A novel TBP-associated factor of SL1 functions in RNA polymerase I transcription

Abstract

In mammalian RNA polymerase I transcription, SL1, an assembly of TBP and associated factors (TAFs), is essential for preinitiation complex formation at ribosomal RNA gene promoters in vitro. We provide evidence for a novel component of SL1, TAF(I)41 (MGC5306), which functions in Pol I transcription. TAF(I)41 resides at the rDNA promoter in the nucleolus and co-purifies and co-immunoprecipitates with SL1. TAF(I)41 immunodepletion from nuclear extracts dramatically reduces Pol I transcription; addition of SL1 restores the ability of these extracts to support Pol I transcription. In cells, siRNA-mediated decreased expression of TAF(I)41 leads to loss of SL1 from the rDNA promoter in vivo, with concomitant loss of Pol I from the rDNA and reduced synthesis of the pre-rRNA. Extracts from these cells support reduced levels of Pol I transcription; addition of SL1 to the extracts raises the level of Pol I transcription. These data suggest that TAF(I)41 is integral to transcriptionally active SL1 and imply a role for SL1, including the TAF(I)41 subunit, in Pol I recruitment and, therefore, preinitiation complex formation in vivo.

Figure 1

Human nucleolar protein TAF_I41 co-purifies with TBP-antibody affinity-purified SL1 and is found at the rDNA promoter in human cells. (A) Human SL1 purification scheme. For analytical purification of SL1, HeLa cell nuclear extract was applied to POROS Heparin Agarose and SP-Sepharose columns, as previously described (Comai et al, 1992; Friedrich et al, 2005), then salt gradient eluted from a POROS Heparin column. The SL1 fractions were pooled, desalted on G25 Sepharose, step-eluted from SP-Sepharose and applied to a TBP-antibody affinity column. SL1 complexes were eluted with an excess of TBP (3G3) peptide and this eluate was applied to a Mono S column, which was developed by a linear salt gradient (KCl at concentrations (M) as indicated). (B) TAF_I41 co-purifies with SL1 protein and activity from HeLa cell nuclear extracts. Fractions obtained by salt gradient elution from the POROS Heparin column (B) were analysed for SL1 protein by immunoblotting, with antibodies specific for SL1 subunits TAF_I110 and TAF_I63, and an antibody generated against three peptides of TAF_I41 (see Materials and methods). The bulk of TAF_I41 eluted with the other SL1 subunits at 0.7 M KCl (fractions 21 and 22). Fraction 22 supported specific SL1-dependent reconstituted Pol I transcription. The 0.7 M KCl fraction was desalted to 0.2 M KCl, passed over SP-Sepharose and a salt-step elution was performed. The bulk of SL1 including TAF_I41 eluted at 0.65 M KCl and supported specific SL1-dependent transcription. This SL1 fraction was loaded onto a TBP-antibody affinity column (input, lane 1), some of which flowed through (FT, lane 4). The column was washed at 0.5 and 1.0 M KCl (lanes 2 and 3). Bound proteins from the washed column were eluted with excess TBP-epitope peptide 3G3 (1 mg/ml) (lane 5) and fractions were immunoblotted for SL1 with antibodies specific for TAF_I110 and TAF_I63, and the TAF_I41-peptides antibody. SL1 was concentrated and purified away from the 3G3-peptide on a Mono S column, developed with a linear salt gradient. The Mono-S purified SL1 fractions 19–25 (fractions 19, 21, 23 and 25 are shown in lanes 7–10) were pooled and tested for the ability to support SL1-dependent transcription with UBF and Pol I (lanes 1–3, arrowhead). (C) TBP and TAF_I41 display similar apparent molecular weights. SL1 (the 0.7 M fraction from the POROS-Heparin column, see A, B) was immunoblotted with antibodies specific for TAF_I110, TAF_I63, TAF_I48 and TBP (top panel, lanes 1, 2, 3 and 5, respectively) and the TAF_I41-peptides antibody (lane 4). A band of similar mobility to those of TBP and TAF_I41, detected by TAF_I48 antibodies, is either a degradation product of TAF_I48 or could represent a protein encoded by a splice variant 2 of TAF_I48 (top panel, lane 3). (D) TBP and TAF_I41 are separable by two-dimensional gel electrophoresis. SL1 (the 0.7 M fraction from the POROS-Heparin column, see A and B) was subject to first dimension isoelectric focusing (pH 3–10), then to second dimension separation (4–12% Bis–Tris gradient gel, Invitrogen). SL1 proteins were immunoblotted, probed first with TAF_I41-peptides antibody (upper panel) and then with antibodies specific for TBP (middle panel). The lower panel shows an overlay of the signals from these immunoblots. (E) Overexpressed TAF_I41 accumulates in nucleoli. HeLa cells were transfected with expression vectors pEGFP-TAF_I41. After 24 h, the cells were fixed and viewed by confocal microscopy. DNA was stained with DAPI. Overlay is a composite of the DAPI (blue) and EGFP-TAF_I41 (green) signals. Scale bar, 10 nm. (F) ChIP analysis indicates that TAF_I41 is present at the promoter of the rRNA genes. ChIP analysis (from HeLa cells) used TAF_I41-peptides antibody (and as a control rabbit preimmune serum) or a TBP mouse monoclonal antibody (and as a control mouse IgG), followed by quantitative real-time PCR with primers specific for the promoter region and the internal transcribed spacer (ITS-2) of the human rDNA (see Materials and methods). The relative levels of rDNA associated with TAF_I41 and TBP at these sites are expressed as percentage of input chromatin and are from two independent experiments.

