IP of RDR2 or NRPD1 have similar activities (, compare lanes 7 and 2). However, RDR2 isolated from the Pol IV null mutant background (nrpd1-3) no longer synthesizes 12–16 nt transcripts (, lane 8). We conclude that RDR2 requires association with Pol IV, or a Pol IV-associated factor, for activity in vitro. In contrast, Pol IV activity is not dependent on RDR2 association.
Affinity-purified Pol V is transcriptionally active in vitro
We tested the activity of Pol V affinity purified from an nrpe1-11
null mutant rescued with wild-type or active site mutant (ASM) forms of FLAG-tagged NRPE1 (see ). NRPE1-FLAG complements the nrpe1-11
mutant but NRPE1(ASM)-FLAG does not (, compare lanes 6 and 8) (Haag et al., 2009
Like Pol IV, no significant Pol V activity was detectable using sheared genomic DNA, chromatin or ssDNA templates (Figure S2
). Unlike Pol IV, no Pol V activity was detected using the tripartite template, (data not shown). However, using the bipartite template, which lacks a non-template DNA strand in need of displacement, Pol V generates transcription products that are similar to those of Pol II ( compare lanes 6 and 7 to lanes 9 and 10). Pol V transcripts are abolished upon mutation of the Pol V active site (lane 8) but their synthesis is insensitive to alpha-amanitin (lanes 4, 7).
Comparison of Pol II, IV and V transcripts generated in vitro
The ability of both Pols IV and V to transcribe bipartite DNA-RNA templates prompted us to test their ability to transcribe bipartite RNA-RNA templates (). Interestingly, Pol IV is able to generate transcripts up to ~27 nt in length (, lanes 3 and 4), but Pol V lacks significant activity using the all-RNA template.