Nucleic Acids Research, 2024, 52, 316–336
https://doi.org/10.1093/nar/gkad1099
Advance access publication date: 22 November 2023
Molecular Biology

A novel HPV16 splicing enhancer critical for viral oncogene
expression and cell immortalization
Johanna Jönsson1 , Lianqing Wang1 ,2 , Naoko Kajitani
12†* and Stefan Schwartz

1 ,†Department of Medical Biochemistry and Microbiology, Uppsala University, BMC-B9, 751 23 Uppsala, Sweden
Center of Translational Medicine, Zibo Central Hospital, 255036 Zibo, China
To whom correspondence should be addressed. Tel: +46 18 4714444; Email: naoko.kajitani@imbim.uu.se
The last two authors contributed equally to this work.

Abstract
High-risk carcinogenic human papillomaviruses (HPVs), e.g. HPV16, express the E6 and E7 oncogenes from two mRNAs that are generated ina mutually exclusive manner by splicing. The HPV16 E7 mRNA, also known as the E6*I/E7 mRNA, is produced by splicing between splice sites
SD226 and SA409, while E6 mRNAs retain the intron between these splice sites. We show that splicing between HPV16 splice sites SD226 andSA409 is controlled by a splicing enhancer consisting of a perfect repeat of an adenosine-rich, 11 nucleotide sequence: AAAAGCAAAGA. Twonucleotide substitutions in both 11 nucleotide sequences specifically inhibited production of the spliced E6*I/E7 mRNA. As a result, productionof E7 protein was reduced and the ability of HPV16 to immortalize human primary keratinocytes was abolished. The splicing-enhancing effectwas mediated by the cellular TRAP150/THRAP3 protein that also enhanced splicing of other high-risk HPV E6*I/E7 mRNAs, but had no effecton low-risk HPV mRNAs. In summary, we have identified a novel splicing enhancer in the E6 coding region that is specific for high-risk HPVs andthat is critically linked to HPV16 carcinogenic properties.

Graphical abstract

Introduction
The International Agency for Research on Cancer (IARC) has
deemed 15 human papillomaviruses (HPVs) carcinogenic tohumans (1,2). HPV16 is the most common carcinogenic, highrisk HPV (HR-HPV) (https://pave.niaid.nih.gov/). In contrast,
low-risk HPVs (LR-HPVs) are not associated with cancer.
During the HPV life cycle, E6 and E7 proteins are requiredfor generation of an intracellular environment that is permissive for HPV DNA replication (3–6). The E7 protein promotes
cell proliferation, primarily by interacting with members of

the pocket protein family including Retinoblastoma protein(pRB) in a manner that liberates transcription factor E2F (7).
The E6 protein on the other hand binds to p53 via E6-AP
and causes ubiquitin-mediated degradation of p53, thereby
preventing apoptosis of the HPV-infected cell (8). HR-HPV
E6 and E7 are notorious in the sense that they are responsible for induction and maintenance of cancer caused by HRHPVs and are defined as viral oncogenes (9–13). HR- and
LR-HPVs differ in the mode of E6 and E7 gene expression.
While LR-HPVs utilize two independent promoters to pro-

Received: March 24, 2023. Revised: October 18, 2023. Editorial Decision: October 28, 2023. Accepted: November 3, 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided theoriginal work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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1 , ,†

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Materials and methods
Plasmids
The following plasmids have been described previously:
pC97ELsL (34), pHPV16AN (35), pX1200 (25), pX1060(25), pX960 (25), pX856F (25), pX765 (28), pX656 (27),pX556 (25), pX478 (27), pCL086 (36), phnRNPK (37),pRBM15b (28), phnRNPM (28), pBCLAF1 (30), pRSVneo(34), pCAGGS-NLS-Cre (38) and pTRAP150 (30).
To construct subgenomic HPV16 plasmids pX550, pX525,
pX502 and pX438, HPV16 sequences were first amplified
by polymerase chain reaction (PCR) from pC97ELsL withprimers B97S and X556A, X550A, X525A, X502A and
X438A, respectively. To generate plasmids pX478AW+M,
pX478AM+W and pX478AM+M, HPV16 sequences werefirst PCR-amplified from pC97ELsL with primers B97S and
X478AWT+mut, X478Amut+WT and X478Amut+mut, respectively. To generate plasmids pX478AM1, pX478AM2,
pX478AM3, pX478AM4 and pX478AM5, HPV16 sequences were first PCR-amplified from pC97ELsL withprimers B97S and X478AM1, X478AM2, X478AM3,
X478AM4 and X478AM5, respectively. The PCR fragments
were digested using Fast Digest enzymes Ptel and Xhol
(Thermo Scientific) and cloned into the pCL086 vector digested with the same restriction enzymes. To generate plasmids pX478sM1, pX478sM4 and pX478midM, Phusion sitedirected mutagenesis (Thermo Fisher Scientific) was usedwith 5 -phosphorylated primers, P-pX478M-Rv and P-sM1Fw, PsM4-Fw and P-middleM-Fw, respectively. To generate
plasmids pX478 SA409OPT and pX478 SA409OPT+M1M4
from plasmid templates pX478 and pX478M1M4, respectively, Phusion site-directed mutagenesis was used with 5 phosphorylated primers P-SA409OPT-Rv and P-SA409OPTFw. Sequences of PCR primers used for plasmid constructions
are listed in Supplementary Table S1.
To generate plasmids with mutations at the identified enhancer in pC97ELsL, a two-step PCR mutagenesis was performed. In the first two PCRs, one outer primer (pC97Sall
or B97+HPV16 Sbfl) and one inner primer (478M1M4AS
PCRmut or 478M1M4S PCRmut) were used in each reaction, with inner primers containing the mutations. Primers
are listed in Supplementary Table S1. This first PCR resulted
in two PCR fragments with overlapping fragments, which
could base-pair. These two PCR fragments were diluted 1:100
and a second PCR using the two outer primers was performed. This resulted in a longer PCR fragment containing
mutations in the center of the fragment. The PCR fragments
were digested with Fast Digest enzymes Sall and Sbfl (Thermo
Scientific), and cloned into the pC97ELsL plasmid digested
with the same enzymes, resulting in pC97M1M4. To generate pC97ELsL M2, the DNA fragment encoding the HPV16
genomic sequence nucleotides 104–881 was purchased from
Eurofins Genomics Llc. The mutant HPV16 sequence wasamplified by PCR using primers pC97SalI and B97+HPV16
SbfI followed by subcloning into SalI and SbfI sites of
pC97ELsL.
The mutations of the enhancer in pHPV16AN wereconstructed by ordering plasmids containing sequences between the two unique restriction sites Bglll and Sbfl in the
pHPV16AN plasmid. The Bglll–Sbfl fragment encompassed
the splicing enhancer and contained the four nucleotide substitutions representing the M1 and M4 mutations. The mutant
fragments were ordered from Eurofins Genomics LIc. These

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duce E6 and E7 mRNAs, the HR-HPVs produce E6 and E7from two alternatively spliced mRNAs generated in a mutually exclusive fashion from the same pre-mRNA (Figure 1A,B) (14). The E6 mRNA is generated by retention of an intron that encodes E6, while the E7 mRNA is generated bysplicing that removes the upstream E6-encoding intron (Figure 1A, B) (https://pave.niaid.nih.gov/). This spliced mRNA
named E6*I/E7 mRNA is diagnostic for HR-HPV types andis the major and most abundant E7-producing mRNA (Figure
1A, B) (15). Indeed, the levels of the E6*I/E7 mRNA gradually
increase during progression of HPV16 infections to cervicalcancer (16–18). Furthermore, E6 and E7 mRNAs are affectedby chemotherapeutic drugs (19), suggesting that they may betargeted for therapy to HPV infections, including malignancies caused by HPV .
The HPV16 splice sites used for production of the spliced
E6*I/E7 mRNA are named SD226 and SA409 in HPV16 (Figure 1A, B) (20–24). Since E6 and E7 are both essential proteinsin the HPV life cycle and are both required for initiation andmaintenance of cancer, it is of paramount importance to maintain an optimal ratio between intron retention and splicing
between SD226 and SA409 to produce sufficient quantities
of both E6 and E7. Therefore, splicing between SD226 andSA409 must be strictly controlled to avoid over- or undersplicing. SA409 has an interrupted polypyrimidine tract (underlined): CCGUUGUGUGAUUUGUUAAUUAG/GUGUA. This
indicates that SA409 is a suboptimal splice acceptor that
may require an additional mechanism to produce E6*I/E7
mRNAs. RNA-binding proteins that inhibit splicing between
SD226 and SA409 and cause intron retention have been identified, i.e. hnRNP A1 (25,26), hnRNP D (27) and hnRNP G(28). In sharp contrast, the cellular trans-acting factor(s) and
HPV RNA elements that are required for splicing between
SD226 and SA409 to produce the major E6*I/E7 oncogenemRNA remain unidentified.
In this work, we sought to define the molecular mechanism for production of the HPV16 oncogenic E6*I/E7
mRNA. We identified a splicing enhancer element named
ENH-1 that spans 32 nucleotides, and is located 35 nucleotides downstream of SA409. It consists of an adenosinerich 11 nucleotide perfect repeat with a 10 nucleotide spacer.
Introducing just twonucleotide substitutions in ENH-1 resulted in the abrogation of the splicing between SD226 andSA409. Additionally, we identified TRAP150/THRAP3 (thyroid hormone receptor-associated protein complex 150 kDa
component/thyroid hormone receptor-associated protein 3)
as a trans-acting factor that interacts with the ENH-1 sequence and mediates the splicing between SD226 and SA409.
TRAP150 has previously been shown to regulate cellular
mRNA alternative splicing and spliced mRNA nuclear export (29–33), but evidence has been lacking regarding which
cis-RNA element TRAP150 interacts with to regulate mRNAalternative splicing. Here we show that TRAP150 interacts
with HPV16 ENH-1 in in vitro RNA oligo pull-down assay
and in living cells, and recruits U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa, U2AF65 to ENH-1.
The effect of TRAP150 on the induction of E6*I/E7 mRNA
splicing was also observed in other HR-HPVs, but not in
LR-HPVs. Our results allow us to propose a model in which
HPV16 ENH-1 and TRAP150 are critical for regulating theexpression of the viral E6 and E7 oncoproteins at the level of
RNA processing and contribute to the carcinogenic propertiesof carcinogenic HPVs.

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Cells
HeLa, 293T, SiHa and C33A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cytiva, previously
Hyclone) supplemented with 10% bovine calf serum (BCS;
Hyclone) and 1% penicillin–streptomycin (Gibco). The normal neonatal human foreskin primary keratinocytes [nHFKs(HEKn, PCS-200–010)] were purchased from ATCC andgrown in EpiLife medium (Gibco) with 1% human keratinocyte growth supplements (HKGS, Gibco) and 0.2%
gentamicin/amphotericin (Gibco). This medium was also used
for propagation of the immortalized human keratinocyte cellline JJM9721 established here by stable transfection of HFKs
with pC97ELsL. The HeLa, SiHa and JJM9721 cell lines usedhere have been subjected to the Cell Line Authentication Test(Eurofins Genomics).

Transfections
Transfection of HeLa cells was performed using Turbofect
according to the manufacturer’s instructions (Thermo Scientific). Transfections of 293T, SiHa and C33A cells were carried out using Fugene6 (Promega). nHFKs (PCS-200–010,
ATCC) were transfected using Xfect (TaKaRa Bio). Cell culture medium was changed 6 h post-transfection. All plasmids were transfected in a minimum of three independent
experiments.

siRNA and siRNA transfections
SiHa and JJM9721 cells were transfected using ONTARGET SMARTpool small interfering RNA (siRNA)
from Dharmacon using Dharmafect1 (Dharmacon), according to the manufacturer’s protocol. The scrambled control(si-scr) was siGENOME Control pool non-targeting #2(D-001206–14–20, Dharmacon™). siRNA to TRAP150 was
ON-TARGETplus Human THRAP3 (GeneID: 9967) siRNASMARTpool (L-019907–00–0020) and siRNA against
BCLAF1 was ON-TARGETplus Human BCLAF1 (GeneID:9774) siRNA SMARTpool (L-020734–00–0005).

RNA extraction and RT–PCR
Total RNA of HeLa was extracted using TriReagent (Sigma)
and the Direct-zol RNA Miniprep kit (Zymogen Research),according to the manufacturer’s instructions. Total RNA ofother cell lines was extracted using TriReagent (Sigma) and
chloroform (Sigma), according to the manufacturer’s instructions. DNase I (Sigma) treatment was performed in solution at
37◦ C for 1 h, followed by phenol–chloroform–isoamyl alcohol (IAA) extraction and ethanol precipitation. A 1 μg aliquotof total RNA was reverse transcribed at 37◦ C for 50 min in
a 20 μl reaction using the M-MLV reverse transcriptase (Invitrogen) and random hexamer primers (Thermo Scientific).
A 1 μl aliquot of the newly synthesized cDNA was used ina PCR amplification assay. Reverse transcription–PCR (RT–
PCR) products were pre-stained with GelRed and subjectedto DNA agarose electrophoresis. RT–PCR primers are listed
in Supplementary Table S2.

