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. 2004 Jul;24(14):6338-49.
doi: 10.1128/MCB.24.14.6338-6349.2004.

Multiple protein-protein interactions by RNA polymerase I-associated factor PAF49 and role of PAF49 in rRNA transcription

Kazuo Yamamoto  1 , Mika Yamamoto, Ken-ichi Hanada, Yasuhisa Nogi, Toshifumi Matsuyama, Masami Muramatsu
Affiliations

Multiple protein-protein interactions by RNA polymerase I-associated factor PAF49 and role of PAF49 in rRNA transcription

Kazuo Yamamoto et al. Mol Cell Biol. 2004 Jul.

Abstract

We previously demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. Here, we report the isolation and characterization of another Pol I-associated factor, PAF49. Mouse PAF49 shows striking homology to the human nucleolar protein ASE-1, so that they are considered orthologues. PAF49 and PAF53 were copurified with a subpopulation of Pol I during purification from cell extracts. Physical association of PAF49 with Pol I was confirmed by a coimmunoprecipitation assay. PAF49 was shown to interact with PAF53 through its N-terminal segment. This region of PAF49 also served as the target for TAF(I)48, the 48-kDa subunit of selectivity factor SL1. Concomitant with this interaction, the other components of SL1 also coimmunoprecipitated with PAF49. Specific transcription from the mouse rRNA promoter in vitro was severely impaired by anti-PAF49 antibody, which was overcome by addition of recombinant PAF49 protein. Moreover, overexpression of a deletion mutant of PAF49 significantly reduced pre-rRNA synthesis in vivo. Immunolocalization analysis revealed that PAF49 accumulated in the nucleolus of growing cells but dispersed to nucleoplasm in growth-arrested cells. These results strongly suggest that PAF49/ASE-1 plays an important role in rRNA transcription.