Figure 2

TAF_I41 is a component of transcriptionally active SL1. (A) EGFP-TAF_I41 co-immunoprecipitates with SL1 and UBF from human cells. Immunoprecipitation (IP) with GFP-specific antibodies was from HEK293 whole-cell extracts 48 h post-transfection of the cells with pEGFP-TAF_I41 (lane 8), empty expression vector (lane 4) or pEGFP-RRN3 (lane 6). Immunoblotting used antibodies specific for the Pol I largest subunit A190, UBF and SL1 subunits TAF_I110, TAF_I63 and TBP. Input (In), 10% of extract used for IP (lanes 3, 5 and 7). SL1 (lane 1), and Pol I and UBF (lane 2) were loaded as markers. (B) Endogenous TAF_I41 and Flag-TAF_I41 co-immunoprecipitate with SL1 from human cells. IP with Flag-specific antibodies was from HEK293 whole-cell extracts 48 h post-transfection of the cells with pcDNA-Flag-TAF_I41 (lane 7), empty expression vector (lane 3) or pcDNA-Flag-TAF_I63 (lane 5). Immunoblotting used antibodies specific for SL1 subunits TAF_I110, TAF_I63, TAF_I48, TBP and TAF_I41-peptides antibody. Input (In), 80% of extract used for IP (lanes 2, 4 and 6). Purified SL1 (lane 1) was loaded as a marker. (C) TAF_I41-associated proteins from cells support SL1-dependent Pol I transcription. Immunoprecipitated complexes of 40 (lanes 3, 6 and 9), 80 (lanes 4, 7 and 10) and 160 ng (lanes 5, 8 and 11) prepared in B, from HEK293 cells transfected with pcDNA-Flag-TAF_I41 (lanes 6–8), empty vector (lanes 3–5) or pcDNA-Flag-TAF_I63 (lanes 9–11), were tested for SL1-dependent activity in a specific transcription assay with Pol I, UBF and the rDNA promoter. Controls are transcription in a HeLa nuclear extract (lane 1) and from a partially purified SL1 fraction (the 0.7 M fraction from the POROS-Heparin column, see A, B; lane 2) added to the reconstituted transcription assay. (D) Endogenous TAF_I41 co-immunoprecipitates with TBP-containing complexes from human cells and the TAF_I41-TBP-containing complexes support rDNA promoter-specific Pol I transcription. TBP-containing complexes immunoprecipitated from HeLa cell nuclear extracts using TBP-specific monoclonal antibody (3G3) beads were eluted from the beads using an excess of epitope peptide, then incubated with TAF_I41-peptides antibody in a second IP. The SL1-dependent transcription activity of the TBP-TAF_I41-containing complexes was determined in an in vitro transcription assay with Pol I and UBF (lane 4). In the control sample, IgG was used in the second immunoprecipitation reaction in place of the TAF_I41-peptides antibody (lane 5). The SL1 activity of the input Hela nuclear extracts is shown in lane 3. Lanes 1 and 2 contain control reconstituted transcription reactions with Pol I and UBF, with and without SL1, respectively.