RNA fractionation
Nuclear and cytoplasmic fractions were obtained using the
NE-PERTM Nuclear and Cytoplasmic Extraction Reagent(Thermo Fisher Scientific) according to the manufacturer’s
protocol. Nuclear and cytoplasmic RNA were extracted by
TriReagent and chloroform as described for RNA extraction,
followed by DNase I treatment and cDNA synthesis.

Real-time quantitative PCR (qPCR)
A 1 μl aliquot of cDNA was used in the qPCR analysis using StepOnePlus (Applied Biosystems) with the SsoAdvanced SYBR Green Supermix (Bio-Rad) in line with the manufacturer’s instructions. To quantify human telomerase reverse transcriptase (hTERT) mRNA levels, primers hTERT-F
and hTERT-R were used. TRAP150 mRNA levels were determined using primers TRAP150QFw and TRAP150QRv.
BCLAF1 mRNA levels were determined using primers
BCLAF1qFw and BCLAF1qRv. Intron retained (IR) E6 andspliced 226∧ 409(E6*I) mRNA levels were determined using
primers E6QF and 234AS or E6*I-F and E6*I-R, respectively.

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plasmids were digested with BgllI and Sbfl (Thermo Scientific)
to release the mutant DNA sequence that was subsequently
inserted into pHPV16AN plasmid digested with the same enzymes, resulting in pHPV16ANM1M4.
To construct pC16HAE6E7F [HPV16 E6/E7 gene
(GeneID: K02718) expressing a plasmid in which E6 isfused with an HA-tag at the 5 end and E7 with a FLAG-tag
at the 3 end], HPV16 HA-E6-E7-FLAG sequences were
PCR-amplified with BHAE6S and XhoI-FLAG-E7-R, followed by digestion with BssHII and XhoI, and inserted into
pCL086. HA-E6-E7-FLAG-encoding plasmids representing
different HPV types were constructed by PCR amplificationof the E6E7-encoding region of various HPV types. HPV6b,
HPV11, HPV31, HPV18 and HPV45 sequences were PCRamplified with the following primer pairs: ClaI-HA-6b-f and
Xho-flag-6b-r, ClaI-HA-11-f and Xho-flag-11-r, ClaI-HA-31-f
and Xho-flag-31-r, ClaI-HA-18-f and Xho-flag-18-r, and ClaIHA-45-f and Xho-flag-45-r. The primer sequences are listed in
Supplementary Table S1. PCR fragments were digested with
ClaI and XhoI, and inserted into plasmid pCL0806, resulting
in plasmids pC6bHAE6E7F, pC11HAE6E7F, pC31HAE6E7F,
pC18HAE6E7F and pC45HAE6E7F.
The chimeric HPV16 E6- and E7-encoding plasmids inwhich the splicing enhancer between HPV16 nucleotide
positions 444 and 475 in plasmid pC16HAE6E7F was
replaced with the corresponding sequence from HPV6b,
HPV31, HPV18 or HPV45, were generated by the Phusion site-directed mutagenesis kit (Thermo Scientific) using the following pairs of 5 -phosphorylated primers: PEnh1.16to6b-F and P-Enh1.16to6b-R, P-Enh1.16to31-F and
P-Enh1.16to31-R, P-Enh1.16to18-F and P-Enh1.16to18-R,
or P-Enh1.16to45-F and P-Enh1.16to45-R. The sequencesof the primers are listed in Supplementary Table S1. Thesemutagenesis reactions resulted in plasmids pC.Enh1.16to6b,
pC.Enh1.16to31, pC.Enh1.16to18 and pC.Enh1.16to45. To
generate pC.Enh1.16to6b-17A#2, -19A#2 and -21A from
pC.Enh1.16to6b, Phusion site-directed mutagenesis was performed using 5 -phosphorylated primers, P-Enh1.16to6bR
and P-Enh.1.6b-17#2-Fw, -19A#2-Fw or -21A-Fw, respectively. To generate pC.Enh1.16to6b-17A#3, -19A#3 and 21A#2, Phusion site-directed mutagenesis was performed using P-Enh1.16to6b-R2 and P-Enh.1.6b-17#2-Fw, -19A#2-Fw
or -21A-Fw, respectively.
All plasmids used in this study have been sequenced to confirm their identity and are available upon request.

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luminescence substrate (Thermo Scientific) and detection in
ChemiDoc MP (Bio-Rad) or with IR-conjugated secondary
antibodies followed by detection in Odyssey CLx Imager (LICOR). Antibodies are listed in Supplementary Table S4.

RNA oligo-mediated protein pull-down assay

RNA–protein immunoprecipitation (RIP) assay

HeLa nuclear extracts were prepared according to a previously described method (39). To extract cytoplasmic proteins,
cells were lysed using buffer I [10 mM HEPES pH 7.9, 10
mM KCl, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.2%
NP-40 and protease inhibitor] followed by centrifugation. The
supernatants were stored as cytoplasmic extracts, while thenuclear pellets were resuspended in buffer II (10 mM HEPES
pH 7.9, 400 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM
DTT and protease inhibitor) to extract nuclear proteins. For
RNA oligo pull-down assay with TRAP150-depleted lysates,
cells were transfected with siRNAs targeting TRAP150 or
BCLAF1 or with scrambled control RNA followed by preparation of nuclear extracts at 72 h post-transfection. In somecases, the nuclear extracts were further purified on HiTrap Qcolumns. Flow-through fractions were collected, while proteins in the column were subjected to step-wise elution with
50 mM, 200 mM or 1 M KCl. A 60 μg aliquot of nuclear extract was mixed with 1 mg (100 μl) of magnetic
streptavidin-coated beads (Dynabeads M-280 Streptavidin,
Invitrogen) pre-incubated with 200 pmol of single-stranded
biotin-labeled RNA oligonucleotides in Binding buffer (10mM HEPES pH 7.9, 2.5 mM MgCl2 , 0.5% Triton X-100,
150 mM NaCl and 1 mM DTT). The sequences of the RNA
oligos are listed in Supplementary Table S3. Incubation withRNA oligo-covered beads was performed at room temperature for 40 min followed by washing 10 times with 500 μlof ice-cold binding buffer (10 mM HEPES pH 7.9, 2.5 mM
MgCl2 , 0.5% Triton X-100, 150 mM NaCl, 1 mM DTT).
Proteins were eluted using 2× Laemlli buffer and subjected
to sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by either western blotting or silver
staining.

Cells were resuspended in 1 ml of non-denaturing cell lysis
buffer (20 mM Tris–HCl pH 8, 137 mM NaCl, 1% IGEPALCA 630, 2 mM EDTA, 2× protease inhibitor, RNase inhibitor)and passed through a 19-gauge needle to sheer the DNA. The
samples were incubated with over-end rotation at 4◦ C for 30
min followed by centrifugation to remove the cell debris. Five
percent of each lysate was stored for later to be used as input samples. In the immunoprecipitation, 50 μl of protein G
Dynabeads (Thermo Scientific) and 1 μg of antibody were incubated with 500 μl of pre-cleared cell lysate with over-end rotation at 4◦ C for 2 h. Antibodies used are listed in Supplementary Table S4. Following incubation, the beads were washedeight times using wash buffer (20 mM Tris–HCl pH 8, 137
mM NaCl, 1% IGEPAL CA 630, 2 mM EDTA) and RNA wasextracted using using Trizol-chloroform, followed by DNase I
treatment (Sigma), purified by phenol–chloroform–IAA andprecipitated by ethanol. The precipitated RNA was resuspended in 22 μl of ddH2 O and 10 μl was reverse transcribedusing M-MLV reverse transcriptase (Invitrogen) and random
hexamer primers (Thermo Scientific) according to the manufacturer’s protocol. A 1 μl aliquot of cDNA was subjected to
PCR amplification using the primers indicated in each figure.

Preparation of proteins for mass spectrometryProteins pulled down using RNA-mediated protein pull-down
assay were separated by SDS–PAGE and stained using silver
stain (SilverQuest™ staining Kit, Invitrogen). Bands appearing stronger in pull-downs with wild-type HPV16 RNA oligos compared with protein bands observed after pull-downwith mutant HPV16 RNA oligos were excised from the gels.
The bands were subjected to proteolytic cleavage and liquid
chromatography–mass spectrometry (LC-MS) analysis at the
SCIBLU Proteomics Resource Center at Lund University.

Preparation of proteins and western blotting
Total cellular protein was extracted by resuspension of cells inradioimmunoprecipitation assay (RIPA) buffer (50 mM Tris
pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2× protease inhibitor and 1 mM DTT)
followed by over-end rotation at 4◦ C for 30 min and centrifugation to remove cell debris. To perform western blotting, protein extracts were subjected to SDS–PAGE and western blotting as described previously (39). The filters were stained withprimary antibodies and subsequently with horseradish peroxidase (HRP)-conjugated secondary antibodies followed bysignal development with the Super Signal West Femto chemi-

Co-immunoprecipitation (co-IP)
Transfected 293T cells were lysed using 1 ml of nondenaturing lysis buffer (20 mM Tris–HCl pH 8, 137 mM
NaCl, 1% NP-40, 2 mM EDTA, 2× protease inhibitor) for
20 min with over-end rotation at 4◦ C followed by centrifugation to remove the cell debris. When RNase treatment was
indicated, RNase A (final 5 μg/ml, Thermo Scientific) was
added followed by incubation for 15 min at room temperature
prior to centrifugation. In parallel, Protein G Dynabeads were
washed twice followed by incubation with 1 μg of antibody
and three washing steps. The cell lysate was pre-cleared by incubation with Protein G Dynabeads without antibody for 30
min at 4◦ C with over-end rotation; 5% of the cell extract wasstored as input sample. The remaining cell lysate was divided
into control, to which IgG was added, and pull-down sample, to which either anti-FLAG or anti-U2AF65 antibody wasadded and incubated overnight, after which they were washed
six times prior to SDS–PAGE and western blotting.

Induction of immortalization of primary humankeratinocytes
Immortalization of nHFKs was assayed by transfection of
HPV16 subgenomic plasmids followed by selection of cells
resistant to calcium- and serum-dependent differentiation, as
described previously (40). Briefly, subconfluent nHFKs growing on 100 mm plates were either untransfected or transfectedwith pC97ELsL or pC97M1M4 using Xfect transfection
reagent according to the manufacturer’s protocol (as briefly
described above). At 24 h after transfection, cells were transferred to 150 mm plates and grown in EpiLife medium. After cells reached confluency, the cells were selected in DMEM
supplemented with BCS (10%) and penicillin–streptomycin(1%) to induce differentiation. The cells were grown in the

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The locations of primers are indicated in Figure 1B. The results
were normalized to glyceraldehyde phosphate dehydrogenase(GAPDH) mRNA levels determined using primers gapdhs andgapdha. qPCR primers are listed in Supplementary Table S2.

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Water-soluble tetrasodium (WST)-1 cell viability
assay
SiHa cells were seeded in 96-well plates at a density of 4 ×10 ,8 × 103 or 16 × 103 cells/well. Cells were transfected thefollowing day, either with control vector (pUC19) or with increasing concentrations of pTRAP150. Cell viability was measured at 24 or 48 h post-transfection. Cell culture medium was
replaced with fresh growth medium at 2 h prior to the WST1 assay. WST-1 assay reagent (Roche) was added to a final
concentration of 10% of the cell culture medium followed byincubation for 0.5–4 h at 37◦ C in 5% CO2 . The absorbance ofthe formazan product was measured at 450 nm using a TriStar
Plate Reader (Berthold Tech).
3

UV-cross-linking and RNA oligo-mediated protein
pull-down assay
The method for RNA oligo-mediated protein pull-down assay
was modified as the RNA oligo and nuclear extract proteins
were UV-cross-linked prior to washing and elution steps. A 60
μg aliquot of HeLa nuclear extract was incubated with 200
pmol of biotinylated single-stranded RNA oligonucleotide in
Buffer D (final concentration: 15 mM HEPES pH 7.6, 80 mM
KCl, 2.4 mM MgCl2 , 0.15 mM EDTA, 8.5% glycerol and 0.2
mM DTT) for 10 min at room temperature. The mixtures wereUV-cross-linked with 800 mJ/cm2 at 254 nm. The UV-crosslinked mixtures were then incubated with 1 mg of Dynabeads
M280-Streptavidin in Binding buffer-2 (10 mM HEPES pH
7.9, 2.5 mM MgCl2 , 4 mM KCl, 0.5 mM EGTA, 0.5 mM
EDTA, 0.5% Triton X-100, 3% glycerol, 150 mM NaCl and
1 mM DTT) for 30 min at room temperature. Following incubation, the beads were washed eight times using Binding
buffer with different concentration of sodium chloride (10mM HEPES pH 7.9, 2.5 mM MgCl2 , 0.5% Triton X-100,
150/300/600/1000 mM NaCl as indicated in the figures and
1 mM DTT). After washings, the proteins were eluted using
2× Laemmli buffer and subjected to SDS–PAGE and western
blotting. For RNA oligonucleotide sequences, see Supplementary Table S3.

