JVI Accepted Manuscript Posted Online 25 February 2015 J. Virol. doi:10.1128/JVI.03434-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

Human papillomavirus type 16 oncoprotein expression is

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controlled by the cellular splicing factor SRSF2 (SC35).

3 4 5 6

Melanie McFarlane, Alasdair I MacDonald, Andrew Stevenson and Sheila

7

V Graham*

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MRC-University of Glasgow Centre for Virus Research; Institute of Infection,

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Immunity and Inflammation; College of Medical Veterinary and Life Sciences,

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University of Glasgow, Garscube Estate, Glasgow, G61 1QH, Scotland, UK

11 12

Running title: HPV16 oncoprotein RNA is stabilised by SFSR2.

13 14

*Corresponding author.

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Rm 253, Jarrett Building, Garscube Estate, University of Glasgow, Glasgow,

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G61 1QH, Scotland, UK.

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Tel: 44 141 330 6256; Fax: 44 141 330 5602;

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e-mail: [email protected]

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Key words: SRSF2 (SC35), HPV16, E6/E7 oncoproteins, RNA processing

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Abstract word count: 208

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Text Word count: 4857

1

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Abstract

24

High risk human papillomaviruses (HR-HPV) cause anogenital cancers,

25

including cervical cancer, and head and neck cancers. Human papillomavirus

26

type 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven

27

by two viral oncoproteins, E6 and E7, which are expressed through alternative

28

splicing of a polycistronic RNA to yield four major splice isoforms (E6 full

29

length, E6*I, E6*II, E6*X). Multiple RNA production from a single gene is

30

controlled by SR splicing factors (SRSFs) and HPV16 infection induces

31

overexpression of a subset of these, SRSFs1, 2 and 3. In this study we

32

examined whether these proteins could control HPV16 oncoprotein

33

expression. siRNA depletion experiments revealed that SRSF1 did not affect

34

oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in

35

E6E7 expression depletion of SRSF2 resulted in a significant loss of E6E7

36

RNAs resulting in reduced levels of the E6-regulated p53 proteins and E7

37

oncoprotein itself. SRSF2 contributed to the tumour phenotype of HPV16-

38

positive cervical cancer cells as its depletion resulted in decreased cell

39

proliferation, reduced colony formation and increased apoptosis. SRSF2 did

40

not affect transcription from the P97 promoter that controls viral oncoprotein

41

expression. Rather, RNA decay experiments showed that SRSF2 is required

42

to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key

43

regulator of HPV16 oncoprotein expression and cervical tumour maintenance.

44

2

45

Importance

46

Expression of the HPV 16 oncoproteins E7 and E6 drives HPV-associated

47

tumour formation. Although increased transcription may yield increased levels

48

of E6E7 mRNAs, it is known that the RNAs can have increased stability upon

49

integration into the host genome. SR splicing factors (SRSFs) control splicing

50

but can also control other events in the RNA life cycle including RNA stability.

51

Previously, we demonstrated increased levels of SRSFs 1-3 during cervical

52

tumour progression. Now we show that SRSF2 is required for expression of

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E6E7 mRNAs in cervical tumour, but not nontumour cells, and may act by

54

inhibiting their decay. SRSF2 depletion in W12 tumour cells resulted in

55

increased apoptosis, decreased proliferation and decreased colony formation

56

suggesting that SRSF2 has oncogenic functions in cervical tumour

57

progression. SRSF function can be targeted by known drugs that inhibit SRSF

58

phosphorylation suggesting a possible new avenue in abrogating HPV

59

oncoprotein activity.

60 61 62 63

3

64

Introduction

65 66

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelia. At

67

least thirteen so-called “high risk” (HR) HPV infect the anogenital epithelium

68

and can cause persistent lesions that may progress to cancer (1). For

69

example, around 500,000 women worldwide experience anogenital HPV

70

infection and nearly 300,000 die per annum

71

Increasingly, HPV infection is also being linked to oropharyngeal cancer

72

whereby incidence of this disease is increasing rapidly (2). HPV type 16

73

(HPV16) is the most prevalent HR-HPV. HPV-associated tumourigenesis is

74

driven by increased expression of HPV E6 and E7 oncoproteins (3).

75

promotes ubiquitin-mediated degradation of p53 to inhibit apoptosis,

76

modulates transcription of cell cycle-related genes, induces telomerase

77

activity, controls cell shape and polarity, and activates cap-dependent

78

translation (4). E7 binds and degrades Rb to promote S phase entry and cell

79

division, controls transcription of cell cycle-related genes and acts as a mitotic

80

mutator (4) .

from cervical cancer.

E6

81 82

HPV E6 and E7 oncoproteins are expressed from a polycistronic transcript

83

that for HPV16, can potentially produce four different alternatively spliced

84

mRNAs (E6 full length (E6fl), E6*I, E6*II and E6*X (also called E6*III)) (5,6).

85

The putative E6* proteins all share the first 44 amino acids of full length E6

86

with C-terminal truncations or frame shifts into the E7 open reading frame (5).