Figure 3

Human TAF_I41 antibodies deplete SL1 activity from human and mouse nuclear extracts. (A) Human TAF_I41 antibodies deplete SL1 subunits from HeLa cell nuclear extract. HeLa nuclear extracts were incubated with hTAF_I41 antibody D (raised against a recombinant human TAF_I41 deletion mutant, see Materials and methods; lanes 1 and 2) or control (IgG; lanes 3 and 4) antibodies overnight at 4°C. Immunoprecipitates (IP, lanes 2 and 4) and 10% of the immunodepleted extracts (ID, lanes 1 and 3) were immunoblotted with antibodies specific for TAF_I110, TBP and TAF_I41-peptides antibody. The signal in the ID for TAF_I110 (lanes 1 and 3) was too high and this resulted in a ‘white-out' following chemiluminescence. (B) Human TAF_I41 antibodies deplete SL1 transcriptional activity from HeLa cell nuclear extract. Extracts from (A), immunodepleted (ID) using hTAF_I41 antibody D (lanes 1 and 2) or control (IgG, lanes 3 and 4), were analysed in a specific transcription assay with the human rDNA promoter, in the absence (lanes 1 and 3) or presence of added purified SL1 (Superose 6 fraction; 1 μl, lanes 2 and 4). Transcripts were analysed by S1 nuclease protection. (C) Human TAF_I41 antibodies immunoprecipitate mouse TBP from mouse 3T3 nuclear extract. Mouse 3T3 nuclear extract (25 μl) was incubated with hTAF_I41 antibody D (lanes 4 and 5) or control (IgG, lanes 2 and 3) overnight at 4°C. Immunoprecipitated complex (IP) and 10% of the immunodepleted extract (ID) were immunoblotted with antibodies specific for TBP. Partially purified mouse SL1 (TIF-IB) (lane 1) was loaded as a marker. (D) Human TAF_I41 antibodies partially deplete Pol I transcription activity from mouse cell nuclear extract. In all, 15% of the extracts immunodepleted by hTAF_I41 antibody D (lane 2) or control (lane 1) antibodies were analysed in a specific transcription assay using the mouse rDNA promoter. Transcripts were analysed by S1 nuclease protection with a 5′-end labelled oligonucleotide complementary to the first 40 nt of the mouse pre-rRNA. Lane 3 contains the control transcription reaction with mouse nuclear extract (mNE).