Sequence alignment
Jalview version 2.11.2.5 was used for multiple sequence alignments.

Quantitation
Image Lab 6.0.1 software was used to determine band intensity on chemiluminescent western blot and RT-PCR images.
ImageJ 2.3.0 software was used to determine band intensity
on immunofluorescent western blot images.

Statistics and reproducibility
Each experiment was carried out at least in triplicate withreproducible results. Quantitative analysis and Student ttests (unpaired, two-tailed test) were performed using Prism
GraphPad Prism 9.

Results
Identification of a cis-regulatory RNA element(ENH-1) that is required for HPV16 226∧ 409 splicing
and production of the high-risk HPV-specificE6*I/E7 mRNA
The mRNAs encoding the E6 and E7 oncogenes of HRHPVs are produced in a mutually exclusive manner. An IR
E6 mRNA encodes the complete E6 coding sequence (CDS),
whereas the spliced E6*I/E7 mRNA encodes a truncated E6(E6*I) as well as the complete E7 CDS (Figure 1A, B). In
HPV16, the E6*I/E7 mRNA is produced by splicing between
the 5 -splice site SD226 and the 3 -splice site SA409, both situated within the E6 CDS (226∧ 409 splicing, hereafter) (Figure 1A, B; Supplementary Figure S1A–C). To identify cisRNA elements that are required for HPV16 226∧ 409 mRNA
splicing, a series of HPV16 deletion mutants with HPV16 sequences of varying length downstream of SA409 were constructed (Figure 1C) and transfected into the cervical cancer
cell line HeLa. The activation of 226∧ 409 splicing was monitored using RT-PCR with primers that amplify both IR E6
and 226∧ 409 cDNA of different amplicon sizes (Figure 1C).
In the smallest plasmid that ends at HPV16 nucleotide position 438, 226∧ 409 splicing was not observed (Figure 1D),
indicating that enhancer elements regulating splice acceptor
SA409 were located downstream of nucleotide 438. When
HPV16 sequences present in the plasmid were extended further with another 40 nucleotides to HPV16 nucleotide position 478, splicing to SA409 increased from 0% to 80% (Figure 1D, E), indicating that a strong splicing enhancer element

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selection medium for 8 weeks, at which time point selection
was terminated. The selected cells were resistant to inductionof keratinocyte differentiation and showed continuous proliferation. At that stage, only cells transfected with pC97ELsL
were proliferative and were transferred back into serum-free
EpiLife medium (= passage 15 at that stage). The cells weregrown and passaged alongside non-transfected nHFKs (= passage 4 at that stage) to obtain a population doubling curve todemonstrate immortalization of the transfected cells. The population doubling level (PDL) was calculated using the formula:
PDL = 3.32[log (total viable cells at harvest/total viable cells
at seed)]. In parallel, cells were harvested at extra passages
0, 2, 4 and 9 for the evaluation of hTERT expression as determined by RT-qPCR. The pC97ELsL-immortalized cell line
was named JJM9721.
Subconfluent nHFKs (passage 5) growing on 100 mm
plates were transfected with pUC (neomycin selection
control), pRSVneo, pHPV16AN, pHPV16ANM1M4,
pHPV16AN+ pCAGGS-nlscre (Cre recombinase) or
pHPV16ANM1M4+pCAGGS-nlscre (Cre recombinase),
respectively. At 24 h post-transfection, the cells were split
1:4 into a new plate to avoid high cell confluency, followedby neomycin selection with 40 μg/ml G418 for 3 days andin 10 μg/ml for an additional 3 days. After selection andexpansion, the cells were frozen as liquid nitrogen stocks withthe same cell number (passage 8 at that time). After thawing,cells were seeded on 6-well plates followed by recovery on
100 mm plates for cultivation and subjected to selection using
DMEM with 10% BCS as described above. After 11 weeks of
selection, colony formation caused by HPV16 plasmid transfection was documented by cell fixation and staining withcrystal violet solution [0.05% w/v Crystal Violet (Sigma),
1% formaldehyde, 1% methanol, phosphate-buffered saline(PBS)] for 20 min at room temperature. Transfected cells
frozen in liquid nitrogen were revived and passaged 0, 2
and 4 times, followed by RNA extraction and evaluation ofhTERT expression determined by RT-qPCR.

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Figure 1. Identification of a splicing enhancer in the HPV16 E6-coding region that is required for production of 226∧ 409-spliced E7 mRNA. (A) Schematic
representation of the HPV16 genome. The early genes E6, E7, E1, E2, E4 and E5 are depicted as white boxes and the late genes L2 and L1 as grayboxes. The early and late promoters P97 and P670, respectively, are indicated as arrows. pAE and pAL indicate early and late polyadenylation signals,
respectively. The splice donors are indicated with black ovals and the splice acceptors as white ovals. The number beneath each splice donor andacceptor indicates their nucleotide position in the HPV16 reference genome (GenBank ID: K02718.1). LCR, long control region. (B) Representative
alternatively spliced mRNA variants produced from the HPV16 genome are shown. The IR E6 and 226∧ 409-spliced HPV16 mRNAs are highlighted in red.
Arrows indicate HPV16-specific reverse transcription PCR (RT-PCR) and real time quantitative PCR (RT-qPCR) primers in this study. The right panel
describes the RNA isoform structures of the HPV16 IR E6 mRNA (upper image) and the 226∧ 409-spliced E6*I/E7 mRNA (lower image). The IR E6 mRNA
consists of the E6 cistron from HPV16 nucleotide number 104–559, a two nucleotide non-coding spacer region (nucleotides 560–561) and the E7 cistron(nucleotides 562–858) both with their own start and termination codons, followed by the 5 end of downstream E1 cistrons. The 226∧ 409-spliced mRNA
consists of the E6*I cistron including nucleotides 104–226 spliced to nucleotides 409–417, a 144 nucleotide non-coding spacer region (nucleotides
418–561) and the E7 cistron (nucleotides 562–858). 226∧ 409 splicing results in an out-of -frame fusion of the E6 coding region of the first exon to thesecond exon. (C) Schematic representation of the HPV16 subgenomic plasmid pX1200 and an overview of HPV16 sequences present in the various
deletion mutant plasmids. The E6, E7 and part of the E1 gene are depicted as white boxes. The splice donors are indicated with black ovals and thesplice acceptors as white ovals. All plasmids start at nucleotide position 97 of the HPV16 genome but end at various positions indicated in the plasmid
names to the left. Black arrow, human cytomegalovirus (CMV) immediate early promoter. Positions of the RT-PCR primers 97S and 438A are indicated.
(D) RT-PCR on RNA extracted from HeLa cells transfected with plasmids displayed in (C). RT-PCR primers 97S and 438A were used to detect both IR E6(top band) mRNAs and mRNAs spliced between SD226 and SA409 (226∧ 409, lower band). gapdh was used as a control. M, marker. (E) The percentage
of 226∧ 409-spliced HPV16 mRNAs is plotted against the endpoint of each HPV16 deletion mutant displayed in (C). The band intensities of IR E6 and226∧ 409 mRNAs obtained from (D) were quantified by Image Lab 6.1 Software (BioRad). The percentage of spliced 226∧ 409 mRNA was calculated by
dividing the values of 226∧ 409 by the sum of IR E6 + 226∧ 409 values multiplied by 100. Mean values and standard deviations are shown. The putative
splicing enhancer between HPV16 nucleotides 438 and 478 is indicated. All data are representative of three biologically independent experiments.

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was present within the 40 nucleotides between HPV16 nucleotide positions 438 and 478. The splicing enhancer wastentatively named enhancer-1 (ENH-1). Additionally, our results suggested the existence of multiple cis-RNA regulatory
elements that induce or reduce splicing to SA409. One of these
RNA elements has been characterized as a splicing silencer element encoding a UAG-containing sequence located between
HPV16 nucleotides 599 and 601 (25). However, in the present
study, we focus on the potential splicing enhancer ENH-1 as
it is currently unclear how splicing of the HPV16 226∧ 409(E6*I) mRNA is activated.

The region between HPV16 nucleotide positions 438 and 478
contained a sequence of 11 nucleotides perfectly repeated
twice (Figure 2A, B: pX478). When mutating either the second, first or both of these 11 nucleotide repeats, splicing toSA409 was significantly reduced (Figure 2C). Next, two nucleotides at a time were mutated in both repeats simultaneously (Figure 2B, M1–M5). Splicing to SA409 was reduced
in all mutant plasmids compared with wild-type control, indicating a role in splicing for all 11 nucleotides in both repeats (Figure 2D), although the nucleotide positions mutatedin pX478M2 appeared to be less important for splicing enhancement. When mutating two nucleotides of M1 in the firstor of M4 in the second 11 nucleotides (sM1 or sM4, respectively), splicing to SA409 was significantly reduced (Figure2E). Mutating the 10 nucleotides between the first and second
11 nucleotide motifs (midM) also reduced splicing to SA409(Figure 2E). These results indicated the importance of the identified ENH-1 region for HPV16 226∧ 409 mRNA splicing regulation.

Four nucleotide substitutions in the identified
ENH-1 RNA splicing element reduced HPV16
E6*I/E7 mRNA splicing and oncoprotein production
In order to verify the results of substitutions in ENH-1 obtained with a shorter HPV16 subgenomic plasmid, pX478(Figure 2A–E), four nucleotide substitutions were introducedin the subgenomic pC97ELsL plasmid which encodes all
HPV16 genes and all HPV16 splice sites, but is driven by
the CMV promoter (Figure 2F) (34). Combining the sM1 andsM4 mutations analyzed in Figure 2E, two nucleotides weremutated in the first 11 nucleotide repeat and two in the second
11 nucleotide repeat of pC97ELsL, resulting in pC97M1M4(Figure 2G). RT-PCR results on RNA extracted from HeLa
cells transfected with wild-type pC97ELsL or the pC97M1M4
mutant showed a near total loss of splicing specifically toSA409 in the pC97M1M4 mutant, confirming that ENH-1
is required for splicing to the SA409 splice site (Figure 2I).
The primers used for RT-PCR are displayed in Figure 2H. RTPCR parallelly performed with reverse transcriptase-negative
samples did not produce amplified DNA, validating the experimental system (Supplementary Figure S1D). The reduced
utilization of SA409 caused by the M1M4 mutations was
also observed using alternative RT-PCR primers (Supplementary Figure S1E). In contrast, other HPV16 mRNAs, such as
those spliced 880∧ 3358, were unaffected by the M1M4 mutations (Figure 2J). Introducing the M2 mutation in ENH-1
of pC97ELsL showed only a minor reduction of splicing toSA409, as expected from the results presented in Figure 2D(Supplementary Figure S1F, G). We concluded that the M1M4
mutations caused a clear inactivation of the splicing enhancer
ENH-1 and they were therefore used hereafter to investigate
the ENH-1 function further.
The altered HPV16 mRNA splicing should be reflected at
the protein level. Therefore, E6 and E7 protein production
from plasmids pC97ELsL and pC97M1M4 were also analyzed (Figure 2K). The M1M4 mutant caused a marked reduction in E7 protein production compared with the wildtype pC97ELsL. These results were consistent with the resultsfrom the RT-PCR analysis and were confirmed using different cell lines (C33A and 293T) in the absence or presence ofproteasomal inhibitor MG-132 (Supplementary Fig. S1H). Interestingly, the E6 protein levels decreased as well (Figure 2K),
despite a relatively modest effect of the M1M4 mutations on
the levels of the IR E6 mRNAs (Figure 2I). The M1M4 mutations resulted in E113V/Q122L amino acid substitutions in
the HPV16 E6 protein (Supplementary Figure S2A) (8,41,42).
To determine if these substitutions affected the antigenicity of
the E6 protein and thereby skewed the western blot resultswith the anti-HPV16 E6-specific antibody, we generated plasmids producing FLAG-tagged E6 protein with or without theM1M4 mutations (Supplementary Figure S2B). The reduced
detection of the E6 protein from pC97M1M4 was not due tothe change in the antigenicity since the same reduction was observed when N-terminal FLAG-tagged-M1M4 mutant E6 protein was detected by FLAG antibody (Supplementary Figure
S2C). These results indicated that the splicing enhancer element ENH-1 may affect post-splicing mRNA processing steps
as well. In fact, the cytoplasmic IR E6 mRNA levels were reduced even though the nuclear IR E6 mRNAs were increased
when ENH-1 was mutated to M1M4 (Supplementary Figure
S3A, B). The RNA export efficiency of both IR E6 mRNAs and226∧ 409 E6 mRNAs was reduced when ENH-1 was mutated(Supplementary Figure S3C). As expected, the effects of theM2 mutation on splicing and nucleocytoplasmic distribution
were relatively minor compared with pC97ELsL (Supplementary Figure S4). These results indicated that ENH-1 may influence several steps of HPV16 RNA processing. We concluded
that the novel splicing enhancer ENH-1 located downstreamof the HPV16 3 -splice site SA409 was required for E6*I/E7
mRNA expression and E6 and E7 protein production.

