Simian virus 40 large T antigen interacts with human TFIIB-related factor and small nuclear RNA-activating protein complex for transcriptional activation of TATA-containing polymerase III promoters

Abstract

The TATA-binding protein (TBP) is common to the basal transcription factors of all three RNA polymerases, being associated with polymerase-specific TBP-associated factors (TAFs). Simian virus 40 large T antigen has previously been shown to interact with the TBP-TAFII complexes, TFIID (B. Damania and J. C. Alwine, Genes Dev. 10:1369-1381, 1996), and the TBP-TAFI complex, SL1 (W. Zhai, J. Tuan, and L. Comai, Genes Dev. 11: 1605-1617, 1997), and in both cases these interactions are critical for transcriptional activation. We show a similar mechanism for activation of the class 3 polymerase III (pol III) promoter for the U6 RNA gene. Large T antigen can activate this promoter, which contains a TATA box and an upstream proximal sequence element but cannot activate the TATA-less, intragenic VAI promoter (a class 2, pol III promoter). Mutants of large T antigen that cannot activate pol II promoters also fail to activate the U6 promoter. We provide evidence that large T antigen can interact with the TBP-containing pol III transcription factor human TFIIB-related factor (hBRF), as well as with at least two of the three TAFs in the pol III-specific small nuclear RNA-activating protein complex (SNAPc). In addition, we demonstrate that large T antigen can cofractionate and coimmunoprecipitate with the hBRF-containing complex TFIIIB derived from HeLa cells infected with a recombinant adenovirus which expresses large T antigen. Hence, similar to its function with pol I and pol II promoters, large T antigen interacts with TBP-containing, basal pol III transcription factors and appears to perform a TAF-like function.

FIG. 1

Pol III transcription initiation complexes formed on the Ad2 VAI and U6 promoters. Transcription factor TFIIIC binds the A and B boxes on the intragenic promoter of the adenovirus VAI gene and recruits transcription factor TFIIIB, which is comprised of TBP and hBRF and, by analogy to the yeast system, may also recruit the human TFC5 factor. The human U6 snRNA promoter is similar to pol II promoters lying upstream of the coding sequence and containing a TATA box located approximately 25 nucleotides upstream of the initiation site. A PSE and an octamer binding site are located upstream of the TATA element. The transcription factor TFIIIB utilizes TBP to interact with the TATA box. A second TBP-containing transcription complex, SNAPc, binds to the PSE. In addition, the Oct-1 transcription factor is capable of binding the upstream octamer motif.

FIG. 2

T antigen does not activate transcription of the VAI gene. CV-1 cells were transfected with a VAI RNA-expressing plasmid, pSP72-VAI, together with 0, 1, 3, or 5 μg of a plasmid expressing SV40 T antigen (pRSV-Tex). Input amounts of DNA were maintained by using a filler plasmid, pRSV3BglII. Two micrograms of pα4X(A+C), which produces α-globin mRNA, was also cotransfected as an internal transfection efficiency control. At 42 h after transfection, the total RNA was harvested, and VAI RNA and α-globin RNA production was analyzed by RNase protection as described in Materials and Methods. The protected VAI and α-globin RNAs were 161 and 132 nucleotides, respectively. The undigested VAI (316 nucleotides) and α-globin (220 nucleotides) antisense probes are also shown.

FIG. 3

T antigen activates transcription of the U6 RNA gene. CV-1 cells were transfected with the U6 RNA producing plasmid pSP72-U6 sense, along with 0, 1, 3, or 5 μg of plasmids expressing full-length large T antigen and small t antigen (WT T+t), full-length T antigen alone (WT T only), or various mutants of T antigen (Table 1). The α-globin RNA producing plasmid pα4X(A+C) was used as the internal control. At 42 h posttransfection, the total RNA was harvested and U6 RNA and α-globin RNA production was analyzed by RNase protection as described in Materials and Methods. The protected U6 (184 nucleotides) and α-globin (132 nucleotides) bands are indicated.

FIG. 4

T antigen interacts in vitro with components of SNAPc and hBRF. The three SNAPc components SNAP 43, 45, and 50 were produced by in vitro transcription and translation; the input lanes indicate 20% of the total amount of in vitro-transcribed and -translated ³⁵S-labeled proteins used in each binding reaction. Each protein was tested for in vitro binding with the GST moiety alone or with a GST fusion with full-length T antigen as described in Materials and Methods. In addition, in vitro-transcribed and -translated T antigen was tested for binding to the GST moiety alone and to a GST fusion with full-length hBRF.

FIG. 5

Purification of hBRF. Nuclear extract from HeLa cells infected with a recombinant adenovirus vector expressing T antigen was fractionated on a phosphocellulose and Sepharose-Q column (see Materials and Methods). Fractions from a Sepharose-Q column, previously shown to contain TFIIIB activity, were analyzed for hBRF by Western analysis using an anti-hBRF antibody. The hBRF elutes in two peaks (fractions 70 to 80 and fractions 88 to 96). In, input.

FIG. 6

T antigen cofractionates with hBRF. The hBRF-containing fractions examined in Fig. 5 were examined for the presence of T antigen (T Ag) by Western blotting using anti-T antigen antibody Pab419. Large T antigen eluted in one peak from the Sepharose-Q column (fractions 88 to 98). Std., standard.

FIG. 7

T antigen and hBRF coimmunoprecipitate. Fractions 88 to 96 from the Sepharose-Q column, containing both hBRF and T antigen, were pooled and dialyzed into 0.1 M KCl buffer D. The samples were then subjected to immunoprecipitation reactions. (A) Immunoprecipitation reaction using an anti-T antigen antibody or an anti-hnRNPC1 (anti-C1) control antibody. The immunoprecipitates were resolved on a gel and subjected to Western analysis using an anti-hBRF antibody. (B) Immunoprecipitation reaction using an anti-hBRF antibody or an anti-hnRNPC1 control antibody. The immunoprecipitates were resolved on a gel and subjected to Western analysis using an anti-T antigen (T Ag) antibody.