87

E6*I is the most abundant isoform in cervical cell lines (7-10) and patient

88

samples (11,12) and has been suggested to encode E7 (6). Although

4

89

detectable in tumour samples (12), the biological function of E6*II and E6*X

90

has not been investigated.

91 92

SR proteins (serine-arginine-rich splicing factors, SRSFs) can regulate most

93

of the processes in the life cycle of an mRNA, including transcription, RNA

94

processing, RNA export, RNA stability and translation (13). SR proteins are

95

key players in the regulation of constitutive and alternative splicing.

96

Constitutive splicing is the process whereby introns are removed from pre-

97

mRNAs and exons are spliced together to form a protein-coding mRNA.

98

Alternative splicing is a mechanism used by mammalian and viral genomes to

99

maximise coding potential (14). A single gene is transcribed to give a single

100

primary transcript but from this precursor RNA different mature mRNA

101

isoforms can be generated by differentiation inclusion or exclusion of exons

102

and introns. Each isoform can encode a different protein. There are nine

103

classical SR proteins named SRSF1-9. Apart from RNA processing-related

104

functions, SR proteins have also been shown to be involved in chromatin

105

remodelling, transcriptional

106

nucleolar stress, cell cycle progression, apoptosis control and protein

107

sumoylation (15,16). Unsurprisingly, due to their diverse functions, many SR

108

proteins are over expressed in a range of tumours (17-21). Importantly,

109

SRSF1 (ASF/SF2), SRSF3 (SRp20) and SRSF9 (SRp30c) have been shown

110

to possess oncogenic properties (22-27). Increased SRSF levels can result in

111

production of alternatively spliced RNA isoforms encoding key anti-apoptotic,

112

cell proliferation and epithelial-mesenchymal transition (EMT)-inducing

113

proteins (28).

regulation, genome stability maintenance,

5

114

HPV16 oncoprotein expression is controlled at several SRSF-regulated

115

posttranscriptional levels including constitutive and alternative RNA splicing,

116

RNA stability and translation (6,29,30). Increased expression levels of

117

SRSFs1-3 in cervical tumour cells and samples from patients with HPV-

118

positive cervical lesions (20) prompted an investigation of a possible

119

oncogenic roles of SR proteins in cervical cancer. Here we examine the effect

120

of SRSF depletion on E6E7 mRNA production in cervical tumour cells. Data

121

revealed that SRSF1 and SRSF3 depletion had very little effect on E6E7

122

expression. However, SRSF2 (SC35) depletion caused a marked reduction in

123

levels of E6E7 mRNAs leading to reduced levels of E7 protein and induction

124

of p53 indicating a reduction in E6 protein levels. SRSF2 depletion resulted in

125

reduced cell proliferation, decreased anchorage independent growth and

126

induction of apoptosis in the tumour cells. SRSF2 did not control transcription

127

of E6E7 RNAs but rather controlled their stability. These data suggest that

128

SRSF2 is a key cellular factor controlling HPV16 oncoprotein expression by

129

protecting the RNAs encoding them from decay. Since SR protein activity is a

130

proven relevant antiviral drug target (31) these findings provide insight into

131

new therapeutic avenues against HPV-associated oncogenesis.

132

6

133

Materials and Methods

134 135

Cell culture and reagents

136

W12E and W12G cells are clones 20863 and 20861 respectively as described

137

in Jeon et al., 1995. W12t and W12ti cells were cloned in our laboratory from

138

W12G cells and were originally named W12GPX and W12GPXY(32). W12ti,

139

293T, CaSki and SiHa cells were passaged in DMEM (Invitrogen), 10% foetal

140

calf serum (FCS) (Invitrogen) and penicillin (50 U/ml)/ streptomycin (50 µg/ml)

141

(Invitrogen). W12E, W12G and W12t cells (32) were cultured in the same

142

medium but with addition of 0.1 M cholera toxin and 0.4 µg/ml hydrocortisone.

143

W12E and W12G cells were grown (2x105 cells per 10 cm plate) on mitomycin

144

C-treated J2 3T3 mouse fibroblasts (33). Differentiation was achieved as

145

previously described (10,33). 3T3 cells were grown in DMEM, 10% donor calf

146

serum with antibiotics as described above. All cells were cultured at 37°C in a

147

5% CO2 humidified incubator.

148 149

Transfection

150

Cells were seeded at 2x105 per well in a six well plate 24 h prior to

151

transfection in antibiotic-free medium.

152

RNAi MAX (Invitrogen) or plasmid (100 ng) and Lipofectamine (Invitrogen)

153

were diluted in Opti-MEM serum-free medium (Invitrogen). siRNAs against

154

SRSF2 were a siGENOME Dharmacon SMART-pool (M-019711-00-0005)

155

consisting of four siRNAs or a single siRNA whose efficacy has been

156

previously demonstrated (34). SMARTpool siRNAs were also used to deplete

157

SRSF3

(M-030081-00-0005).

siRNA (10 µM) and Lipofectamine

SRSF1

was

depleted

using

Sigma

7

158

SASI_Hs02_00313260. Cells were transfected for 48 h according to the

159

manufacturer’s

160

transfection with siGLO (Dharmacon) or a GFP expression plasmid were

161

between 70-80% for W12E/G, CaSki and C33A, 80-90% for W12ti cells and

162

>90% for 293T cells.

protocol.