Replacement of the HPV16 ENH-1 withcorresponding sequences from other high-risk
HPVs maintained 226∧ 409 splicing, whereas
replacement with corresponding sequences fromlow-risk HPVs did not
E6*I/E7 mRNA splicing is specific for carcinogenic HRHPVs. To determine if ENH-1 is conserved among HR-HPVs,
the HPV16 ENH-1-encoding region was aligned with other
HPV types. The alignment revealed that none of HR-HPVs
except HPV16 encoded a perfect 11 nucleotide repeat sequence (Supplementary Figure S5A, B). However, the adenine(A) richness of the sequences corresponding to the first andthe second 11 nucleotide repeats of HPV16 was conserved in
the HR types (Supplementary Figure S5A, B). The A content
of the first 11 nucleotide sequence was on average 73% for the
HR-HPVs, whereas the A content of the second 11 nucleotide
sequence was on average 66% for the HR-HPVs (Supplementary Figure S5C). Intriguingly, sequences corresponding to the

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The cis-acting RNA splicing element downstreamof the HPV16 splice site SA409 (ENH-1) contains an
11 nucleotide perfect repeat

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Figure 2. A novel splicing enhancer (ENH-1) consists of a perfectly repeated 11 nucleotide sequence that is required for 226∧ 409 splicing and for
production of the E6*I/E7 mRNA. (A) Schematic representation of the HPV16 plasmid pX478. HPV16 sequences (GenBank ID: K02718.1) between
nucleotide positions 97 and 478 were expressed from the CMV promoter. Large black arrow, human CMV immediate early promoter; pAL,
polyadenylation site; black oval, SD226; white oval, SA409; small black arrows, RT-PCR primers 97S and 438A used in (C–E). The box with (+) indicatesthe position of the novel HPV16 splicing enhancer ENH-1. (B) Nucleotide sequence of the HPV16 region between nucleotide positions 441 and 478 thatencompasses the HPV16 splicing enhancer ENH-1. The upper sequence represents the wild-type HPV16 sequence in pX478 and sequences below
represent various mutants. The 11 nucleotide repeats in the wild-type sequence are indicated in green lowercase letters and nucleotide substitutions in
red uppercase letters. Plasmid names are indicated to the left. (C–E) RT-PCR on RNA extracted from HeLa cells transfected with the indicated plasmids
displayed in (B). The top band represents HPV16 IR E6 mRNA and the lower band mRNAs spliced between SD226 and SA409 (226∧ 409) as indicated to
the right. gapdh was used as a control. M, marker. (F) Schematic representation of the HPV16 subgenomic plasmid pC97ELsL. The early genes E6, E7,
E1, E2, E4 and E5 are depicted as white boxes and the late genes L2 and L1 as gray boxes. Part of the L1 gene has been replaced with the poliovirus 2Ainternal ribosome entry site (IRES) and secreted luciferase (sLuc). pAE and pAL indicate HPV16 early and late polyadenylation signals, respectively. The
splice donors are indicated as black ovals and the splice acceptors as white ovals. The number beneath each splice donor and acceptor indicates their
nucleotide position in the HPV16 reference genome (GenBank ID: K02718.1). Black arrow, human CMV immediate early promoter. (G) An enlargementof the HPV16 E6 region containing splice donor SD226 and splice acceptors SA409 and SA526 in pC97ELsL. The identified splicing enhancer ENH-1 isindicated as a green box with a plus sign. The nucleotide sequence of wild-type ENH-1 present in pC97ELsL and the M1M4 mutant present in
pC97M1M4 is shown. The 11 nucleotide repeats in the wild-type ENH-1 sequence are in green lowercase letters and mutated nucleotides are marked in
red uppercase letters. Numbers above the sequences indicate their nucleotide positions in the HPV16 reference genome (GenBank ID: K02718.1). (H)Schematic representation of alternative splicing in the HPV16 E6/E7 region. IR E6 and mRNAs spliced between the HPV16 splice donor SD226 andsplice acceptors SA409 (226∧ 409), SA526 (226∧ 526) or SA742 (226∧ 742) are shown. Splice donors and splice acceptors are indicated as black and whiteovals, respectively. Primer positions of RT-PCR primers 97S and 880A used in (I) are indicated as arrows. (I) RT-PCR on RNA extracted from HeLa cells
transfected with pC97ELsL or with pC97M1M4. RT-PCR was performed with primers 97S and 880A that amplify alternatively spliced E6/E7 mRNAs as
indicated in (H). (J) RT-PCR on the same RNA samples as in (I), but with primers 773S and E42A that amplify HPV16 mRNAs spliced from spliced donor
SD880 to splice acceptor SA3358 (880∧ 3358, see Figure 1B for location of RT-PCR primers in HPV16). gapdh was used as a control. M, marker. (K)Western blot on cell extracts from 293T cells transfected with pC97ELsL (wild-type ENH-1) or pC97M1M4 (mutant ENH-1) and treated withdimethylsulfoxide (DMSO) or MG132 proteasome inhibitor 3 h before harvest. HPV16 E6 and E7 proteins were detected by anti-HPV16 E6 (GeneTex,
GTX132686) and anti-HPV16 E7 antibody (GeneTex, GTX133411), respectively. Actin was used as control.

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restored to the levels obtained with wild-type HPV16 ENH-1,
supporting previous results that demonstrated that the exact
nucleotide sequence of each 11 nucleotide repeat is importantfor function of HPV16 ENH-1 and that the 10 nucleotide‘spacer’ in between the 11 nucleotide repeats contributes tofunction (Figure 2E). Taken together, we concluded that theentire 32 nucleotide HPV16 ENH-1 element was critical for
function. This is a relatively long RNA sequence to be a splicing enhancer, suggesting that multiple trans-acting factors interact with 16ENH-1.

TRAP150 interacts with the HPV16 ENH-1 enhancer
RNA sequence and increases HPV16 226∧ 409
splicing and production of E7 protein
In order to identify the trans-acting factor(s) of HPV16 ENH1, we performed an in vitro RNA oligo pull-down assay withRNA oligonucleotides containing either the wild-type or theM4 mutant ENH-1 sequence (Supplementary Figure S6A, B).
Bands on silver-stained gels that represented proteins interacting preferentially with the wild-type ENH-1 sequence weresent for MS for identification (Supplementary Figure S6C, left
panel). A number of RNA-binding proteins were identified,including TRAP150, RBM15 and hnRNP M (Supplementary
Figure S6C, right panel). Additional RNA oligo pull-down
experiments were performed and subjected to western blotting, using antibodies specific for the candidate proteins, toconfirm specific interactions of ENH-1 RNA with the various proteins listed in Supplementary Figure S6C (right panel).
Western blots of TRAP150, RBM15 and hnRNP M specifically pulled down by ENH-1 RNA are shown in Supplementary Figure S6D. Pull-down of the various proteins was less efficient when RNAs with only one of the 11 nucleotide repeats
were used compared with RNAs with both 11 nucleotide repeats (Supplementary Figure S6D). These results emphasized
the significance of the entire ENH-1 region as a cis-RNA regulatory element required for splicing. Finally, we confirmedthat TRAP150 was specifically and efficiently pulled down by
an RNA oligo encoding wild-type ENH-1 compared with theenhancer mutant M1M4 (Figure 4A, B) that was used in the
HPV16 expression plasmids (Figure 2).
To determine the effect of various proteins pulled down
specifically with wild-type ENH-1 RNA on HPV16 226∧ 409
mRNA splicing, plasmids expressing the identified proteins
hnRNP M, hnRNP K, RBM15 and TRAP150 were transfected into HeLa cells with HPV16 plasmid pC97ELsL. The
only protein enhancing splicing to SA409 was TRAP150 (Figure 4C). TRAP150 was previously reported to interact with
PSF to antagonize splicing activated by PSF (32). Interestingly,
PSF was included in our candidate proteins obtained fromMS results (Supplementary Figure S6C) and was specificallypulled down by wild-type ENH-1 RNA oligo (Supplementary
Figure S6D). In contrast to TRAP150, PSF inhibited splicing
to SA409 (Supplementary Figure S6E). PSF overcame the effect of TRAP150 when both proteins were co-overexpressed(Supplementary Figure S6E). Similarly to PSF, RBM15 also inhibited 226∧ 409 splicing (Figure 4C) and overcame the effectof TRAP150 (Supplementary Figure S7A, B). On the other
hand, the relatively minor splicing inhibitory effect exerted
by hnRNP M was overcome by TRAP150 (Supplementary
Figure S7C, D). These results indicated that competitive relationships between 16ENH-1 trans-acting factors TRAP150,

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two 11 nucletide repeats in LR-HPVs were also A rich, but to alower extent (65% and 44% for the two 11 nucleotide repeats,
respectively). The A content of the corresponding sequencesin LR-HPVs was significantly lower than the A content of
the HR-HPVs, in particular for the second 11 nucleotide repeat (Supplementary Figure S5C). The spacer sequence between the two 11 nucleotide repeats had an A content of 28%
and 32% in HR- and LR-HPVs, respectively (Supplementary
Figure S5C). Our results suggested that the high A content
of the 11 nucleotide repeat sequence was a defining property of the splicing enhancer of the HR-HPVs (Supplementary
Figure S5D).
To determine if sequences corresponding to HPV16 ENH1 in other HR-HPVs possessed 226∧ 409 splicing enhancer
activity, 32 nucleotides of HPV16 sequences encompassing
HPV16 ENH-1 (nucleotides 444–475) were replaced by corresponding sequences from other HR-HPVs (HPV31, HPV18
and HPV 45) or LR-HPV (HPV6b) in the HPV16 E6E7encoding plasmid pC16HAE6E7F (Figure 3A, B). The influence of the HR-HPV or LR-HPV sequences on HPV16226∧ 409 splicing was monitored by RT-PCR. The results
revealed that 226∧ 409 splicing was maintained at a relatively high level between 14% and 37% when HPV16 ENH1 was replaced by sequences from other HR-HPVs (Figure 3C, D), while 226∧ 409 splicing was reduced from 34%
to 3% when the HPV16 ENH-1 was replaced by LR-HPV(HPV6b) sequences (Figure 3C, D). Considering the sensitivity of 226∧ 409 splicing to just four nucleotide substitutions in
HPV16 ENH-1 (Figure 2D, I), it was to some extent surprisingthat replacement of HPV16 ENH-1 with the relatively divergent sequences from other HR-HPVs also supported splicing
to SA409. By contrast, LR-HPV sequences did not significantly
enhance splicing. We concluded that sequences from different
HR-HPVs that correspond to HPV16 ENH-1 possessed splicing enhancing activity, whereas the corresponding sequence
from an LR-HPV did not.
To further investigate the defining properties of HPV16
ENH-1, the A content in the splicing-incompetent LR6bENH-1 sequence (pCEnh1.16to6b) was gradually increased (Figure 3E). In 6bENH-1, the first 11 nucleotide
region consists of 73% A nucleotides just like 16ENH-1,
whereas the second 11 nucleotide region has an A content of
18% compared with 73% in HPV16. Next, the A content in
the second 11 nucleotide region of 6bENH-1 was gradually
increased, resulting in a total of 17, 19 or 21 A nucleotides.
However, these mutations did not appear to significantly affect splicing (Figure 3F). As a next step, two nucleotide positions in the sequence of HPV6b that represented the first 11
nucleotide repeat, positions 7 and 10, were mutated (Figure3E). The rationale for this mutagenesis was that position 7 inthe sequence of HPV6b was a ‘T’ just like in the HPV16 ENH1 M4 mutation and that position 10 was ‘A’ in HPV6b, but
a strictly conserved ‘G’ in the high-risk types (Supplementary
Figure S5B, D). Thus, the ‘T’ and ‘A’ at positions 7 and 10,
respectively, of the first 11 nucleotide repeat of ENH-1, werechanged to ‘A’ and ‘G’, respectively, increasing the A content
of the second 11 nucleotide repeat in HPV6b in combination
with mutations of two key positions in the first 11 nucleotide
repeat in HPV6b, resulting in activation of splicing between
HPV16 splice sites SD226 and SA409 on mRNAs producedby the pCEnh1.16to6b plasmid (Figure 3F). These results confirmed the importance of the two A-rich, 11 nucleotide sequences in splicing regulation. However, splicing was not fully