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Figures

FIG. 1.
FIG. 1.
Amino acid sequence alignment of PAF49 with the human and lamprey orthologues. (A) Residues identical between murine PAF49 (mPAF49) and its potential human counterpart ASE-1 (hASE-1) (48) and lamprey CAST (45) are indicated by dark shading. Similar residues are also highlighted by gray shading, according to the grouping (A, I, L, V), (F, W, Y), (E, D), (G, P), (K, R), (N, Q), (S, T), (C), (H), and (M). Sequences missing in PAF49 are represented by dashes. The sequences obtained by peptide microsequencing of PAF49 are indicated above the sequence of PAF49 as sequence 1, 2, or 3. Asterisks under the sequence of CAST indicate clusters of basic aminoacids. The position of the amino acid insertion (GE) found in human CAST is indicated by an open triangle above the sequence of PAF49 (see text for details). (B) Neighbor-joining tree of PAF49/ASE-1 sequences. The tree is based on the amino acid alignment in panel A and is rooted at the midpoint.
FIG. 2.
FIG. 2.
PAF49 is contained in Pol IB. (A) Schematic diagram of the purification procedure for Pol IA and Pol IB from whole-cell extracts (WCE). Abbreviations: PC, phosphocellulose; DEAE, DEAE-Sepharose; CM, CM-Sepharose; Q, Mono-Q; GG, glycerol gradient fractionation. The values represent the molar concentration of KCl used to develop the column. (B) The flowthrough fraction (Pol IA; lane 1) and 0.3 M KCl fraction (Pol IB; lane 2) from CM-Sepharose were analyzed by Western blotting with anti-RPA194, anti-PAF53, anti-PAF49, and anti-RPA40 antibodies. (C) The glycerol gradient fractions (2 to 10, recovered from the bottom to the top of the sediments, respectively) were analyzed by Western blotting with anti-RPA194, anti-PAF53, anti-PAF49, and anti-RPA40 antibodies.
FIG. 3.
FIG. 3.
Physical association of PAF49 with Pol I core enzyme. Mouse nuclear extracts (indicated in lane 1 as input) were incubated with a control IgG (lane 2), affinity-purified anti-PAF49 antibody (lane 3), anti-RPA16 antibody (lane 4), or anti-RPB11 antibody (lane 5), and then precipitated with protein A-Sepharose beads. The presence of the Pol I-specific largest subunit (upper panel) or the Pol II-specific largest subunit (lower panel) in each immune complex was determined by Western blotting with anti-RPA194 antiserum and anti-CTD monoclonal antibody, respectively. IB, immunoblot.
FIG. 4.
FIG. 4.
Far-Western analysis of PAF49 and PAF53. (A) Purified Pol IB was blotted onto a nitrocellulose membrane and then incubated with 35S-labeled PAF49 (lane 2) or PAF53 (lane 3) prepared by in vitro coupled transcription-translation reaction. A control labeling reaction with an empty plasmid template was used as control probe (lane 4). Same Pol I sample was also loaded onto the SDS-polyacrylamide gel, and individual subunits of Pol I were stained by Coomassie brilliant blue (lane 1). The positions of formerly identified subunits and PAFs are indicated at the left. (B) The far-Western blotting experiment was performed with purified GST-PAF53 and 35S-labeled PAF49 (lane 2) or PAF53 (lane 3). The purity of GST-PAF53 was indicated by Coomassie staining (lane 1).
FIG. 5.
FIG. 5.
PAF49 binds to PAF53 through its N-terminal region. (A) Schematic presentation of GST-PAF49 fusion proteins: —, GST protein alone; FL, full-length PAF49 fused to GST; ΔN, GST fusion with PAF49 lacking the N-terminal 198 residues; ΔC, GST fusion with PAF49 lacking the C-terminal 141 residues. (B) GST pulldown assay with GST-PAF49 mutants and PAF53. The recombinant fusion proteins GST-PAF49FL (lane 3), ΔN (lane 4), and ΔC (lane 5) as well as GST alone (lane 2) were immobilized on glutathione beads and then incubated with 35S-labeled PAF53. The bound protein was detected by autoradiography after SDS-PAGE. Ten percent of the input PAF53 was electrophoresed in lane 1.
FIG. 6.
FIG. 6.
PAF49 interacts with TAFI48 in vitro and in vivo. (A) GST-PAF49 (lane 3), GST-PAF53 (lane 4), and GST-RPA40 (lane 5) as well as GST alone (lane 2) were immobilized on glutathione beads and then incubated with 35S-labeled UBF1 and the SL1 subunits TAFI95, TAFI68, and TAFI48. After extensive washing of the beads, bound proteins were eluted by boiling in SDS sample buffer and applied to an SDS-polyacrylamide gel. Ten percent of the input labeled protein was also included in lane 1. (B) GST-PAF49FL (lane 2), ΔN (lane 3), and ΔC (lane 4) were immobilized on glutathione beads and then incubated with 35S-labeled TAFI48. The bound protein was detected by autoradiography. Ten percent of the input labeled TAFI48 was also included (lane 1). (C) NIH 3T3 cells were cotransfected with plasmids expressing Flag-tagged TAFI48 and various forms of HA-tagged PAF49 as well as the empty vector. The expression of HA-tagged full-length PAF49 (FL, lane 2), the N-terminal truncation mutant (ΔN, lane 3), and the C-terminal truncation mutant (ΔC, lane 4) was confirmed in nuclear extracts by Western blotting. The extracts were then immunoprecipitated with anti-Flag M2 affinity beads (lanes 5 to 8). The positions of the full-length and C-terminally truncated forms of PAF49 in the immune complexes are indicated by arrowheads at the right. The amounts of Flag-tagged TAFI48 in the immune complexes are shown in the lower panel (lanes 5 to 8). (D) Nuclear extracts were incubated with (lane 3) or without (lane 2) anti-PAF49 antibodies, followed by precipitation with protein A beads. Each immune complex was analyzed by Western blotting with anti-TAFI95 antibody (upper panel), anti-TAFI68 antibody (middle panel), or anti-TBP antibody (lower panel). About 6% of the input nuclear extracts were also examined on the left (lane 1).
FIG. 7.
FIG. 7.
Inhibition of rRNA transcription in vitro by anti-PAF49 antibody and in vivo by overexpression of the PAF49 mutant. (A) Nuclear extracts (30 μg) were incubated with affinity-purified anti-PAF49 IgG (lane 7, 0.8 μg; lane 8, 1.6 μg; lanes 9 to 11, 3.2 μg) or control IgG (lane 2, 0.8 μg; lane 3, 1.6 μg; lanes 4 to 6, 3.2 μg) in the absence (lanes 1 to 4 and 7 to 9) or the presence of 4.2 μg of recombinant PAF49 (lanes 5 and 10) or bovine serum albumin (lanes 6 and 11). Then, the template DNA containing the rRNA promoter and nucleotides was added for in vitro transcription. The promoter-specific transcripts were analyzed on a denaturing polyacrylamide gel and visualized by autoradiography. (B) NIH 3T3 cells were transfected with expression plasmids for Flag-tagged PAF49 or its deletion mutants. Total RNA were recovered after incubation for 24 h and analyzed by primer extension assay with a 5′-labeled pre-rRNA primer and β-actin primer. (C) Summary of the primer extension assay results. The relative transcription rate of pre-rRNA was obtained by normalizing the amounts of pre-rRNA to those of β-actin mRNA and compared with the control value. Values are represented as averages of three independent experiments with the standard deviation. *, P < 0.01, Student's t test (ΔC versus full-length PAF49). (D) The amounts of ectopically expressed PAF49 and its deletion mutants were determined by Western blotting with anti-Flag M2 monoclonal antibody.
FIG. 8.
FIG. 8.
PAF49 accumulates in the nucleolus of growing cells. NIH 3T3 cells were grown in culture medium containing 10% (A to C) or 0.1% (D to F) calf serum. The cells were then fixed and incubated with anti-UBF mouse monoclonal antibody and anti-PAF49 rabbit polyclonal antibodies, followed by fluorescent secondary antibodies. Images were obtained separately for UBF (red; A and D) and for PAF49 (green; B and E) or merged (C and F); yellow spots indicate colocalization of UBF and PAF49.
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