Transfection

efficiencies

calculated

by

co-

163 164

RNA extraction

165

All protocols followed the manufacturer’s instructions unless otherwise stated.

166

Cells were scraped into TRIzol reagent (Invitrogen) and total RNA was

167

extracted. Polyadenylated RNA was isolated using an oligo-dT-based mRNA

168

extraction kit (Oligotex, Qiagen). DNA was removed from all RNAs using the

169

Promega RQ1 DNase kit.. DNase-treated RNA was reverse transcribed using

170

the Superscript III kit (Invitrogen).

171 172

PCR

173

cDNA was amplified using 200 nM primers, 200 µM dNTPs, 1.5 mM MgCl2

174

and 2 units Taq polymerase (Invitrogen). Primers were chosen at the 5’ end of

175

the E6 open reading frame and the 3’ end of the E7 open reading frame so as

176

to

177

GAGAACTGCAATGTTTCAGGACCC-3’ (HPV16 genome nts 94-117); E7

178

reverse 5’-GAACAGATGGGGCACACAATTCC-3’ (HPV16 genome nts 845-

179

823). PCR products were separated on a 6% polyacrylamide gel and post-

180

stained with ethidium bromide.

amplify

all

E6E7

isoforms:

E6

forward

5’-

181 182

8

183

qRT-PCR

184

Standard curves were generated as recommended (Applied Biosystems

185

instruction manual). cDNA (100 ng) was amplified for each reaction and

186

carried out in triplicate. E6 forward primer: 5’-TCATGTTTCAGGACCCACAG-

187

3’. E6 reverse primer: 5’-CTGTTGCTTGCAACAGAGCTGC-3’. E6 probe

188

sequence: 5’-CCACAGTTATGCACAGAGCTGC-3’. GAPDH was used as the

189

internal standard control. GAPDH forward: 5’-CGCTCTCTGCTCCTCCTGTT-

190

3’. GAPDH reverse: 5’-CCATGGTGTCTGAGCGATGT-3’. GAPDH probe: 5’-

191

CAAGCTTCCCGTTCTCAGCC-3’. Reaction mixes (25 µl) contained 1x

192

Mastermix (Stratagene), 900 nM primers, 100 nM probe, 300 nM reference

193

dye (Stratagene).

194

Applied Biosystems 7500 Fast System.

qPCR reactions were performed and analysed on an

195 196

Western blotting

197

Cells were harvested into NP40 lysis buffer (0.5% NP40, 150 mM NaCl, 50

198

mM Tris-HCl pH 8.0 with protease and phosphatase inhibitor cocktails (Roche

199

Diagnostics)). Lysates were cleared by centrifugation at 10,000 g for 10 min

200

at 4oC. Protein concentration was determined by Bradford assay. SDS-PAGE

201

and western blotting was carried out exactly as described (35). Primary

202

antibodies

203

supernatant

204

Biotechnology), p53 (1:500, BD Pharmingen clone DO-7), Rb (1:2000, Cell

205

Signalling Technology clone 4H1), SRSF1 (1:1000, Zymed clone 96), SRSF2,

206

SRSF3 (Pharmingen, 1:1000), (1:250, Zymed clone 7B4) and HPV16 E6

were clone

as

follows: Mab104,

anti-phospho-SRSF used

neat),

(ATTC

GAPDH

hybridoma

(1:1000,

AMS

9

207

(1:200, Santa Cruz sc1583). Secondary antibodies (Sigma) were used at a

208

dilution of 1:2000.

209 210

Annexin V assay

211

Cells were transfected 48 h before harvesting for staining. Control cells were

212

treated with UV light at 500 J/m2 24 h prior to harvesting for staining. Cells

213

were trypsinised, counted and resuspended at 1x106 cells/ml in 500 µl

214

annexin binding buffer (100 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2). Next,

215

100 µl of cells was mixed with 5 µl annexin V 488nm (Invitrogen) and 1 µg/ml

216

propidium iodide, and incubated at room temperature for 15 min in the dark.

217

Finally, 400 µl annexin binding buffer was added before analysis on a BD

218

Biosciences FACScalibur machine.

219 220

Colony formation assay

221

Cells were transfected 24 h prior to plating on soft agar. Cells were counted

222

and

223

agarose, and then 2.5 ml was plated out onto 6 cm plates containing 0.5%

224

agarose in 1xDMEM, 10% FCS. The plates were incubated at 37 °C in 5%

225

CO2 for 12-14 days. The cells were stained with 0.5 ml 0.005% crystal violet

226

for 1 h before being dried and photographed.

2x104 cells were resuspended in 1xDMEM, 10% FCS and 0.35%

227 228

Luciferase assay

229

293T cells were transfected as above with a control vector pGL3promoter

230

(Promega) or with an HPV16 LCR expression vector (36) and either siGLO or

231

SRSF2 siRNA. Cell here harvested after 48 h and luciferase reporter activity

10

232

(Promega luciferase assay kit) measured using a Thermolabs Luminoskan

233

Ascent plate reader.