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Figure 3. HPV16 226∧ 409 splicing is enhanced by corresponding sequences from HR-HPV types but not from LR-HPV types. (A) Sequence alignment ofdifferent HPVs based on the HPV16 sequence between nucleotides 407 and 484 that encompass the novel splicing enhancer ENH-1. HPV16 (GenID:
K02718) and HPV31 (GeneID: J04353) are HR, alpha-9 HPV types, HPV18 (GeneID: X05015) and HPV45 (GeneID: X74479) are HR, alpha-7 HPV types,
and HPV6b (GeneID: X00203) is an LR, alpha-10 HPV type. The splice acceptor used for generation of the E6*I/E7 mRNA in each HR-HPV is indicated asa triangle and labeled SA409, SA413, SA416 and SA413 for HPV16, HPV31, HPV18 and HPV45, respectively. The well-conserved amino acid motif‘CXXC’ in HPV E6 (61) is highlighted as a gray box. HPV16 ENH-1 is located 23 nucleotides downstream of CXXC, and sequences corresponding to
HPV16 ENH-1 in the other HPV types are highlighted in uppercase letters in color and enclosed in a green box. Numbers above the upper sequencerefer to nucleotide positions in HPV16. (B) (Top) Schematic representation of the HPV16 E6 and E7 encoding plasmid pC16HAE6E7F. HPV16 E6 and E7
coding sequences (nucleotides 104–855) were inserted in a CMV promoter-driven plasmid, fusing E6 with HA sequence at the 5 end and E7 with FLAG
sequence at the 3 end. HA-tag and FLAG-tag sequences are shown as blue boxes. HPV16 splice sites SD226, SA409, SA526 and SA742 are indicatedas black (SD) or white (SA) ovals. HPV16 ENH-1 is indicated as a green box with a (+). Arrows indicate positions of RT-PCR primers HA-F and FLAG-R.
(Below) Schematic representation of replacements of HPV16 ENH-1 encoding sequences between HPV16 nucleotides positions 444 and 475 in
pC16HAE6E7F with corresponding sequences of different HPVs, thereby generating chimeric plasmids: pC16Enh1.16to6b (16to6b), pC16Enh1.16to31(16to31), pC16Enh1.16to18 (16to18) and pC16Enh1.16to45 (16to45). (C) RT-PCR on RNA extracted from HeLa cells transfected with the indicatedplasmids shown in (B). RT-PCR was performed with primers HA-F and FLAG-R that amplify cDNAs representing IR E6 mRNAs or alternatively spliced
between HPV16 SD226 and SA409 (226∧ 409), SA526 (226∧ 526) and SA742 (226∧ 742). RT-PCR was performed in the presence (RT+) or absence (RT–) ofreverse transcriptase. gapdh was used as a control. M, marker. (D) Percentage spliced 226∧ 409 mRNA was calculated by quantification of band
intensities obtained from (C). Percentage 226∧ 409 splicing was calculated by dividing the band intensity of the quantified band representing
226∧ 409-spliced mRNA by the sum of band intensities representing IR E6 mRNAs and all spliced variants (IR E6 + 226∧ 409 + 226∧ 526 + 226∧ 742)
× 100. Mean values and standard deviations are shown. (E) Schematic representation of the mutagenesis of the HPV6b sequences in the
HPV16–HPV6b chimeric plasmid ‘16to6b’ to determine whether the A number elevation of the HPV6b sequence corresponding to the second 11
nucleotide repeat sequence in HPV16 would convert the HPV6b sequence to a splicing enhancer. In addition to A number elevation, nucleotide positions
that are strictly conserved in HR-HPV types, e.g. HPV16, but not in LR-HPV types, e.g. HPV6b, were also changed. The mutations introduced in the
HPV6b sequence are indicated in red uppercase letters. The A content (%) in the first and second A-rich 11 nucleotide repeat in each plasmid is indicatedat the right. (F) RT-PCR on RNA extracted from HeLa cells transfected with the indicated plasmids displayed in (E). RT-PCR was performed with primers
HA-F and FLAG-R that simultaneously detect IR E6 mRNAs and 226∧ 409-, 226∧ 526- or 226∧ 742-spliced mRNAs.

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Figure 4. Identification of TRAP150 as an ENH-1-binding factor that induces E6*I/E7 mRNA production and E7 protein production. (A) Nucleotide
sequence of RNA oligos used in in vitro RNA pull-down experiments. The wild-type sequence of ENH-1 (WT) is indicated in green lowercase letters andmutations in M1M4 are marked in red uppercase letters. (B) Western blotting on proteins from in vitro RNA oligo pull-downs. Filters were stained using
antibody specific to TRAP150. (C) RT-PCR on RNA extracted from HeLa cells transfected with subgenomic HPV16 plasmid pC97ELsL and empty pUC
vector or the indicated plasmids expressing hnRNP M, hnRNP K, RBM15 or TRAP150. RT-PCR was performed with primers 97S and 880A that detect
alternatively spliced HPV16 mRNAs from the E6 and E7 coding regions. See Figure 1B for primer locations. gapdh was used as a control. M, marker; RT,reverse transcriptase. (D) Western blot on extracts from 293T cells transfected with empty pUC plasmid or pC97ELsL and pUC (–), pTRAP150 orpRBM15 as indicated. Filters were stained using antibodies specific to either HPV16 E6, E7 or cellular actin. MW, molecular weight marker. (E) RT-PCR
on RNA extracted from HeLa cells transfected with subgenomic HPV16 plasmid pC97ELsL or pC97M1M4 together with empty pUC plasmid or
pTRAP150. RT-PCR was performed with primers 97S and 880A that detect alternatively spliced HPV16 mRNAs from the E6 and E7 coding regions (IRE6, 226∧ 409, 226∧ 526 and 226∧ 742), or with 773S and E42A that detect mRNAs spliced from HPV16 SD880 to SA3358 (880∧ 3358) (for position of
RT-PCR primers, see Figure 1B). gapdh was used as a control. M, marker. (F) Percent spliced 226∧ 409 mRNA calculated by first quantifying the RT-PCR
bands representing the various E6 and E7 splice variants in (E). Percent spliced 226∧ 409 was calculated by dividing the band intensity of 226∧ 409 by thesum of the band intensities of all bands (IR E6 + 226∧ 409 + 226∧ 526 + 226∧ 742) × 100. Mean values, standard deviations and Student t-test are shown.
***P < 0.005. (G) Schematic overview of HPV16 full genome plasmid pHPV16AN. The HPV16 genome in pHPV16AN is flanked by loxP sites. The
episomal form of the HPV16 genome can be released by co-transfection with a plasmid expressing the Cre recombinase. (H) RT-PCR on RNA extracted
from HeLa cells transfected with HPV16 plasmid pHPV16AN and empty pUC plasmid or pTRAP150. RT-PCR was performed with primers 97S and 880A
that detect alternatively spliced HPV16 mRNAs from the E6 and E7 coding regions (IR E6, 226∧ 409, 226∧ 526 and 226∧ 742). See Figure 1B for primer
locations. gapdh was used as a control. M, marker. (I) RT-PCR on RNA extracted from HeLa cells transfected with HPV16 plasmid pHPV16AN or
pHPV16ANM1M4 with four nucleotide substitutions in the ENH-1 splicing enhancer. The exact mutations are shown in (A). Transfections were
performed in the presence of Cre recombinase to release the episomal form of the HPV16 genome, as described in the Materials and methods (also,
see G). RT-PCR was performed with primers 97S and 880A that detect alternatively spliced HPV16 mRNAs from the E6 and E7 coding regions (IR E6,226∧ 409, 226∧ 526 and 226∧ 742). gapdh was used as a control. M, marker.

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TRAP150 enhances HPV16 226∧ 409 mRNA splicing
via the HPV16 splicing enhancer ENH-1
To determine if 226∧ 409 splicing activation by TRAP150
required an intact ENH-1 element, TRAP150 was overexpressed in transient transfections with either pC97ELsL or
the M1M4 mutant pC97M1M4. Splicing to SA409 increased
when TRAP150 was co-transfected with pC97ELsL (Figure 4E), in a statistically significance manner (Figure 4F),
as expected. In contrast, this increase in 226∧ 409 splicing
by TRAP150 overexpression was not seen with the M1M4
mutant pC97M1M4 (Figure 4E, F). Furthermore, TRAP150
overexpression did not affect splicing between HPV16 splicesites SD880 and SA3358 (880∧ 3358) (Figure 4E). We concluded that TRAP150 enhanced 226∧ 409 splicing via ENH-1.
Next, we wished to determine if the ENH-1 was required forthe 226∧ 409 splicing of mRNAs produced from the episomal
form of the full-length HPV16 genome from which transcription is driven by the HPV16 long control region (LCR). To thisend, we utilized plasmid pHPV16AN in which the full-length
HPV16 genome is flanked by loxP sites and can be excised byoverexpression of the Cre recombinase (Figure 4G). The enhancing effect of TRAP150 on HPV16 226∧ 409 splicing was
reproduced with pHPV16AN (Figure 4H). Consistent with
pC97ELsL results, M1M4 mutations in pHPV16AN abrogated 226∧ 409 splicing (Figure 4I), confirming the importanceof ENH-1 for 226∧ 409 splicing. We concluded that TRAP150
enhances production of the HPV16 spliced 226∧ 409 mRNA
also from the epsisomal HPV16 genome.

TRAP150 enhances E6*I/E7 mRNA splicing ofvarious high-risk HPVs, but does not affect E6/E7
mRNAs produced by low-risk HPVs
Next, we investigated if TRAP150 is a common trans-acting
factor of E6*I/E7 mRNA splicing of other HR-HPVs. First,
the E6/E7 genes of three HR-HPVs in addition to HPV16(HPV31, HPV18 and HPV45) and two LR-HPV (HPV6band HPV11) were subcloned into the same backbone plasmid as pC16HAE6E7F (Figure 3B), resulting in the plasmids
displayed in Figure 5A. These plasmids were transfected into
HeLa cells and alternative splicing was analyzed by RT-PCR
using primers annealing to mRNAs from all the analyzed plasmids. Consistent with previous studies, LR-HPVs (6b and 11)
did not produce spliced E6/E7 mRNAs, whereas all HR-HPVs
analyzed here produced spliced E6*I/E7 mRNAs as well as IR
E6 mRNAs (Figure 5B). To determine the effect of TRAP150
overexpression on these mRNAs, we first confirmed the effect of TRAP150 on HPV16 mRNA splicing using plasmid
pC16HAE6E7F. The results revealed that TRAP150 enhanced
production of HPV16 226∧ 409-spliced E6*I/E7 mRNAs in adose-dependent manner (Figure 5C, D). TRAP150 enhanced
production of spliced E6*I/E7 mRNAs from the HR-HPVs,
but did not affect the E6/E7mRNAs produced from the LRHPVs (Figure 5E, F), demonstrating that TRAP150 specifically enhances production of spliced E6*I/E7 mRNAs fromcarcinogenic HR-HPVs.

TRAP150 interacts with HPV16 mRNAs and with
U2AF65/U2AF2 in human cells
Next, we wished to determine if the interactions between
TRAP150 and HPV16 mRNAs could be observed in cells
and if this is dependent on ENH-1 using RIP. HPV16 plasmid
pC97ELsL was co-transfected with FLAG-tagged TRAP150expressing plasmid and cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. Immunoprecipitated
RNA was extracted and analyzed by RT-PCR or RT-qPCR
using HPV16 E6*I/E7 cDNA-specific primers. The interactions between FLAG-TRAP150 and HPV16 226∧ 409-spliced
E6*I/E7 mRNAs were demonstrated in FLAG-TRAP150expressing samples specifically (Figure 6A, upper panel). Similar results were obtained with different primer pairs detecting
880∧ 3358-spliced HPV16 mRNAs (Figure 6A, bottom panel).
These interactions were reduced when ENH-1 was mutated,either in pC97ELsL (Figure 6B) or in pX478 (Figure 6C), revealing a 14- and 479-fold decrease, respectively (Supplementary Figure S9A, B). The interactions between TRAP150 and
ENH-1 RNA appeared to be indirect when evaluated by UVcross-linking RNA oligo pull-down assay (Supplementary Figure S9C). In a control reaction using BRUWT2 RNA oligo encoding the U-rich HPV16 early 3 -untranslated region (UTR)
sequence that was previously demonstrated to interact with
CPSF30 (43), CPSF30 was UV-cross-linked and pull-downed
by RNA oligo even after the most stringent wash condition(1000 nM NaCl) (Supplementary Figure S9D). In contrast, theinteraction of TRAP150 with ENH-1 RNA oligo was significantly reduced by high salt washes, suggesting that TRAP150
may not interact with ENH-1 RNA directly, but via an as
yet unidentified factor(s) (Supplementary Figure S9E). On theother hand, the interaction of PSF with ENH-1 RNA oligo
was weak but binding was maintained even after the moststringent wash conditions, suggesting that PSF binds to RNA
directly, which was consistent with previously reported results(29,32).
The general splicing factor U2 snRNP auxiliary factor 65 kDa (U2AF65/U2AF2) is known to promote prespliceosome assembly at splice acceptors. As shown in Figure
6D, FLAG-TRAP150 co-immunoprecipitated endogeneous
U2AF65. This interaction was confirmed with a reversed coIP in which co-IP was performed with U2AF65 antibody andwestern blot to FLAG-TRAP150 (Figure 6E). The interactions
between U2AF65 and TRAP150 increased when TRAP150
was overexpressed (Figure 6E). This interaction was not abolished but was reduced by RNase treatment, indicating thatU2AF65 interactions with TRAP150 could occur in the absence of RNA too (Figure 6F). In addition, U2AF65, as wellas U2AF35 could be pulled down with the ENH-1-encoding
RNA oligo but not with the mutant ENH-1 (M4)-encoding
RNA oligo (Figure 6G), supporting the idea that U2AF components were included in the ENH-1 RNA interactome. Theinteractions between U2AF65 and ENH-1 RNA oligo werereduced when TRAP150 levels were lowered by siRNA transfections prior to preparation of the nuclear extract (Supplementary Figure S10A). These results suggested that TRAP150

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PSF, RBM15 and hnRNP M regulate splicing between HPV16
SD226 and SA409.
TRAP150 overexpression resulted in significantly increased
E7 protein levels and reduced E6 protein levels (Figure 4D),
which was in line with the activation of 226∧ 409 splicing in
the E6 coding region (Figure 4C) and the reduction of cytoplasmic IR E6 mRNA levels by TRAP150 overexpression(Supplementary Figure S8A, B). In contrast, RBM15 reduced
E7 protein levels (Figure 4D), as a consequence of its splicing inhibitory effect (Figure 4C). We concluded that TRAP150
enhanced HPV16 226∧ 409 mRNA splicing, thereby increasing HPV16 E7 protein production at the expense of the E6
protein.