Figure 4

siRNA-mediated downregulation of TAF_I41 expression in cells leads to a decrease in Pol I transcription levels. (A) TAF_I41-specific siRNA reduces TAF_I41 protein levels in cells. Nuclear extracts (5, 10 and 20 μg, respectively) of HEK293 cells transfected with TAF_I41-specific siRNA (lanes 1–3) or scrambled siRNA (control; lanes 4–6) were immunoblotted using TAF_I41-peptides antibody and GAPDH-specific antibody. Lane 7 contains SL1 as a marker. (B) TAF_I41-specific siRNA reduces Pol I transcription levels in cells. Pre-rRNA levels in total RNA extracted from HEK293 cells transfected, in triplicate, with TAF_I41-specific siRNA (lanes 1–3) or scrambled siRNA (control; lanes 4–6) were analysed by S1 nuclease protection with an oligonucleotide complementary to the first 40 nt of the 47 S pre-rRNA. Combined data are represented in the graph. (C) siRNA-mediated reduction of hTAF_I41 TAF_I48 and RRN3 mRNA levels. Messenger RNA levels in total RNA from SJSA cells transfected with TAF_I41-, TAF_I48- and RRN3-specific or scramble (control) siRNAs were determined by RT–PCR and normalized to β-actin mRNA levels. (D) siRNA-mediated reduction of TAF_I41, TAF_I48 or RRN3 gene expression reduces pre-rRNA synthesis in cells. Pre-rRNA levels in total RNA extracted from the siRNA-treated SJSA cells (see C) were analysed by S1 nuclease protection as in (B). (E) Pol I transcription in extracts of TAF_I41-specific siRNA-treated cells can be partially restored by addition of SL1. Nuclear extracts (30 μg) of HEK293 cells transfected with siRNAs specific for TAF_I41 (lanes 1–3), or scrambled (control; lanes 4–6) were analysed for specific transcription activity in the absence (lanes 1 and 4) or presence of purified SL1 (Superose 6 fraction; 0.5 μl, lanes 2 and 5, or 1 μl, lanes 3 and 6).

Figure 5

Reduced occupancy of SL1 and Pol I at the rDNA in TAF_I41-siRNA-treated cells. ChIP analysis of control or TAF_I41-siRNA-treated HEK293 cells (72 h post-transfection) with antibodies specific for TAF_I110 (SL1) or A135 (second largest subunit of Pol I) and with an array of primers sets for the human rDNA repeat (Pr1 and Pr2: UCE and core promoter sequences; Tr1 and Tr2, the transcribed 18S and 28S rRNA gene sequences; T, sequences downstream of terminator; IGS1 and IGS1, sequences in pseudo-gene CDC27 and p53-binding moiety in the intergenic spacer of the rDNA repeat; see Materials and methods). The levels of rDNA associated with TAF_I110 (SL1) and A135 (Pol I) at each site (relative to control antibodies) are expressed as percentage of input chromatin.

Figure 6

TAF_I41 interacts with UBF and can squelch UBF-dependent activation of Pol I transcription. (A) Recombinant His-TAF_I41 interacts directly with in vitro translated UBF. His-TAF_I41 (lanes 2 and 5), baculovirus-expressed in insect cells and purified on NiNTA beads, was incubated with in vitro translated (IVT) ³⁵S-radiolabelled UBF (lanes 1–3) or luciferase (negative control protein; lanes 4 and 5). Empty NiNTA beads were included as a control (lane 3). Beads were washed and bound proteins analysed by autoradiography. (B) GST-TAF_I41 interacts directly with recombinant UBF. GST-TAF_I41 (lane 2) or GST (lane 1) proteins on beads were incubated with purified recombinant baculovirus-Sf9 cell-expressed human UBF1. Beads were washed and bound proteins analysed by immunoblotting with UBF-specific antibodies. (C) A direct interaction between TAF_I41 and UBF is detectable by Far-Western analysis. In vitro translated ³⁵S-radiolabelled TAF_I41 was incubated with membrane-bound, then denatured and renatured UBF protein (Far-WB; lane 2). Binding was analysed by autoradiography. In control lane 1, denatured–renatured UBF was detected by UBF-specific antibodies following Western blotting (WB). (D) TAF_I41 squelches UBF-activated but not basal SL1-directed Pol I transcription. Preinitiation complexes (PICs) of SL1 (Superose 6 fraction) and Pol I were assembled on an immobilized human rDNA promoter template (IT-rDNA, Fr4; Panov et al, 2001) for 30 min on ice. The PICs were washed in TM10/0.075 M KCl, then GST-TAF_I41 (50 ng) was added concomitantly with 0, 1, 10 and 20 ng of UBF (lanes 9, 10, 11 and 12, respectively) and NTPs and transcription was analysed by S1 nuclease protection. In the control samples, in place of GST-TAF_I41, no protein (lanes 1–4) or GST (50 ng; lanes 5–8) was used.