Activation of RNA polymerase III transcription in cells transformed by simian virus 40

Abstract

RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses.

FIG. 1

The SV40-transformed cell lines Cl38 and Cl49 express elevated levels of TFIIIC2 subunits. (A) Whole-cell extracts (57 μg of protein) prepared from Cl38 (lane 1), A31 (lanes 2 and 4), and Cl49 (lane 3) cells were resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western immunoblotting with anti-TFIIICα antibody Ab4. Lanes 2 and 4 show extracts prepared from two separate batches of A31 cells. (B) CHep-1.0 fraction (15 μg of protein) containing TFIIIC2 (lane 1) and whole-cell extracts (38 μg of protein) prepared from HeLa (lane 2), Cl38 (lane 3), A31 (lane 4), and Cl49 (lane 5) cells were resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western immunoblotting with anti-TFIIICβ monoclonal antibody clone 46. (C) Whole-cell extracts (50 μg of protein) prepared from A31 (lane 1) and Cl38 (lane 2) cells, CHep-1.0 (12 μg of protein) TFIIIC2 fraction (lane 3), and A25(0.15) (1.6 μg of protein) TFIIIB fraction (lane 4) were resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western immunoblotting with anti-BRF antibody 330. (D) Whole-cell extracts (50 μg of protein) prepared from Cl38 (lane 1), A31 (lane 2), and Cl49 (lane 4) cells and PC-D (5.6 μg of protein) TFIID fraction (lane 3) were resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western immunoblotting with anti-TBP antibody SL30.

FIG. 2

The SV40-transformed cell lines Cl38 and Cl49 express elevated levels of transcripts encoding TFIIICα and TFIIICβ. (A) cDNAs generated by reverse transcription of 3 μg of RNA from A31 (lane 1), Cl38 (lane 2), and Cl49 (lane 3) cells were PCR amplified by using TFIIICβ (top) and GAPDH (bottom) primers. Amplification products were resolved on a 2% agarose gel. (B) cDNAs generated by reverse transcription of 3 μg of RNA from A31 (lane 1), Cl38 (lane 2), and Cl49 (lane 3) cells were PCR amplified by using TFIIICα (top) and BRF (bottom) primers. Amplification products were resolved on a 2% agarose gel.

FIG. 3

SV40 large T antigen can counteract the repressive effect of RB on Pol III transcription. Template pVAI (250 ng of DNA) was transcribed by using 2 μl of Mono Q-purified TFIIIB and 2 μl of CHep-1.0 fraction in the presence of 100 ng of either GST (lanes 1 to 3) or GST-RB (lanes 4 to 6) and 20 ng (lanes 2 and 5) or 40 ng (lanes 3 and 6) of immunoaffinity-purified recombinant large T antigen (TAg).

FIG. 4

Cl38 and Cl49 cell extracts contain higher TFIIIB activity than A31 cell extracts. (A) Template pVAI (250 ng of DNA) was transcribed by using 2 μl of HeLa-derived PC-C fraction and buffer alone (lane 2) or PC-B fraction (6.4 μg of protein) derived from HeLa (lane 1), Cl38 (lane 3), or A31 (lane 4) cell extracts. (B) Template pVA1 (500 ng DNA) was transcribed by using 4 μl of HeLa-derived PC-C fraction supplemented with 10 ng of recombinant TBP and 6 μl of heat-treated Mono Q-purified TFIIIB (lane 1), buffer alone (lanes 2 and 6), or 7.5 μg of heat-treated extract from Cl38 (lane 3), A31 (lane 4), or Cl49 (lane 5) cells.

FIG. 5

The BRF subunit of TFIIIB coimmunoprecipitates with endogenous RB present in A31 extracts, but this interaction is compromised in extracts of Cl38 and Cl49 cells. (A) Reticulocyte lysate (15 μl) containing in vitro-translated BRF was immunoprecipitated (IP) with anti-RB antibody G3-245 in the presence of buffer alone (lane 2) or 150 μg of whole-cell extract prepared from SAOS2 (lane 3), Cl38 (lane 4), or A31 (lane 5) cells. Proteins retained after extensive washing were resolved on an SDS–7.8% polyacrylamide gel and then visualized by autoradiography. Lane 1 shows 10% of the input reticulocyte lysate containing in vitro-translated BRF. (B) Reticulocyte lysate (15 μl) containing in vitro-translated BRF was immunoprecipitated with anti-RB antibody C-15 in the presence of buffer alone (lane 1) or 150 μg of whole-cell extract prepared from A31 (lane 2) or Cl49 (lane 3) cells. Proteins retained after extensive washing were resolved on an SDS–7.8% polyacrylamide gel and then visualized by autoradiography.

FIG. 6

The association between endogenous RB and endogenous TFIIIB is greater in A31 cells than in Cl38 and Cl49 cells. (A) Cl38 (lanes 1 and 2) and A31 (lanes 3 and 4) cell extracts (150 μg of protein) were immunoprecipitated (IP) with anti-TAF_I48 antibody M-19 (lanes 1 and 3) or anti-RB antibody C-15 (lanes 2 and 4). Precipitated material was resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western blotting with anti-BRF antiserum 128. (B) Cl49 (lanes 1 and 2) and A31 (lanes 3 and 4) cell extracts (150 μg of protein) were immunoprecipitated with anti-TAF_I48 antibody M-19 (lanes 1 and 3) or anti-RB antibody C-15 (lanes 2 and 4). Precipitated material was resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western blotting with anti-BRF antiserum 128.

FIG. 7

Transfected HPV E7 activates Pol III transcription in vivo in a manner that is dependent on its RB-binding domain. NIH 3T3 cells were transfected with pVAI (2 μg), pCAT (2 μg), and 6 μg of empty vector (lane 1) or vector encoding wild-type E7 (E7 WT; lane 2), PRO2 mutant E7 (lane 3), Δ21-35 mutant E7 (lane 4), or GLY24 mutant E7 (lane 5). (A) VA_I primer extension products. (B) VA_I transcription levels that have been normalized to the levels of CAT expression to correct for transfection efficiency and are expressed relative to the value obtained for the empty vector control (designated 1.0).