234 235

mRNA stability assay

236

W12ti cells were transfected with control siRNA or with SRSF2 siRNA for 24 h

237

then actinomycin D at 10 µg/ml (or control vehicle (H2O) alone) was added to

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inhibit de novo RNA synthesis. RNA was harvested at 0, 1 and 4 h post-drug

239

addition. DNase 1-treated RNA was converted to cDNA and amplified by

240

semi-quantitative RT-PCR using the E6 primers described in the PCR section.

241

PCR products were separated on a 6% acrylamide gel and post-stained with

242

ethidium bromide.

243 244 245

11

246

Results

247 248

HPV16 oncoproteins are expressed from a set of alternatively spliced

249

mRNA isoforms in non-tumour and tumour cervical epithelial cells.

250

HPV16 expresses at least four HPV16 E6 mRNA isoforms predicted to be

251

generated by alternative splicing (Figure 1A) (6). Some of these isoforms

252

have been detected previously in cell lines and patient tissues (7,12,37-41).

253

However, it is unclear whether all isoforms can be expressed simultaneously

254

and/or are differentially expressed in non-tumour versus tumour cells. To

255

investigate expression of the E6 RNA isoforms we used the W12 cell line, an

256

epithelial cell line derived from an HPV16-infected low grade cervical lesion

257

(42). Previous studies demonstrated that W12 cells express at least some E6

258

RNA isoforms (10,43). A subclone, W12E (clone 20863) contains ~100

259

episomal copies of the HPV16 genome, and if treated as a non-continuous

260

cell line (i.e. not passaged in culture) maintains a non-tumour phenotype

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capable of differentiation in monolayer culture (10,33). Another subclone,

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W12G (clone 20861) also displays a non-tumour phenotype but with reduced

263

differentiation capacity. This is despite the cells containing HPV16 genome

264

copies integrated into the host genome, often a hallmark of cervical cancer

265

cells (33). Previously, we derived two tumour lines sequentially from W12G

266

cells (32). W12t cells (formerly called W12GPX) are cervical tumour cells

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while W12ti (formerly called W12GPXY) have an invasive tumour phenotype

268

in vitro and in vivo (32). W12ti cells express 5-fold more total E6 RNA than

269

W12G cells (Figure 1B).

270

12

271

It proved very difficult to design appropriate cross-splice junction primers and

272

probes with similar efficiencies in qRT-PCR for each of the E6 RNA isoforms.

273

So a semiquantitative PCR strategy was designed to amplify simultaneously

274

all E6-encoding mRNA isoforms. Two minor E6 isoforms, E6^E4 and E6^L1,

275

were not included in the analysis because neither is expressed in W12G,

276

W12t or W12ti cells that contain integrated HPV16 genomes. Figure 1C tracks

277

3 and 5 show the result of amplification of E6E7 RNA isoforms from the non-

278

tumour W12E and W12G cell lines. Bands corresponding in size to E6fl, E6*I

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and E6*II were detected. These three bands were also detected in the tumour

280

cells W12t and W12ti. However, a band corresponding in size to E6*X was

281

also detected (tracks 7 & 9). No products were amplified in the absence of

282

reverse transcriptase. PCR products were sequenced to verify coding

283

potential. The band above E6fl in track 7 may represent the use of cryptic

284

splice sites within the E6 gene region. These data suggest that all four E6

285

isoforms can be simultaneously expressed in HPV positive cells.

286 287

E6 expression is controlled by SRSF2

288

Previously, we demonstrated that the three smallest SR proteins, SRSF1-3

289

are overexpressed during cervical tumour progression (20), suggesting that

290

they may possess cervical tumour promoting activities. Figure 2A-C shows

291

that these three proteins are overexpressed in W12t and W12ti tumour cells

292

(tracks 3 & 4) compared to a much lower expression level in the W12E and

293

W12G nontumour cells (tracks 1 & 2). Increased SR protein levels could

294

contribute to the increased levels of E6E7 RNAs in the tumour cells (Figure

295

1B). To determine whether HPV16 oncoprotein expression was controlled by

13

296

SRSF1, 2 or 3, expression of each SR protein was individually knocked down

297

using commercially available siRNA pools in W12ti tumour cells. Levels of

298

siRNA depletion of greater than 80% were achieved in every experiment

299

(Figure 3A). Knock down of SRSF1 (Figure 3B, track 6) had little effect on

300

E6E7 RNA isoform levels. SRSF3 depletion resulted in some reduction in

301

levels of all of the E6 RNA isoforms as observed in another study (29) (Figure

302

3B track 10). However, SRSF2 knock down resulted in a significant reduction

303

in levels of all E6 RNA isoforms (Figure 3B, track 12). In case this was due to

304

the siRNA pool used, the experiment was repeated using a single siRNA

305

known to effectively deplete SRSF2 (34). Very similar results were obtained

306

with a significant reduction in levels of E6 RNAs (data not shown). The E6*X

307

band is present but at low levels in the control tracks 4 and 8. Its apparent

308

absence in tracks 2, 6 and 10 could be due to the lower amounts of E6E7

309

RNAs in these PCR reactions. qRT-PCR was used to quantify total E6 RNA

310

levels in W12ti cells following SRSF depletion. Figure 3C shows that SRSF1

311

depletion caused around a 20% reduction in total E6 RNA levels. SRSF3

312

depletion reduced RNA levels by around 60% while SRSF2 depletion reduced

313

E6 RNA levels by over 90% compared to control transfected cells.

314 315

In case the effect of knock down of SRSF2 on E6 RNA levels was an artefact

316

of the cell line used, experiments were repeated in SiHa (data not shown) and

317

CaSki cells (Figure 4B). SRSF2 depletion also reduced total E6 RNA levels in

318

these cell lines. qRT-PCR analysis of total E6 levels in CaSki cells revealed

319

around a 60% reduction following SRSF2 depletion (Figure 4C). This

320

demonstrates that SRSF2 control of E6 isoform expression is not restricted to

14

321

the W12ti cell line. SRSF2 depletion in non-tumour W12G cells (the parental

322

line for W12ti) where it is not overexpressed (Figure 2A, 3E), had no

323

detectable effect on E6 RNA isoform expression (Figure 4D) but qRT-PCR

324

indicated a small increase, rather than a decrease in total E6 RNA levels.

325

These data suggest that overexpression of SRSF2 may be required to

326

maintain the high levels of viral oncoprotein RNA expression seen in the

327

cervical tumour cells.

328 329

SRSF2 knock down results in increased p53 and induction of apoptosis

330

E6 degrades the tumour suppressor p53 (44). Because it is difficult to detect

331

E6 oncoprotein using currently available antibodies we assessed changes in

332

p53 protein levels upon SRSF2 depletion as a read-out for E6 protein levels.

333

Levels of p53 increased significantly (p=0.021) in W12ti cells upon SRSF2

334

depletion suggesting that SRSF2 is required for E6 oncoprotein expression

335

(Figure 4A). E6 mRNA isoform E6*I encodes E7 oncoprotein (6). Reflecting

336

loss of the RNA isoform E6*I (Figure 3B), E7 protein levels also decreased in

337

cells treated with siRNA against SRSF2 (Figure 4B). However, no

338

corresponding increase in pRb was detected (Figure 4B).

339 340

Loss of E6 expression in the tumour cells should stabilise p53 leading to

341

apoptosis (4). Annexin V assays were carried out to determine if SRSF2

342

depletion caused cells to enter apoptosis (Figure 5A). Cells were treated with

343

control siRNA or siRNAs against SRSF2 (Figure 5C) or siRNA against E6

344

itself, then stained with fluorescent annexin V and propidium iodide followed

345

by flow cytometry analysis. Proliferating and senescing cells do not take up

15

346

either stain and are represented in the lower left hand quadrant of the two-

347

dimensional plots. Early apoptotic cells are present in the lower right hand

348

quadrant because they display annexin staining but are impermeable to

349

propidium iodide. Cells in the upper right hand quadrant are stained with both

350

annexin V and propidium iodide and are late apoptotic or dead cells. Material

351

in the upper left hand quadrant is likely dead cells/cell debris. As a positive

352

control for apoptosis, cells were treated with 500 J/m2 UV-B 24 hours prior to

353

harvesting. After this treatment the majority of cells were found in the early

354

(48.8%) or late (43.4%) apoptotic quadrants (Figure 5A, UVB). Treating W12ti

355

cells with a non-targeting siRNA (siCntrl) resulted in some apoptosis likely due

356

to the toxic effects of the siRNA transfection reagent (Figure 5A, siCntrl).

357

Depletion of E6 (and therefore derepression of p53) with a specific E6 siRNA

358

caused the majority of cells to enter apoptosis (31.5% early apoptosis and

359

58.6% late apoptosis) as expected (Figure 5A, siE6).

360

depletion of SRSF2 the majority of cells also entered the apoptotic quadrants

361

(58.1% early apoptosis, 34.3% late apoptosis) (Figure 5A, siSRSF2). Table 1

362

summarises the data from three separate experiments. Figure 5B shows the

363

mean and standard deviation from the mean of percentage live versus

364

apoptotic cells from three separate experiments. It is clear that depleting

365

tumour cells of SRSF2 mimicked the effect of direct E6 depletion by driving

366

cells into apoptosis.

Following siRNA

367 368

SRSF2 depletion causes a reduction in tumour cell proliferation and

369

anchorage-independent growth

16

370

If SRSF2 is required to maintain high expression levels of HPV16 oncoprotein

371

and its depletion causes apoptosis, SRSF2 depletion would be expected to

372

cause a reduction in cell proliferation. Cell proliferation was thus measured by

373

counting live cells (trypan blue exclusion) over a 72 hour time course in W12G

374

(HPV-positive non-tumour), W12ti (HPV-positive tumour) and C33a (HPV-

375

negative tumour) cells after transfection with siRNA against SRSF2.

376

Following SRSF2 depletion, the proliferation rate of the W12ti and C33a

377

tumour cells decreased by over 50% (Figure 6A, Table 2) while there was no

378

significant effect on W12G cell growth (Figure 6B, Table 2). However, flow

379

cytometry analysis indicated that SRFS2 knock down did not significantly

380

inhibit cell cycle progression. Only a small increase in G1 phase with a

381

corresponding decrease in G2 phase cells was noted (Table 3).

382 383

Anchorage-independent growth is characteristic of tumour cells and can be

384

measured by observing colony formation of the tumour cells in soft agar. To

385

determine whether SRSF2 overexpression is required to maintain anchorage-

386

independent growth, W12ti tumour cells were treated with control siRNA or

387

siRNA against SRSF2 then grown in soft agar and the number and size of the

388

colonies were counted. SRSF2 depletion resulted in a significant reduction

389

(p=0.025) in the ability of the W12ti tumour cells to form colonies (Figure 6C).

390

These data implicate SRSF2 in maintenance of the W12ti tumour phenotype.

391 392

SRSF2 knock down does not affect transcription of the E6E7 gene

393

region but is required to stabilise E6E7 oncoprotein RNAs

17

394

SRSF2 can regulate transcription elongation directly (45,46), but it can also

395

regulate expression of key transcription factors such as SP1 that trans-

396

activates the HPV16 P97 promoter (29). Therefore, we tested the hypothesis

397

that the depletion of E6E7 RNAs upon SRSF2 knock down in W12ti cells was

398

a result of SRSF2-mediated downregulation of E6E7 transcription from the

399

viral P97 promoter. 293T cells were transfected with either control siRNA or

400

siRNA against SRSF2 together with a luciferase reporter construct under

401

control of the HPV16 long control region (LCR) (genome nts 7101 (-803) to

402

+137: This region contains the P97 promoter and a transcription initiation site)

403

(36). The HPV16 LCR was less transcriptionally active than the control SV40

404

promoter in the pGL3 promoter plasmid. However, there was no detectable

405

effect of SRSF2 depletion on luciferase production from the test HPV16 P97

406

promoter indicating neither transcription initiation nor elongation was

407

attenuated (Figure 6A).

408 409

An alternative explanation for the reduction in E6 mRNA production in the

410

absence of SRSF2 could be that SRSF2 is required to protect E6 RNA

411

against decay. HPV16 oncogene expression in cervical tumour cells is

412

enhanced by increased stability of the E6E7 mRNAs (30). Moreover, SRSF2

413

can stabilise the mRNA encoding the microtubule protein tau by binding to an

414

SRSF2 binding site in exon 10 (47). In order to test whether SRSF2 regulates

415

E6E7 RNA stability, the protein was siRNA-depleted in W12ti cells, followed

416

by treatment 24 h post transfection with 10 µg/ml actinomycin D, which

417

blocks de novo RNA synthesis allowing a time course analysis of the decay of

418

steady state levels of RNA already present in the cell. E6 RNA isoform levels

18

419

were detected by semiquantitative PCR (in order to detect all isoforms

420

simultaneously which is not possible with qPCR) over a 4 h time course of

421

drug treatment in W12ti cells depleted of SRSF2. Although starting levels of

422

E6 mRNA isoforms were very low when levels of SRSF2 were reduced as

423

expected (35 PCR cycles were performed in Figure 7B as compared to 30

424

cycles in Figure 7C), the data in Figure 7B reveal that in the absence of

425

SRSF2, E6E7 transcript stability is significantly reduced between 1-4 h after

426

addition of actinomycin D when compared to E6 RNAs in cells with

427

undepleted levels of SRSF2 (Figure 7C). All E6 RNA isoforms were equally

428

depleted over the four hour period (Figure 7B). E6*X was not detected at this

429

level of amplification in the absence of SRSF2. GAPDH levels were not

430

significantly reduced upon actinomycin D treatment (Figure 7C) nor upon

431

depletion of SRSF2 (Figure 7B). Taken together, these observations suggest

432

that in cervical tumour cells SRSF2 enhances E6E7 RNA stability.

433

19

434

Discussion

435 436

SR proteins control viral and cellular splicing as well as other RNA processing

437

events such as nuclear export, RNA stability, and translation (13,31).

438

Moreover, SRSF1, 3 and 9 are overexpressed in a number of cancers and

439

possess oncogenic properties (18,22,27,48,49). Previously, we reported

440

increased levels of the three smallest SR proteins SRSFs 1-3 in our W12

441

model of cervical tumour progression and in patient samples (50). Another

442

study has revealed that SRSF3 can control E6E7 expression in cervical

443

cancer cells and in undifferentiated keratinocytes (29) and has oncogenic

444

properties in HPV18-positive HeLa cells (18). Here we investigated if SRSFs

445

1, 2 or 3 regulated expression of viral oncoprotein RNA isoforms in the

446

HPV16-positive W12 cervical cancer cells.

447

control constitutive and alternative splicing a change in splice isoform

448

production was expected. However, depletion of none of these factors altered

449

the relative levels of oncoprotein RNA isoforms. SRSF1 knock down had only

450

a small effect on E6E7 RNA levels. SRSF3 depletion caused a 60% reduction

451

in E6E7 RNA levels as observed previously (29). However, SRSF2 depletion

452

resulted in very significantly reduced levels of all E6 mRNA isoforms

453

suggesting that although SRSF3 can control viral oncoprotein expression,

454

possibly by stimulating production of transcription factors that can trans-

455

activate the HPV16 P97 promoter (29), SRSF2 makes a greater contribution

456

and is absolutely required for viral oncoprotein expression.

Because these proteins can

457

20

458

E6E7 RNA levels were also reduced in CaSki cells when SRSF2 was

459

depleted. Although the reduction was less than that in W12ti cells, the large

460

number of HPV16 integrant genomes in CaSki cells (600 copies) may result in

461

high E6E7 RNA expression levels that are not able to be as fully repressed as

462

in W12ti cells. Furthermore, SRSF2 depletion in W12G cells where there is

463

much reduced SRSF2 expression compared to W12ti cells (50) (Figure 2) did

464

not result in any reduction in E6 RNA levels. Indeed a small increase (0.5-

465

fold) was observed perhaps due to SRSF1-regulation of transcription factors

466

that control the P97 viral promoter or to mRNA decay proteins. These data

467

suggest that SRSF2 control of E6/E7 RNAs is tumour cell-specific and may be

468

due to the high levels of the splicing factor that are present in cervical cancer

469

cells.

470 471

SRSF2 depletion resulted in decreased levels of E6E7 RNA but also gave a

472

functionally significant E6 oncoprotein-associated effect: p53 levels increased

473

after SRSF2 knockdown indicating reduced levels of E6 protein. As expected

474

in the presence of increased p53, cells treated with SRSF2 siRNA displayed

475

significantly increased levels of apoptotic cells compared to mock transfected

476

cells. Knockdown of SRSF2 has been shown to arrest the cell cycle at the

477

G2/M checkpoint in non virally-infected cells (15) (51). Our data did not

478

indicate significant cell cycle arrest even in the face of an SRSF2 depletion-

479

mediated decreased in E7 levels (Table 3). A reduction in E7 expression

480

would normally result in increased levels of the cell cycle checkpoint protein

481

pRb leading to G1 cell cycle arrest. Surprisingly, levels of pRb were not

482

increased after SRSF2 depletion (Figure 4B). This can be explained if SRSF2

21

483

controls pRb expression directly. SRSF2 siRNA-mediated reduction in pRb

484

levels would stimulate cell cycle progression and could ameliorate the effects

485

of increased p53 caused by decreased E6 levels. This may explain why

486

SRSF2 depletion resulted in less late apoptotic/dead cells than siRNA

487

depletion of E6 itself (Figure 5A). Alternatively, if, like other SR proteins,

488

SRSF2 can exert oncogenic effects by altering the levels of tumour-promoting

489

alternatively spliced isoforms of cellular mRNAs then SRSF2 depletion could

490

potentially result in changes in other cellular proteins that could also

491

antagonise the apoptotic effect of reduced levels of viral oncoprotein E6.

492

As well as its roles in splicing regulation, SRSF2 can control transcription

493

elongation either directly by affecting recruitment of the RNA Polymerase II C-

494

terminal domain ser-2 kinase complex, P-TEFb (52) or indirectly by regulating

495

expression of transcription factors, for example SP1 (29). However, as seen

496

here SRSF2 depletion did not significantly alter transcription of a luciferase

497

reporter gene under control of the viral P97 promoter that regulates expression

498

of E6 and E7. SR proteins can control other steps in the life cycle of an

499

mRNA, and SRSF2 is known to regulate RNA stability. This can be

500

accomplished directly by SRSF2 binding to an SRSF2 cognate binding site,

501

for example in exon 10 of the mRNA encoding the microtubule protein tau

502

(47), or indirectly through recruitment of RNA surveillance pathways, for

503

example to SRSF2-regulated alternatively spliced isoforms of its own RNAs,

504

to degrade the transcripts (53). Our results suggest that SRSF2 is involved in

505

processing of the viral oncoprotein RNAs at some stage in the RNA

506

biogenesis

507

transcriptionally-regulated disappearance of the RNAs. The most likely

pathway

because

loss

of

the

protein

caused

a

non-

22

508

mechanism is that SRSF2 is required for E6E7 RNA stability. The E6E7

509

bicistronic mRNA is unusual in that it does not possess a 5' untranslated

510

region and there are several splice acceptor sites close to stop codons

511

meaning that it should normally be targeted for nonsense mediated decay

512

(NMD) (54). SR proteins can regulate targeting of RNAs to the NMD pathway

513

(55). Therefore SRSF2 may recruit factors to E6E7 RNAs in the nucleus to

514

protect from premature stop codon-mediated decay in the cytoplasm.

515

Alternatively, during cervical tumour progression when the HPV genome is

516

inserted into the host genome 3’ processing of the oncoprotein RNAs no

517

longer occurs at the viral polyadenylation site, but at some genomic

518

polyadenylation site downstream from the point of genome integration.