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Figure 5. (A) Schematic representation of plasmids encoding E6 and E7 sequences from HPV6b (LR), HPV11 (LR), HPV16 (HR), HPV31 (HR), HPV18(HR) or HPV45 (HR) named pCHPV6bHAE6E7F, pCHPV11HAE6E7F, pCHPV16HAE6E7F, pCHPV31HAE6E7F, pCHPV18HAE6E7F and pCHPV45HAE6E7F,
respectively. The E6 open reading frame was fused with an HA-tag sequence in the 5 end, and the E7 open reading frame was fused with a FLAG-tag
sequence in the 3 end. The green box indicates splicing enhancer-1 (ENH-1) in HPV16 and corresponding sequences in the other HR-HPV types. Foralignment of ENH-1 sequences, see Supplementary Figure S5. Black ovals represent splice donors and white ovals splice acceptors. Numbers below
the schematics refer to nucleotide positions of splice sites in the genomes of each HPV type, and numbers to the right represent start and end points ofthe E6 and E7 sequences present in the plasmids displayed in the figure. Nucleotide positions refer to reference genomes of the various HPV types aspresented in the Materials and methods. Arrows indicate positions of RT-PCR primers HA-F and FLAG-R. (B) RT-PCR on RNA extracted fromm HeLa
cells transfected with plasmids displayed in (A). Plasmids express E6 and E7 encoding mRNAs of the HPV types indicated above each lane of the gel
image. RT-PCR was performed with primers HA-F and FLAG-R that amplify cDNAs representing IR E6 mRNAs and spliced E6*I/E7 mRNAs common to
all HR-HPV types. A weak band representing the E6*II mRNA is observed in cells transfected with the HPV16 plasmid. gapdh was used as a control. M,
marker. (C) RT-PCR on RNA extracted from HeLa cells transfected with HPV16 plasmid pC16HAE6E7F and serially diluted pTRAP150 plasmid as
indicated in (D). RT-PCR was performed with primers HA-F and FLAG-R. cDNAs detecting HPV16 IR E6 mRNAs, spliced E6*I/E7 mRNAs (226∧ 409) andE6*II mRNAs (226∧ 526) as indicated. gapdh was used as a control. M, marker. (D) Quantification of the RT-PCR results displayed in (C). Percent spliced
226∧ 409 mRNAs was calculated by dividing band intensities of 226∧ 409 by the sum of the band intensities of all bands (IR E6 + 226∧ 409 + 226∧ 526) ×100. The results revealed a TRAP150 dose-dependent enhancement of HPV16 mRNA splicing. Mean values and SDs are shown. (E) Effect of TRAP150
on E6*I mRNA production of different HPVs was determined. RT-PCR on RNA extracted from HeLa cells transfected with the indicated plasmids
encoding the E6 and E7 genes of LR-HPV types HPV6b and HPV11, or the E6 and E7 genes of HR-HPV types HPV16, HPV31 HPV18 or HPV45 in theabsence or presence of TRAP150 expression plasmid. RT-PCR was performed with primers HA-F and FLAG-R that detect alternatively spliced HPV
mRNAs from the E6 and E7 coding regions (IR E6 mRNA and spliced E6*I/E7 mRNA). gapdh was used as a control. M, marker. (F) Quantification of the
RT-PCR results displayed in (E). Ratios between 226∧ 409-spliced E6*I/E7 mRNA and IR E6 mRNA (E6*I/IR E6) were calculated. Mean values andstandard deviations are shown. Student t-test results are shown: n.s. = no significance. ***P < 0.001. ****P < 0.0001.

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Figure 6. (A) RIP assay. RNA–protein complexes were immunoprecipitated with anti-FLAG-specific antibody from 293T cells transfected with emptypUC plasmid (–) or with HPV16 subgenomic expression plasmid pC97ELsL in the absence (–) or presence (+) of FLAG-tagged TRAP150-expressing
plasmid as indicated. RNA was extracted and RT-PCR was performed using RT-PCR primers E6*I-F and E6*I-R that specifically detect 226∧ 409-spliced
HPV16 mRNAs, or with RT-PCR primers 773S and E42A that detect 880∧ 3358-spliced HPV16 mRNAs (for location of RT-PCR primers, see Figure 1B). (B)RIP assay. RNA–protein complexes were immunoprecipitated with anti-FLAG-specific antibody from 293T cells transfected with empty pUC plasmid (–)
or with HPV16 subgenomic expression plasmid pC97ELsL or pC97M1M4 in the absence (–) or presence (+) of FLAG-tagged TRAP150-expressing
plasmid pTRAP150 as indicated. RNA was extracted and RT-PCR was performed as described in (A). (C) RIP assay and RT-PCR were performed using
cell lysates from 293T cells transfected with empty pUC plasmid (–) or with HPV16 subgenomic expression plasmid pX478 or pX478M+M (Figure 2B) in
the absence (–) or presence (+) of pTRAP150 as described in (B). (D and E) Co-IP of FLAG-TRAP150 and U2AF65 was performed on cell extracts from293T cells transfected with HPV16 subgenomic plasmid pC97ELsL and either empty pUC plasmid (–) or pFLAG-TRAP150 that expresses FLAG-tagged
TRAP150 (+). Immunoprecipitations were performed with mIgG (normal mouse IgG) or anti-FLAG antibody against FLAG-TRAP150 followed by western
blotting with antibody to U2AF65 (D), or with anti-U2AF65 antibody followed by western blotting with antibody to TRAP150 (E). Input samples represent
5% of the sample volume used for immunoprecipitation. MW, molecular weight marker. (F) Co-IPs were performed as in (D) and (E) with the exceptionthat cell lysates were treated with RNase A (final concentration of 5 μg/ml) prior to IP with anti-FLAG antibody, followed by western blotting with
antibody to U2AF65. MW, molecular weight marker. (G) Western blotting on proteins from in vitro RNA oligo pull-down samples using RNA oligo
encoding HPV16 wild-type (WT) or mutant (M4) ENH-1 sequence (for sequences of RNA oligos, see Supplementary Figure S6). Filters were stainedwith antibodies specific for U2AF65 or U2AF35. MW, molecular weight marker. (H) Optimization of the polypyrimidine tract of the HPV16 3 -splice site
SA409 to evaluate the significance of ENH-1 for SA409 recognition. Schematic representation of the HPV16 E6 expression plasmid pX478 that produces
IR E6 mRNAs and 226∧ 409-spliced mRNAs. Primer positions of the RT-PCR primers used in (I) are indicated as arrows. The CMV immediate early
promoter and the polyadenylation signal (pAL) are indicated. Schematic drawing of HPV16 SA409 with a polypyrimidine tract and ENH-1. pX478 wasused as the wild-type HPV16 SA409 polypyrimidine tract and ENH-1-encoding plasmid. The SA409 polypyrimidine tract is interrupted by multiple
adenosine or guanosine insertions in the consecutive pyrimidines. This suboptimal polypyrimidine tract was optimized by converting interrupting
adenosine or guanosine to ‘T’, resulting in pX478 SA409OPT. We also optimized the polypyrimidine tract of SA409 in pX478 M1M4 in which ENH-1 hadbeen mutationally inactivated by the M1M4 mutations, resulting in plasmid pX478 SA409OPT+M1M4. Numbers below the HPV16 sequences indicate
nucleotide position in the reference HPV16 genome. Introduced nucleotide substitutions are indicated in red uppercase letters. Blue uppercase letters
indicate SA409 while green letters indicate the 11 nucleotide repeats in ENH-1. (I)The plasmids presented in (G) were transfected in HeLa cells, followed
by RNA extraction and RT-PCR to determine the IR E6 and spliced 226∧ 409 mRNAs. RT-PCR were performed with primers 97S and 438A. gapdh wasused as control. M, marker; RT(+), reverse transcriptase-positive samples; RT(–), reverse transcriptase-negative samples.

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TRAP150 alters HPV16 E6/E7 mRNA splicing in
HPV16-positive cervical cancer cells, thereby
reducing cell viability
Usage of the HPV16 3 -splice site SA409 results in an mRNA
coding for the E6*I and E7 proteins. The 226∧ 409-spliced
E6*I/E7 mRNA is the major producer of E7 protein (15). Because IR E6 mRNA production and spliced 226∧ 409 mRNA
production are mutually exclusive, one may assume that E6
and E7 protein levels are affected in an opposite manner by
any change in E6/E7 mRNA splicing efficiency. We propose ahypothesis, demonstrated in Figure 7A, according to the results obtained so far. TRAP150 enhances 226∧ 409 mRNA
production through the interaction with ENH-1 and the recruitment of U2AF, thereby up-regulating E7 protein levels,
while simultaneously reducing IR E6 mRNA levels and E6
protein production. Although the increased E7 protein levels
may enhance cell proliferation, this may be counteracted by reduced E6 protein production and restoration of p53 followed
by apoptosis. Thus, a balanced splicing of HPV16 226∧ 409
would be of paramount importance in the HPV16 replication
cycle as well as during carcinogenesis.
In an attempt to address this hypothesis, we transfected SiHa cells (HPV16-positive cervical cancer cells) with
TRAP150 plasmid. The results revealed that TRAP150 reduced cell viability in a TRAP150 dose-dependent manner(Figure 7B). The reduction of cell viability by TRAP150 overexpression was not observed in HPV-negative cell lines 293T
and C33A (Supplementary Figure S11), indicating that the observed reduction of cell viability in SiHa by TRAP150 could
be explained by altered HPV16 E6/E7 mRNA splicing andE6 and E7 expression. Next we monitored HPV16 E6 andE7 mRNA levels in SiHa cells after TRAP150 overexpression
or knockdown. Initial experiments with overexpression or
knockdown of TRAP150 in SiHa cells revealed relatively minor effects on HPV16 E6/E7 mRNA splicing. Previous studies had shown that the effects of TRAP150 on mRNA splicing could be enhanced by BCLAF1 (33), a protein closely
related to TRAP150, and that the expression of TRAP150

and BCLAF1 could compensate for each other, suggesting thatknockdown of both proteins may be required to obtain a measuerable effect (33,44). TRAP150 and BCLAF1 share 66%
similarity in their primary amino acid sequence (44). In fact,
BCLAF1 interacted with HPV16 ENH-1 but less well with the
mutant RNA sequence (Figure 7C), indicating that BCLAF1
may also be a regulator of HPV16 mRNA splicing mediatedby interactions with ENH-1. We therefore performed overexpression of TRAP150 and BCLAF1 simultaneously. Production of spliced HPV16 226∧ 409 mRNAs from HPV16
plasmid pC97ELsL in HeLa cells was further enhanced when
TRAP150 was co-expressed with BCLAF1 compared with
TRAP150 or BCLAF1 alone (Figure 7D), supporting the ideaof an additive effect on HPV16 mRNA splicing. Next, either
TRAP150 or BCLAF1 or both proteins were knocked down
by siRNA transfection. The production of IR E6 mRNAs in
SiHa cells was significantly increased when both TRAP150
and BCLAF1 were knocked down by siRNAs, as comparedwith knockdown of TRAP150 or BCLAF1 alone (Figure
7E, F). The influence on spliced 226∧ 409 mRNA production was further confirmed with RT-qPCR using primers specific for 226∧ 409 mRNAs, showing a reduction of 226∧ 409
mRNA production in SiHa cells in which both TRAP150 and
BCLAF1 were knocked down (Figure 7G). Taken together,
these results suggested redundant activities of TRAP150 and
BCLAF1 in SiHa cells. As an additional complexity, we foundthat the expression of TRAP150 mRNA was increased after knockdown of BCLAF1 in SiHa cells, and vice versa(Figure 7H). These results further explained the necessity toknock down both TRAP150 and BCLAF1 to observe an effect on HPV16 mRNA splicing (Figure 7E, F). Consistent withthese results, viability of SiHa cells was reduced when both
TRAP150 and BCLAF1 were knocked down by siRNA (Figure 7I). Taken together, these results supported an importantrole for TRAP150 in the control of HPV16 oncoprotein expression via mRNA splicing in HPV16-positive cervical cancer cells.

HPV16 genomes with mutations in the splicing
enhancer ENH-1 fail to immortalize human primary
keratinocytes
The cell-immortalizing activity of HPV16 using plasmids
pC97ELsL and the ENH-1 mutant pC97M1M4 was investigated by transfection of nHFKs with pC97ELsL or
pC97M1M4. The expected expression pattern of HPV16
spliced E6 and E7 mRNAs was reproduced in nHFKs transiently transfected with pC97ELsL or pC97M1M4 (Supplementary Figure S12A). Untransfected nHFKs were used asa control when nHFKs transfected with HPV16 plasmid
pC97ELsL or pC97M1M4 were assayed for cell immortalization. Cells that became resistant to serum-induced differentiation and cell death were selected by cultivation in DMEM
supplemented with 10% BCS (Figure 8A) (40). After 60 daysof selection, the untransfected cells were dead, as were thepC97M1M4-transfected cells (Figure 8B). The lower pH colorof the cell culture medium and the appearance of cell colonies
indicated that only the pC97ELsL-transfected cells were actively dividing (Figure 8B). The pC97ELsL-transfected cells
were returned to normal keratinocyte medium after selection,
split into two plates that were grown in parallel as JJM9721#1
and JJM9721#2 (passage 15 at that point) (Figure 8C). Alongside these cells, two parallel cultures of nHFKs were grown

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interacts with HPV16 pre-mRNA at the ENH-1 enhancer tofacilitate recruitment of U2AF65 to the HPV16 mRNAs andenhance splicing to SA409. HPV16 SA409 is a suboptimal 3 splice site with an interrupted polypyrimidine tract (Figure
6H), prompting us to speculate that the ENH-1 RNA interactome would be obsolete if SA409 was artificially optimized.
To test this idea, the polypyrimidine tract upstream of SA409
was optimized in the pX478 plasmid (Figure 6H, SA409OPT).
As expected, the optimal polypyrimidine tract in SA409OPT
significantly increased splicing to SA409 (SA409OPT) (Figure 6I). Once the polypyrimidine tract in SA409 was optimized, ENH-1 was no longer required, as demonstrated
by the equally efficient splicing of mRNAs produced fromSA409OPT and SA409OPT+M1M4 (Figure 6H, I). Taken together, these results indicated that the ENH-1 interactome including TRAP150 was required to activate wild-type SA409
since it is suboptimal and that SA409 suboptimality was required for regulation of SA409 by ENH-1 and TRAP150.
Interestingly, all of the E6*I splicing acceptors that were investigated in this study (HPV31, 18 and 45) possess interrupted polypyrimidine tracts (Supplementary Figure S10B),
which emphasizes the importance of cis-RNA elements andtrans-acting factos for regulation of HR-HPV E6*I splicing.

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Figure 7. Significance of TRAP150 regulation of HPV16 E6/E7 mRNA splicing in SiHa cells. (A) Schematic model of how TRAP150 and ENH-1 enhance
HPV16 226∧ 409 mRNA splicing and promote production of the major E7-producing mRNA named E6*I/E7 (226∧ 409). Interaction of TRAP150 with theENH-1 RNA sequence facilitates U2AF complex formation, thereby activating HPV16 splice site SA409. Increased production of the 226∧ 409-spliced
E6*I/E7 mRNA enhances production of HPV16 E7 oncoprotein that promotes cell proliferation. The increase of the 226∧ 409-spliced mRNA occurs at
the expense of the IR E6 mRNA which is required for production of the HPV16 antiapoptotic E6 oncoprotein. (B) Cell viability of HPV16-positive SiHa
cells (cervical cancer cells) transfected with various amounts of pTRAP150 plasmid was evaluated by WST-1 assay. Fold change over control is shown.
Mean values and SDs are shown. n.s., no significance; **P < 0.01; ***P < 0.001. (C) Western blotting on proteins from in vitro RNA oligo pull-down
samples using RNA oligo encoding HPV16 wild-type (WT) or mutant (M1M4) ENH-1 sequence (for sequences of RNA oligos, see Figure 4A). Filters
were stained with antibodies specific for U2AF65 or U2AF35. MW, molecular weight marker. (D) Effect of TRAP150 and BCLAF1 overexpression on
HPV16 226∧ 409 mRNA splicing was evaluated by RT-qPCR on RNA extracted form HeLa cells transfected with HPV16 subgenomic plasmid pC97ELsL
with empty pUC plasmid (–) or with TRAP150- or BCLAF1-expressing plasmids pTRAP150 or pBCLAF1, or both. RT-qPCR was performed with primers
E6*I-F and E6*I-R that specifically amplify cDNAs representing HPV16 mRNAs spliced between SD226 and SA409 (226∧ 409 mRNAs). The mRNA
expression levels were normalized to gapdh. ****P < 0.0001. (E) RT-PCR on RNA extracted form SiHa cells transfected with siRNAs to TRAP150(si-TRAP150) or to BCLAF1 (si-BCLAF1) or with scrambled siRNAs (si-scr). RNA was extracted at 72 h post-transfection. RT-PCR was performed withprimers 97S and 438A that amplify cDNAs representing HPV16 IR E6 mRNAs or 226∧ 409-spliced mRNAs (see Figure 1B for location of RT-PCR
primers). gapdh was used as a control. M, marker. (F) Quantification of bands representing HPV16 IR E6 mRNAs on the gel image in (C). Quantitation
was performed as described in the Materials and methods. Band intensities were all divided by the band intensities in the si-scr control. Mean values
and SDs are shown. n.s., no significance; ***P < 0.001. (G) Quantification of bands representing HPV16 226∧ 409-spliced mRNAs using the cDNAs
analyzed in (C). Quantitation was performed by RT-qPCR as described in the Materials and methods. RT-qPCR was performed with primers E6*I-F andE6*I-R, and normalized by gapdh. Mean values and standard deviations are shown. n.s., no significance. **P < 0.01. (H) TRAP150 and BCLAF1 mRNAlevels were determined by RT-qPCR on the cDNA used in (C). RT-qPCR results were normalized to gapdh. Fold change of TRAP150 and BCLAF1 mRNAlevels was calculated by dividing by TRAP150 and BCLAF1 mRNA levels in the si-scr transfections. Mean values and standard deviations are shown.
Primer information is available in Supplementary Table S2. (I) Cell viability of HPV16-positive SiHa cells (cervical cancer cells) transfected with siRNAs to
TRAP150 (si-TRAP150) to BCLAF1 (si-BCLAF1) or both or with scrambled siRNAs (si-scr) was evaluated by WST-1 assay. Fold change over control isshown. Mean values and SDs are shown. n.s., no significance; **P < 0.01.

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Figure 8. An intact HPV16 splicing enhancer-1 (ENH-1) RNA element is required for immortalization of human primary keratinocytes. (A) Description of
the procedure for immortalization of primary human keratinocytes. Untransfected primary human keratinocytes or primary human keratinocytes
transfected with HPV16 subgenomic plasmid pC97ELsL harboring a wild-type ENH-1 element or pC97M1M4 with four nucleotide substitutions in
ENH-1 (displayed in Figure 2F and G) were treated with DMEM supplemented with 10% BCS to determine if the HPV16 plasmids could confer calcium
and serum resistance to the cells and cause cell immortalization. (B) After 60 days of selection of untransfected and pC97ELsL- or
pC97M1M4-transfected primary human keratinocytes in DMEM supplemented with 10% BCS, the color of the cell culture medium was inspected andthe presence of cellular colonies in the 15 cm plates was monitored. Upper panel: yellow cell culture medium is indicative of cell growth while red cell
culture medium suggests absence of cell growth. Lower panel: cell colony formation is indicated with white dotted lines surrounding colonies ofcalcium- and serum-resistant cells. (C) Population doubling curve of two parallel cultures of untransfected primary human keratinocytes (nHFK#1 and #2)(passage 4 at start) and of two parallel cultures of pC97ELsL-transfected primary human keratinocytes selected in DMEM supplemented with 10% BCS
causing immortalization (JJM9721#1 and #2) (passage 15 at start). After 30 days, nHFK#1 and #2 had died while JJM9721#1 and #2 kept growing. The
experiment was terminated after 60 days. (D) hTERT expression levels at different passages in nHFK (starting at passage 4) or JJM9721 (starting atpassage 15) cells were determined by RT-qPCR as described in the Materials and methods. hTERT expression levels were normalized by gapdh
expression, and fold changes of hTERT expression were calculated based on each starting passage (described as 0). (E) Levels of IR E6 mRNA weremonitored by RT-qPCR in JJM9721 cells transfected with siRNAs to TRAP150 or with scrambled siRNAs (si-scr). E6QF and 234A primers were used for
IR E6 mRNA-specific RT-qPCR. The IR E6 mRNA levels were monitored at 24 and 48 h post-transfection and normalized to gapdh mRNA levels. (F)Description of the procedure for immortalization of primary human keratinocytes. Primary human keratinocytes were transfected with pRSVneo plasmidor HPV16 genomic plasmids pHPV16AN harboring a wild-type ENH-1 element or with pHPV16ANM1M4 containing four nucleotide substitutions in
ENH-1 (displayed in Figure 4A and G). Two days after transfection, the transfected cells were selected by 40 μg/ml G418 treatment for 3 days followedby 10 μg/ml for 3 days. After the selection, cells were grown until confluent followed by selection of calcium- and serum-resistant cells in DMEM
supplemented with 10% BCS for 11 weeks, after which cells were fixed and stained with crystal violet. (G) hTERT expression levels for several passages
in primary human keratinocytes stably transfected with pRSVneo, pHPV16AN or pHPV16ANM1M4 were monitored by RT-qPCR. hTERT expression
levels were normalized by gapdh expression, and fold changes of hTERT expression were calculated based on each starting passage (described as 0).
(H) Formation of calcium- and serum-resistant cell colonies after transfection of primary human keratinocytes with pRSVneo, pHPV16AN or
pHPV16ANM1M4. The transfected cells were selected in G418 until confluent, followed by selection of calcium- and serum-resistant cells for 11 weeks
in DMEM supplemented with 10% BCS and then fixation and staining with crystal violet. Co-transfection of pHPV16AN or pHPV16ANM1M4 with
Cre-expressing plasmid released the episomal form of the HPV16 genome (see Figure 4G for details).

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Discussion
Although not universally true, the majority of known splicing enhancers are purine rich (45), sometimes adenosine rich(46). The existence of splicing enhancers is not easily predictedbased on sequence analysis as a result of positional effects,
RNA secondary structure, the relative promiscuity of RNAbinding proteins and combinatorial effects (47). Thus, experimental results are required to unequivocally identify splicing enhancers. The HPV16 splicing enhancer element (ENH1) identified here represents a perfect 11 nucleotide repeat
with an adenosine content of 73% (Figure 2A). Alignment
with various HR-HPV sequences revealed that none of themhad perfect bipartite repeats, but all had bipartite high A content regions (Supplementary Figure S5). Compared with thecorresponding sequences in LR-HPVs, the HR-HPV ENH-1
sequences had a significantly higher adenosine content. Thiswas particularly striking in the second 11 nucleotide repeat inwhich HR-HPVs had an average A content of 66% whereas
LR-HPVs had an average A content of 44%. In contrast, theadenosine content of the first 11 nucleotide motif was moresimilar between HR- and LR-HPVs (74% in HR-HPVs versus 65% in LR-HPVs). Finally, the spacer sequence between
the two repeats had a similar adenosine content in LR- andHR-HPVs (average 30%), approaching the generally expected
25% (Supplementary Figure S5). Taken together, these results
suggest that the splicing enhancer in the high-risk types may
have arisen evolutionarily by the accumulation of adenosinein the region corresponding to the second 11 nucleotide repeatin HPV16 and the other HR-HPVs.
Although our data clearly indicate that the A richness of thetwo 11 nucleotide repeats is a defining property of the splicing
enhancer in HR-HPV types, we believe that A richness itself it
is not sufficient to generate a splicing enhancer. Some degreeof higher sequence complexity is clearly needed. For example,
positions 5 and 6 in the 11 nucleotide A-rich repeat appear
to tolerate a variety of substitutions in the various HR-HPV
types (Supplementary Figure S5A, B) and the ‘G’ nucleotide
at position 10 is strictly conserved among the HR-HPV types.
In addition, the 10 nucleotide spacer sequence in between the
11 nucleotide A-rich repeats shows that four nucleotide positions are strictly conserved among high-risk types whereas theremaining six nucleotides may vary. Thus, A richness in combination with strictly conserved A or non-A nucleotides arelikely to define the splicing enhancer. Considering the multiple
RNA processing factors that interact with ENH-1, we speculate that the entire ENH-1 region, including the 10 nucleotide
spacer, act in concert as a ‘landing pad’ for multiple splicing
factors including TRAP150, PSF and RBM15 that contributeto the control of HPV16 226∧ 409 splicing.
In addition to ENH-1, the deletion analysis in Figure 1 revealed that sequences downstream of ENH-1 also appear toaffect splicing between SD226 and SA409. However, mutating
ENH-1 was sufficient to significantly reduce splicing between
SD226 and SA409 in the E6 coding region regardless of thedownstream elements. These results established that ENH-1
is essential for HPV16 226∧ 409 splicing. One may speculatethat the downstream sequences represent splicing-modulatory
elements or act on RNA secondary structure in such a waythat splicing between SD226 and SA409 is affected, but theirtrue significance remains to be determined. The presence ofmultiples positive and negative factors that interact with thecis-RNA regulatory elements (ENH-1 and possibly others)
and the presence of splicing acceptor sites proximal to SA409(SA526 and SA742) support the idea that multiple control
mechanisms are in place, and postulate that dynamic structural changes could occur in both proximal and distant RNA
regions, perhaps influenced by protein–protein, protein–RNA

