1. Volume 48, Issue 5, Pages 811-818 (December 2012)
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 
Jeremy R. Haag, Thomas S. Ream, Michelle Marasco, Carrie D. Nicora, Angela D. Norbeck, Ljiljana Pasa-Tolic, Craig S. Pikaard 
Molecular Cell 
Volume 48, Issue 5, Pages (December 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Pol IV and RDR2 Interact in an RNA-Independent Fashion
(A) Model for the RNA-directed DNA methylation pathway in Arabidopsis thaliana.
(B) RDR2 tryptic peptides detected upon LC-MS/MS analysis of affinity-purified Pol IV are highlighted within the RDR2 sequence. The peptide shaded in green was sequenced individually and as part of a larger peptide.
(C) RDR2 and Pol IV coimmunoprecipitate (see also Tables S1 and S2 and Figure S1). HA-tagged RDR2 or FLAG-tagged NRPD1, NRPE1, DCL3, RDR6, or NRPB2 was immunoprecipitated (IPed) using anti-HA or anti-FLAG antibodies. Immunoblots were then probed with anti-FLAG, anti-RDR2, anti-NRPD1, anti-NRPE1, anti-NRP(D/E)2, anti-DCL3, or anti-RDR6. The anti-Pol I, anti-Pol II, anti-Pol III antibody recognizes the second subunits of Pols I, II or III but not Pols IV or V.
(D) Amino acids at the Metal A sites of Pol I–Pol V largest subunits. Invariant aspartates changed to alanines in NRPD1 and NRPE1 active site mutants (ASM) are shaded.
(E) RT-PCR analysis of solo LTR expression in wild-type, nrpd1 or nrpe1 mutants, or mutants expressing wild-type or ASM mutant NRPD1 or NRPE1 transgenes. Actin and no reverse transcriptase (−RT) controls are included.
(F) Test of RDR2 interaction with wild-type or ASM forms of Pol IV. RDR2-HA, NRPD1-FLAG, or NRPD1(ASM)-FLAG were IPed using anti-HA or anti-FLAG antisera. Immunoblots were probed using anti-FLAG, anti-RDR2, or anti-NRPD2 antibodies.
(G) NRPD1-FLAG was immunoprecipitated from control or RNaseA-treated cell extracts. Immunoblots were probed with anti-NRPD1 or anti-RDR2 antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Pol IV Displays DNA-Dependent RNA Polymerase Activity
(A) Model of the nucleic acids within a Pol II transcription bubble, adapted from Gnatt et al. (2001).
(B) A tripartite oligonucleotide template that mimics essential aspects of a transcription bubble, modeled after Kuhn et al. (2007).
(C) Autoradiogram of 32P-CTP-labeled transcription products catalyzed by Pol II or Pol IV-RDR2 using the tripartite template. Unmutated or active site mutant (ASM) forms of Pol IV were tested in parallel (lanes 2–4). Anti-FLAG IP of nontransgenic tissue extract served as a control (lane 1), revealing two background bands present in all reactions. Reactions conducted in the presence of α-amanitin are shown in lanes 3 and 6. Lane 8 shows a 32P end-labeled aliquot of the 16 nt RNA oligonucleotide used in the tripartite template.
(D) In vitro reactions using dissected components of the tripartite template. (Lanes 1–4) Affinity-purified Pol II, Pol IV-RDR2, or Pol IV(ASM)-RDR2 complexes incubated with the tripartite template. (Lanes 5–8) Transcription using a bipartite template consisting of the RNA and template DNA. (Lanes 9–12) Transcription using the template DNA oligonucleotide only. (Lanes 13–16) Transcription using the RNA oligonucleotide only. An aliquot of 5′ end-labeled 16 nt RNA was loaded in lane 17. Figures S2–S4 show results for other templates tested, the basis for Pol IV/V α-amanitin insensitivity, and sequences of Pol II, IV, and V transcripts generated using the bipartite template.
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4. Figure 3
Pol IV Transcription Is Independent of RDR2, but RDR2 Requires Pol IV
(A) FLAG-tagged Pol IV or Pol IV(ASM) (active site mutant) complexes were immunoprecipitated from wild-type (RDR2; lanes 2 and 3) or rdr2-1 mutant (no RDR2; lanes 4 and 5) backgrounds. Anti-FLAG IP of nontransgenic plant extract served as a negative control. Immunoblots were probed using anti-NRPD1, anti-RDR2, or anti-NRPD2 antibodies.
(B) RDR2-HA was IPed from wild-type Pol IV (lane 2) or nrpd1-3 mutant (no Pol IV, lane 3) backgrounds, and resulting immunoblots were probed using anti-RDR2, anti-NRPD1, or anti-NRPD2 antibodies. Anti-HA IP of nontransgenic plant extract served as a negative control.
(C) In vitro transcription of bipartite templates by Pol IV-RDR2 or by Pol IV or Pol IV(ASM) isolated in an rdr2 mutant background (lanes 2–5). Lanes 7 and 8 compare Pol IV-RDR2 or RDR2 isolated in a Pol IV mutant (nrpd1-3) background. In lanes 2–5, complexes were isolated via anti-FLAG IP of FLAG-tagged NRPD1. In lanes 7 and 8, RDR2-containing complexes were isolated by anti-HA IP of HA-tagged RDR2. Anti-FLAG or anti-HA immunoprecipitations of nontransgenic plant extracts serve as negative controls (lanes 1 and 6). End-labeled 16 nt RNA was loaded in lane 9.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Comparison of Pol II, IV, and V Transcripts Generated In Vitro
(A) In vitro transcription products catalyzed by Pol II, Pol IV, Pol IV active site mutant (ASM); Pol V; or the Pol V active site mutant (ASM) using the bipartite DNA-RNA template. Reactions were conducted in the absence or presence of α-amanitin, as indicated. No added protein and nontransgenic controls are shown in lanes 1 and 2.
(B) In vitro transcription products catalyzed by immunoprecipitated Pol IV, Pol IV active site mutant (ASM), Pol V, or Pol V active site mutant (ASM) using an RNA-RNA bipartite template. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions