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. 2005 Apr;79(7):4270-88.
doi: 10.1128/JVI.79.7.4270-4288.2005.

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein

Xiaomin Zhao  1 , Daniel Oberg, Margaret Rush, Joanna Fay, Helen Lambkin, Stefan Schwartz
Affiliations

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein

Xiaomin Zhao et al. J Virol. 2005 Apr.

Abstract

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.

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Figures

FIG. 1.
FIG. 1.
The HPV-16 genome and the subgenomic expression plasmids pBearly97, pBearly, and pBEL (61). Schematic representation of the HPV-16 genome. Boxes indicate the protein-coding regions. Numbers refer to nucleotide positions in the HPV-16R sequence (6). The major p97 promoter and the differentiation dependent promoter p670 are indicated. 5′ Splice sites (SD), 3′ splice sites (SA), and the early and late polyadenylation signals pAE and pAL, respectively, are shown. The structures of the pBearly97, pBearly, and pBEL (61) expression plasmids are shown. The pBEL-derived plasmids contain a deletion in the late UTR that removes previously identified negative elements (25, 26) in order to increase the chances of obtaining detectable levels of late mRNAs (61). CMV, human cytomegalovirus immediate-early promoter.
FIG. 2.
FIG. 2.
(A) Structure of the pBearly97 expression plasmid. The locations of the CMV probe (61) and probes #1, #2, and #3 are indicated. The generation of probes #1, #2, and #3 is described in Materials and Methods. Primers A, B, C, D, and E represent primers p97S, Probe SA526as, SD880as, E2as, and 3515as, respectively (Table 1). Locations of the primers are shown. Schematic presentation of all predicted splice variants of the mRNAs produced by pBearly97 are shown. The two E1 and E4 mRNAs generated by the use of the 3′ splice site at position 742 are shown (mRNAs 12 and 13). The 3′ splice site at position 742 is circulized. SD, 5′ splice site; SA, 3′ splice site. (B) Northern blots on total or cytoplasmic RNA extracted from HeLa cells transfected with pBearly97. Filters were hybridized with the CMV probe (61) and probes #1, #2, and #3. Arrows indicate four mRNAs species detected by Northern blotting. (C) RT-PCR with multiple primer pairs on total RNA extracted from HeLa cells transfected with pBearly97. Lane 1, primers A and B; lane 2, primers A and C; lane 3, primers A and E. Primers A, B, C, D, and E represent primers p97S, Probe SA526as, SD880as, E2as, and 3515as, respectively (Table 1). The location of the RT-PCR primers is shown in panel A. The 1-kb DNA ladder (Invitrogen) was used as a molecular size marker and is shown on the left. The splice junctions each band spans are indicated. An asterisk indicates the position of the band that represents splicing from the 5′ splice site at position 226 to the 3′ splice site at position 742 identified here. This splice site has recently been seen by two research groups (32, 62). The lower gel shows the same RNA sample as that in lanes 1, 2, and 3 in the upper gel, subjected to RT-PCR with the same primer pairs (A and B, A and C, and A and E) as above but in the absence of reverse transcriptase. −, Reverse transcriptase.
FIG. 2.
FIG. 2.
(A) Structure of the pBearly97 expression plasmid. The locations of the CMV probe (61) and probes #1, #2, and #3 are indicated. The generation of probes #1, #2, and #3 is described in Materials and Methods. Primers A, B, C, D, and E represent primers p97S, Probe SA526as, SD880as, E2as, and 3515as, respectively (Table 1). Locations of the primers are shown. Schematic presentation of all predicted splice variants of the mRNAs produced by pBearly97 are shown. The two E1 and E4 mRNAs generated by the use of the 3′ splice site at position 742 are shown (mRNAs 12 and 13). The 3′ splice site at position 742 is circulized. SD, 5′ splice site; SA, 3′ splice site. (B) Northern blots on total or cytoplasmic RNA extracted from HeLa cells transfected with pBearly97. Filters were hybridized with the CMV probe (61) and probes #1, #2, and #3. Arrows indicate four mRNAs species detected by Northern blotting. (C) RT-PCR with multiple primer pairs on total RNA extracted from HeLa cells transfected with pBearly97. Lane 1, primers A and B; lane 2, primers A and C; lane 3, primers A and E. Primers A, B, C, D, and E represent primers p97S, Probe SA526as, SD880as, E2as, and 3515as, respectively (Table 1). The location of the RT-PCR primers is shown in panel A. The 1-kb DNA ladder (Invitrogen) was used as a molecular size marker and is shown on the left. The splice junctions each band spans are indicated. An asterisk indicates the position of the band that represents splicing from the 5′ splice site at position 226 to the 3′ splice site at position 742 identified here. This splice site has recently been seen by two research groups (32, 62). The lower gel shows the same RNA sample as that in lanes 1, 2, and 3 in the upper gel, subjected to RT-PCR with the same primer pairs (A and B, A and C, and A and E) as above but in the absence of reverse transcriptase. −, Reverse transcriptase.
FIG. 3.
FIG. 3.
(A) Structure of the pBearly expression plasmid. The locations of the CMV, E1, and E4 probes (61) are indicated. Locations of primers 757S (60) and E4A (61) used for RT-PCR are shown. Schematic representation of all predicted splice variants of the mRNAs produced by pBearly are shown. SD, 5′ splice site; SA, 3′ splice site. (B) Northern blots on total or cytoplasmic RNA extracted from HeLa cells transfected with pBearly. Filters were hybridized with the CMV, E1, and E4 probes (61) (Fig. 3A). The E1 mRNA and the mRNAs spliced from 880 to 2709 or 3358 are indicated on the right. (C) RT-PCR with primers 757S (60) and E4A (61) on total RNA extracted from HeLa cells transfected with pBearly or pBEL (61). The position of the mRNA spliced from 880 to 3358 is indicated. Lane 1, RT-PCR on sample lacking RNA. The right-hand gel shows the same RNA samples as in the left-hand gel analyzed by RT-PCR with the same primer pair in the absence of RT.
FIG. 4.
FIG. 4.
(A) Schematic drawing of two deletions in the early 3′ UTR introduced in pBearly to create plasmids pBearlyDU and pBearlyDUTR. Nucleotide numbers refer to the HPV-16R sequence (6). The U-rich region in the 3′ end of the early 3′ UTR is indicated. (B) Northern blot on total RNA extracted from HeLa cells transfected with the indicated plasmids. Filters were hybridized to either CMV probe or E4 probe (61) (for locations of probes, see Fig. 3A). (C) Quantitation of the Northern blot probed with the CMV probe in a phosphorimager.
FIG. 5.
FIG. 5.
(A) Schematic drawing of the deletions in the early 3′ UTR introduced in pBEL (61) to create the indicated plasmids. The pBEL-derived plasmids contain a deletion in the late UTR that removes previously identified negative elements (25, 26) in order to increase the chances of obtaining detectable levels of late mRNAs (61). The same deletions were introduced into pBELM, in which the splicing silencer in the L1 coding region had been inactivated (61). The AAUAAA-to-ACGCGU mutation of the pAE in pBELDP is indicated. Nucleotide numbers refer to the HPV-16R sequence (6). The location of the L1 probe in pBEL is indicated. The locations of the RT-PCR primers 757S (61) and E5stops (Table 1) are shown. The two late mRNAs encoding L2 and L1 are displayed, and the location of the previously identified splicing silencer in L1 is indicated. (B and C) Northern blots on total or cytoplasmic RNA extracted from HeLa cells transfected with the indicated plasmids. Filters were hybridized to the L1 probe (61) (Fig. 5A). The L2/L1 and the L1 mRNAs are indicated. The fold induction of late mRNAs were calculated on the data obtained with pBELM-derived plasmids. The levels of late mRNAs were quantified in a phosphorimager and were divided with the late mRNA levels detected in the pBELM lane to yield fold induction. (D) Northern blots on total RNA extracted from HeLa cells transfected with the indicated plasmids. Filters were hybridized to the CMV probe or the E4 probe (61) (for location of probes, see Fig. 3A). The early mRNAs, the L2/L1 and the L1 mRNAs, are indicated.
FIG. 5.
FIG. 5.
(A) Schematic drawing of the deletions in the early 3′ UTR introduced in pBEL (61) to create the indicated plasmids. The pBEL-derived plasmids contain a deletion in the late UTR that removes previously identified negative elements (25, 26) in order to increase the chances of obtaining detectable levels of late mRNAs (61). The same deletions were introduced into pBELM, in which the splicing silencer in the L1 coding region had been inactivated (61). The AAUAAA-to-ACGCGU mutation of the pAE in pBELDP is indicated. Nucleotide numbers refer to the HPV-16R sequence (6). The location of the L1 probe in pBEL is indicated. The locations of the RT-PCR primers 757S (61) and E5stops (Table 1) are shown. The two late mRNAs encoding L2 and L1 are displayed, and the location of the previously identified splicing silencer in L1 is indicated. (B and C) Northern blots on total or cytoplasmic RNA extracted from HeLa cells transfected with the indicated plasmids. Filters were hybridized to the L1 probe (61) (Fig. 5A). The L2/L1 and the L1 mRNAs are indicated. The fold induction of late mRNAs were calculated on the data obtained with pBELM-derived plasmids. The levels of late mRNAs were quantified in a phosphorimager and were divided with the late mRNA levels detected in the pBELM lane to yield fold induction. (D) Northern blots on total RNA extracted from HeLa cells transfected with the indicated plasmids. Filters were hybridized to the CMV probe or the E4 probe (61) (for location of probes, see Fig. 3A). The early mRNAs, the L2/L1 and the L1 mRNAs, are indicated.
FIG. 6.
FIG. 6.
(A) 3′ RACE on total RNA from HeLa cells transfected with pBELDP and pBEL (Fig. 5A). The primer used for pBELDP in 3′ RACE was 757S (61) (for the location of the primer, see Fig. 5A) and oligo(dTGC) (31). The primers used for pBEL in 3′ RACE were E5stops (Table 1) and oligo(dTGC) (31). The arrow indicates the major amplification product which was cloned and sequenced. The right-hand gel shows the same RNA samples as those in the left-hand gel analyzed by RT-PCR with the same primer pair in the absence of RT. (B) The major cleavage site identified by 3′ RACE on RNA from pBELDP-transfected cells is indicated. (C) Positions of multiple minor cleavage sites identified by 3′ RACE on RNA from pBELDP-transfected HeLa cells. (D) The cleavage sites used by the wild-type pAE. Cleavage sites were identified by 3′ RACE on RNA from pBEL-transfected HeLa cells. Numbers refer to nucleotide positions in the HPV-16R sequence (6). Two or three equally plausible cleavage sites are given when the poly(A) tail is preceded by one or two As.
FIG. 7.
FIG. 7.
(A) Schematic drawing of the in vitro-synthesized RNAs. The sequences in plasmids TUP, TDU, and TDP represent sequences from the HPV-16 early 3′ UTR. Numbers refer to the nucleotide positions in HPV-16R (6). The AAUAAA-to-ACGCGU mutation of the pAE in pTDP is indicated. The T2B2 RNA contains four HuR binding sites (underlined) (43), the T2C1 RNA contains six hnRNP C1/C2 binding sites (underlined) (44, 45), and the TCstF64 RNA contains two CstF-64 binding sites. T7, bacteriophage T7 RNA polymerase promoter. (B) Left panel, UV cross-linking of HeLa nuclear extract to the indicated RNA probes. The L1 RNA is a non-U-rich sequence derived from the HPV-16 L1 coding region and served as control. Arrows indicate four proteins, named A, B, C, and D, detected with the TUP probe but not with the TDU probe. For the right panel, radiolabeled TUP probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled TUP or TDU competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. (C) UV cross-linking of HeLa nuclear extract to the indicated RNA probes. HnRNP C is indicated. (D) Radiolabeled CstF64 probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled TUP, TDU, or TCstF64 competitor RNA as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. CstF-64 is indicated. (E) Radiolabeled TUP probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled T2C1 or T2B2 competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. hnRNP C is indicated. (F) Radiolabeled TUP probe was UV cross-linked to his-tagged hnRNP C1 in the absence or presence of threefold serially diluted unlabeled TUP or TDU competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. His-hnRNP C1 is indicated. (G) UV cross-linking of GST-PTB to the indicated RNA probes.
FIG. 7.
FIG. 7.
(A) Schematic drawing of the in vitro-synthesized RNAs. The sequences in plasmids TUP, TDU, and TDP represent sequences from the HPV-16 early 3′ UTR. Numbers refer to the nucleotide positions in HPV-16R (6). The AAUAAA-to-ACGCGU mutation of the pAE in pTDP is indicated. The T2B2 RNA contains four HuR binding sites (underlined) (43), the T2C1 RNA contains six hnRNP C1/C2 binding sites (underlined) (44, 45), and the TCstF64 RNA contains two CstF-64 binding sites. T7, bacteriophage T7 RNA polymerase promoter. (B) Left panel, UV cross-linking of HeLa nuclear extract to the indicated RNA probes. The L1 RNA is a non-U-rich sequence derived from the HPV-16 L1 coding region and served as control. Arrows indicate four proteins, named A, B, C, and D, detected with the TUP probe but not with the TDU probe. For the right panel, radiolabeled TUP probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled TUP or TDU competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. (C) UV cross-linking of HeLa nuclear extract to the indicated RNA probes. HnRNP C is indicated. (D) Radiolabeled CstF64 probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled TUP, TDU, or TCstF64 competitor RNA as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. CstF-64 is indicated. (E) Radiolabeled TUP probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled T2C1 or T2B2 competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. hnRNP C is indicated. (F) Radiolabeled TUP probe was UV cross-linked to his-tagged hnRNP C1 in the absence or presence of threefold serially diluted unlabeled TUP or TDU competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. His-hnRNP C1 is indicated. (G) UV cross-linking of GST-PTB to the indicated RNA probes.
FIG. 7.
FIG. 7.
(A) Schematic drawing of the in vitro-synthesized RNAs. The sequences in plasmids TUP, TDU, and TDP represent sequences from the HPV-16 early 3′ UTR. Numbers refer to the nucleotide positions in HPV-16R (6). The AAUAAA-to-ACGCGU mutation of the pAE in pTDP is indicated. The T2B2 RNA contains four HuR binding sites (underlined) (43), the T2C1 RNA contains six hnRNP C1/C2 binding sites (underlined) (44, 45), and the TCstF64 RNA contains two CstF-64 binding sites. T7, bacteriophage T7 RNA polymerase promoter. (B) Left panel, UV cross-linking of HeLa nuclear extract to the indicated RNA probes. The L1 RNA is a non-U-rich sequence derived from the HPV-16 L1 coding region and served as control. Arrows indicate four proteins, named A, B, C, and D, detected with the TUP probe but not with the TDU probe. For the right panel, radiolabeled TUP probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled TUP or TDU competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. (C) UV cross-linking of HeLa nuclear extract to the indicated RNA probes. HnRNP C is indicated. (D) Radiolabeled CstF64 probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled TUP, TDU, or TCstF64 competitor RNA as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. CstF-64 is indicated. (E) Radiolabeled TUP probe was UV cross-linked to HeLa nuclear extract in the absence or presence of threefold serially diluted unlabeled T2C1 or T2B2 competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. hnRNP C is indicated. (F) Radiolabeled TUP probe was UV cross-linked to his-tagged hnRNP C1 in the absence or presence of threefold serially diluted unlabeled TUP or TDU competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold. His-hnRNP C1 is indicated. (G) UV cross-linking of GST-PTB to the indicated RNA probes.
FIG. 8.
FIG. 8.
(A) UV cross-linking of recombinant his-tagged hFip1 to the RNA probes TUP and TDU (see Fig. 7A). (B) Radiolabeled TUP probe was UV cross-linked in the absence or presence of threefold serially diluted unlabeled competitor RNAs as indicated. The molar excess of the competitor over probe was 1-, 3-, 9-, 27-, and 81-fold.
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References