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from passage 4 as nHFK#1 and nHFK#2. Cell growth curves
were established for the four cell cultures (Figure 8C). Whilethe nHFKs died after 30 days of culture, the pC97ELsLtransfected cells continued to grow until the experiment was
terminated after 60 days. In addition, up-regulation of hTERT
expression was observed in pC97ELsL-transfected cells, while
hTERT mRNA levels were down-regulated in nHFKs (Figure 8D). The cell line that had been established was named
JJM9721. Our results supported the idea that the pC97ELsLtransfected JJM9721 cells had been immortalized.
To assess the significance of TRAP150 for the regulation of HPV16 oncogene expression in the JJM9721 cells,
TRAP150 was knocked-down with siRNAs (Supplementary
Figure S12B) and the effect on HPV16 IR E6 mRNAs andspliced 226∧ 409 mRNA levels was evaluated by RT-PCR(Supplementary Figure S12C). The results revealed that theknockdown of TRAP150 altered the ratio of IR E6 mRNAs
to 226∧ 409-spliced mRNAs in JJM9721 cells (Supplementary Figure S12D, E). Statistically significant changes wereobserved only for the increase of the IR E6 mRNA at 24 hpost-transfection although there was a tendency towards adecrease in 226∧ 409 mRNA by TRAP150 knockdown. The
effect of the HPV16 IR E6 mRNA levels was also evaluatedby RT-qPCR using primers that were specific for IR E6 mRNAs. The results revealed that there was a statistically significant increase of HPV16 IR E6 mRNAs at both 24 and 48
h post-transfection of the JJM9721 cells (Figure 8E). Taken
together, these results indicated that mutational inactivationof the HPV16 splicing enhancer ENH-1 abolished the abilityof the HPV16 pC97ELsL plasmid to immortalize nHFKs, aprocess mediated by TRAP150.
Finally, the immortalizing activity was further assessedby transfection of nHFKs with HPV16 genomic plasmid pHPV16AN or the corresponding M1M4 mutant
pHPV16ANM1M4 as described in Figure 8F. Co-transfectionof pHPV16AN or pHPV16AN-derived plasmids releases theepisomal form of the HPV16 genome. Compared with the immortalization assay described in Figure 8A, the immortalization assay used for pHPV16AN and pHPV16ANM1M4 included a selection step of pHPV16AN-transfected cells with
G418 (Figure 8F). After selection of pHPV16AN-transfected
cells in G418, cells were propagated further for analysis of either hTERT expression (Figure 8G) or colony formation by
crystal violet staining (Figure 8H). pHPV16AN-transfected
cells showed an up-regulation of hTERT expression (Figure8G) and colony formation (Figure 8H), neither of which wasobserved in cells transfected with the M1M4 mutant (Figure
8G, H). Similar results were obtained when the pHPV16ANderived plasmids were transfected in the absence of Cre plasmid (Supplementary Figure S13). We concluded that an intact
HPV16 ENH-1 RNA element was required for immortalization of nHFKs by the HPV16 genome. Taken together, ourresults established the significance of a HPV16 splicing enhancer (ENH-1) for HPV16 E6 and E7 oncoprotein production and cell immortalization, and indicate that the HPV16
ENH-1 RNA element and its partner TRAP150 are criticalfor HPV16 carcinogenesis.

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down by the wild-type ENH-1 RNA oligo but not with the
mutant RNA oligo (Supplementary Figure S14). Recent studies have revealed novel functions for PABPN1 that include regulation of RNA splicing, and RNA degradation and export(49,50). It is of interest to further investigate if PABPN1 is a
splicing regulator specific for HR-HPV E6/E7 mRNA splicing. Besides PABPN1, the A-to-I editing ADAR1 enzyme acts
on adenosines. ADAR1 appears to be a prognostic factor for
cervical cancer (51). One study reported that HPV16 E7 protein levels in SiHa were enhanced by ADAR1 knockdown (52).
However, this effect was apparently mediated by the innate
immune system rather than A-to-I editing, as no A-to-I editing was detected on HPV transcripts.
HR-HPVs [two HR-HPVs from the alpha-7 species(HPV18 and HPV45) and two from the alpha-9 species(HPV16 and HPV31)] analyzed in this study responded to
TRAP150 overexpression with increased splicing, despite displaying bigger sequence divergence in the corresponding enhancer elements than that between HPV16 wild-type andM1M4 mutant enhancer. One may speculate that TRAP150
targets adenosine-rich, bipartite-like RNA elements, possiblyinterrupted by 1–3 non-adenosine residues. TRAP150 was initially identified as a spliced mRNP-associated protein (53),but an RNA-binding motif in TRAP150 has not been identified to our knowledge. However, the N-terminal RS domainof TRAP150 has been shown to contribute to the regulationof alternative splicing by an unknown mechanism (31). Both
TRAP150 and BCLAF1 contain SR-domains. Many of theserine–arginine-rich (SR) proteins interact with purine-rich
RNA elements to enhance splicing (45). We showed that theeffect of TRAP150 was enhanced by BCLAF1 (Figure 7D). In
this regard, it is of interest to note that BCLAF1 is mutationally inactivated in ∼40% of HPV-positive tonsillar and baseof tongue cancer (54), suggesting that the absence of BCLAF1
may alter HPV16 E6/E7 mRNA splicing in these cancers.
The correlation between TRAP150 and cervical cancer development remains to be elucidated. In this report, we suggest
a model in which TRAP150 promotes HPV16 E6*I/E7 alternative splicing and E7 protein production via interactions
with the identified HPV16 cis-acting RNA element ENH-1,
thereby effectively controlling and upholding an optimal balance of HPV16 E6 and E7 oncogene expression. TRAP150
overexpression did enhance E7 mRNA and protein levels, but
resulted in reduced cell viability, presumably due to reduced
E6 production caused by ‘over splicing’ of the E6/E7 premRNAs (Figure 7A). In fact, it has been reported that both
E6 and E7 protein expression is required for HPV-induced
cell immortalization in vitro (55). RNA-seq results from 169
HPV-positive cervical cancer samples revealed that all cancer
samples had detectable expression of both HPV E6 and E7
mRNAs (56). On the other hand, it has been shown that the
spliced E6*I/E7 mRNA is overexpressed in high-grade lesions
and cancer caused by HR-HPV infections, and therefore potentially could serve as a biomarker for women at risk of progressing to cancer (16,17,57,58). Furthermore, high expression of E6*I/E7 spliced mRNA was shown to predict poor
response to treatment of cervical cancer (59). Based on the results presented here, we suggest that the E6*I/E7 mRNA splicing is increased due to dysregulated expression of TRAP150
and that it may be of interest to determine the utility of
TRAP150 as a biomarker for cancer progression or treatment
response. Indeed, TRAP150 is part of the DNA damage response induced by HPV (43,60). Both TRAP150 and BCLAF1
are highly expressed in the cervical epithelium, with higher

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and RNA–RNA interactions. As a result, the formation of
spliceosomal complexes could shift between the splice acceptors and affect which splice acceptor choice is made within
the HPV16 E6/E7 gene region. Our experimental system inthis study was simplified to primarily focus on the regulationof 226∧ 409 mRNA splicing, limiting our ability to fully comprehend the complexity of HPV16 mRNA splicing.
An enigmatic result that appeared from this study wasthat the four nucleotide substitutions in the splicing enhancer (M1M4) that virtually abolished production of spliced
E6*I/E7 mRNAs without major effects on the IR E6 mRNAs not only reduced production of E7 protein as predicted,
but also dramatically reduced the levels of E6 protein (Figure
2K; Supplementary Figure S1F). RNA analysis after cell fractionation revealed that IR E6 mRNAs carrying the M1M4
mutations displayed an RNA export defect that resulted in
low E6 protein levels (Supplementary Figure S3C). It has
been reported that purine-rich splicing enhancers present on
unspliced mRNAs interact with nuclear factors that retain
these mRNAs in the nucleus, a process that is relieved bysplicing (48). On the other hand, our results demonstrated
that the association of TRAP150 with the M1M4 mutant
HPV16 mRNAs was impaired (Figure 6B, C). TRAP150 has
been reported to facilitate the RNA export of spliced mRNAs by interacting with the mRNA, exon junction complex
components and the export receptors (30,33). These results
may indicate that mutational inactivation of splicing enhancer
ENH-1 deprives the mRNA of TRAP150 or other ENH-1associated factors necessary for further mRNA processing including RNA export. Alternatively, it may indicate that RNA
stability of the M1M4 mutant mRNAs in the cytoplasm wasattenuated by an unidentified mechanism. These results may
imply that TRAP150 has different effects on IR mRNAs andspliced mRNAs, and modulates the RNA levels in the nucleus
and cytoplasm differently. Although our results indicate that
TRAP150 is an alternative splicing regulator, we could notentirely exclude that TRAP150 might affect RNA stability
and/or the efficiency of RNA nuclear export of individual alternatively spliced mRNAs to achieve the same results. Lee etal. reported that TRAP150-associated unspliced pre-mRNAs
are destined to get degraded in the nucleus (30). The observed
decrease in E6 mRNA and protein levels by TRAP150 expression could potentially be explained by TRAP150-mediateddegradation of IR E6 mRNAs in the nucleus. However, quantitation of IR E6 mRNA and spliced E7 mRNA in the absence orpresence of TRAP150 demonstrated that an increase in spliced
E7 mRNA levels compensated for the reduction in IR E6 mRNAs, indicating that the reduction in the levels of IR E6 mRNAs in the presence of TRAP150 was caused by an increasein splicing rather than increased degradation of E6 mRNAs
mediated by TRAP150. Nonsense-mediated decay (NMD) of
HPV mRNAs has not been extensively researched. Further experimentation is required to determine if NMD contributes tothe control of E6/E7 gene expression. It is of interest to determine how HPV16 E6 protein levels were reduced when ENH1 was mutated and how TRAP150 differently regulates IR E6
mRNAs and spliced E7 mRNAs post-splicing.
The presence in the ENH-1 element of multiple consecutive adenosines (A-tracts) led us to speculate that ENH-1 may
be targeted by poly(A)-binding protein (PABP) or adenosine
deaminases acting on RNA (ADAR). In fact, PABPN1 was
identified in our MS analysis following ENH-1 RNA oligo
pull-down assay (Supplementary Figure S6C). Western blotting demonstrated that nuclear PABP (PABPN1) was pulled

Nucleic Acids Research, 2024, Vol. 52, No. 1

Nucleic Acids Research, 2024, Vol. 52, No. 1

staining in the basal and middle cell keratinocyte layers than in
the upper differentiated cells (www.proteinatlas.org). In summary, interactions between TRAP150 and the HPV16 ENH-1
RNA element are critical for HR-HPV pathogenesis, demonstrating that both TRAP150 and the splicing enhancer ENH-1
may be exploited as targets for therapy to treat high-grade lesions or cancer caused by HR-HPV infections, or as diagnostic
or predictive biomarkers.

Data availability

Supplementary data
Supplementary Data are available at NAR Online.

Acknowledgements
We thank Neele Bergemann, Xiaoxu Cui, Chengyu Hao, Yunji
Zheng, Qiaoli Zhai and Chengjun Wu for advice and discussion. We would like to thank Charlotte Welinder andthe Center for Translational Proteomics at Medical Faculty,
Lund University for mass spectrometry analysis. We are grateful to Tanel Punga, Göran Akusjärvi, Catharina Svensson,
Göran Magnusson and Anders Bergqvist and members of
their groups for insightful discussions and input. We thankJoakim Esbjörnsson and Patric Jern for valuble advice.
Author contributions: S.S. and N.K. conceptualized the
project. J.J., N.K. and L.W. performed the experiments. J.J.,
N.K. and S.S. prepared the figures. J.J., L.W. and N.K. performed statistical analysis. S.S., J.J. and N.K. wrote themanuscript.

Funding
The Swedish Research Council [2019–01210 to S.S.]; Swedish
Cancer Society [CAN2018/702 and CAN 2018/1164 to S.S.];
and Stiftelsen Clas Groschinskys Minnesfond [M21126 and
M2320 to N.K].

Conflict of interest statement
None declared.

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Received: March 24, 2023. Revised: October 18, 2023. Editorial Decision: October 28, 2023. Accepted: November 3, 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Forcommercial re-use, please contact journals.permissions@oup.com

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Nucleic Acids Research, 2024, Vol. 52, No. 1