    1. Andrew, E. M., and D. DiMaio. 1993. Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed cells. J. Virol. 67:7705-7710. - PMC - PubMed
    1. Baker, C., and C. Calef. 1997. Maps of papillomavirus mRNA transcripts, p. 3-10. In S. R. Billakanti, C. E. Calef, A. D. Farmer, A. L. Halpern, and G. L. Myers (ed.), Human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences. Theoretical biology and biophysics, Los Alamos National Laboratory, Los Alamos, N.M.
    1. Baker, C. C. 1997. Posttranscriptional regulation of papillomavirus gene expression, p. 11-16. In S. R. Billakanti, C. E. Calef, A. D. Farmer, A. L. Halpern, and G. L. Myers (ed.), Human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences. Theoretical biology and biophysics, Los Alamos National Laboratory, Los Alamos, N.M.
    1. Baker, C. C., and J. S. Noe. 1989. Transcriptional termination between bovine papillomavirus type 1 (BPV-1) early and late polyadenylation sites blocks late transcription in BPV-1-transformed cells. J. Virol. 63:3529-3534. - PMC - PubMed
    1. Barksdale, S. K., and C. C. Baker. 1995. The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5′ splice site in a 3′ untranslated region. Mol. Cell Biol. 15:2962-2971. - PMC - PubMed

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