519

possible that SRSF2 could direct alternative splicing of the chimaeric

520

viral/genomic mRNAs by selecting a downstream exon or 3’ untranslated

521

region (33,56)

522

previously noted (30). SRSF2 could also regulate E6E7 RNAs indirectly by

523

controlling expression of an intermediary factor that controls E6E7 RNA

524

stability. Indeed, the lower numbers of cells that entered late apoptosis upon

525

treatment with SRSF2 siRNA compared to treatment with E6 siRNA might

526

suggest a temporal difference in response of E6E7 expression to depletion of

527

SRSF2 due to an intermediary regulatory event. However, the full mechanism

528

of SRSF2-medated stabilisation of E6 RNA isoforms requires further study.

It is

that would confer stability on these E6E7 mRNAs as

529 530

Our data demonstrate that SRSF2 has oncogenic properties in HPV16-

531

positive cervical cancer cells. When the splicing factor was depleted in W12ti

532

cells there was around a 50% reduction in anchorage independent growth,

23

533

and an initiation of apoptosis. While high levels of SRSF2 in the tumour cells

534

is clearly required for viral oncoprotein expression, we cannot discount the

535

possibility that the increased levels of endogenous SRSF2 seen in cervical

536

tumour progression (50) could change the profile of cellular mRNA alternative

537

splicing leading to production of oncogenic or antiapoptotic protein isoforms

538

as has been demonstrated for SRSF1 (28). It is highly likely that SRSF2 plays

539

a role in cervical tumour progression by acting directly on E6E7 mRNA

540

expression but our data showing that SRSF2 depletion reduces the

541

proliferation of HPV-negative C33a cells underlines the fact that SRSF2

542

depletion also impacts on cellular gene expression.

543

This is the first report, to our knowledge that SRSF2 is an essential cellular

544

regulator of HPV oncogenic expression. This suggests it could be a valid

545

target for therapeutic inhibition in cervical tumours. SR protein function is

546

regulated by phosphorylation and drugs that regulate SR kinases have been

547

used successfully to inhibit the replication of HIV and hepatitis C virus (31).

548

Development of small molecule inhibitors against SR kinases could have the

549

potential to abrogate HPV oncoprotein expression in the early stages of

550

cervical cancer progression.

551

552

Acknowledgements

553

We would like to thank Margaret Stanley (University of Cambridge) for the

554

original W12 cell line, Paul Lambert and Denis Lee (University of Wisconsin)

555

for the W12E and G clones and Trond Aasen, Mike Edward and Malcolm 24

556

Hodgins (University of Glasgow) for collaborations on developing and

557

characterising the W12t and W12ti cells lines. Iain Morgan (Virginia

558

Commonwealth University) and Ed Dornan (University of Glasgow) kindly

559

supplied the HPV16 LCR plasmid and the E2 expression plasmid was

560

obtained from Peter Howley (Havard Medical School). We are grateful to

561

Diane Vaughan for help with flow cytometry and FACS analysis.

562 563

Grant support

564

M. McF was funded by an MRC Doctoral Training Award. The study was also

565

funded by grant number 08-0159 from the Association for International

566

Cancer Research and Wellcome Trust grant number Wtd004098 to SVG.

567

25

568

Live compartment Early apoptosis Late apoptosis Control siRNA 31.2 ±5.8

20.2 ±5.1

20.4 ±2.1

SRSF2 siRNA 8.0 ±2.7

55.1 ±5.8

36.4 ± 2.1

E6 siRNA

7.0 ±2.3

28.1 ± 3.5

49.7 ±8.9

UVB

6.1 ±4.4

45.0 ±4.3

44.3 ±5.7

569

570

Table 1. Flow cytometry analysis of apoptosis of W12ti cells upon

571

depletion of SRSF2.

572

iodide upon treatment with control siRNA, siRNA against SRSF2 or E6, or

573

treated with UVB (500 J/m2 24 h prior to harvesting). The numbers are the

574

mean and standard deviation from the mean from three separate

575

experiments.

Percentage cells positive for annexin V/propidium

26

W12G Hours

W12ti

C33a

post

transfection

control

24

5.87±0.29x10

72

1.25±0.37x10

siSRSF2

control

4

5.77±0.62x10

5

1.1±0.46x10

5

4

siSRSF2

control

4

7.5±0.2x10

5

1.3±0.09x10

6.47±0.37x10

3.58±0.86x10

4

siSRSF2

1.76±0.09x10

5

6

2.81±0.2x10

5

1.73±0.1x10

5

9.57±0.14x10

5

576

577

Table 2. Actual numbers of W12G (HPV-positive nontumour) and W12ti

578

(HPV-positive tumour) and C33a (HPV-negative tumour) cells at 24 and

579

72 hours post transfection with control siRNA or with siRNA against

580

SRSF2. Control, control transfection with siGLO; siSRSF2 transfection with

581

siRNA against SRSF2.

582

583

27

584

Stage

in

cell Mock

Control siRNA

siSRSF2

Average G1

65.5% ±3.2

66.1% ±2.2

71.9% ±6.0

Average S

10.4% ±0.8

12.9% ±2.9

13.8% ±5.3

Average G2

19.9% ±3.1

15.4% ±1.8

12.8% ±3.2

cycle

585

586

Table 3. Flow Cytometry. The mean number (and standard deviation from

587

the mean) of W12ti cells in each stage of the cell cycle in three independent

588

experiments. Mock, cells with no siRNA; Control siRNA, cells transfected with

589

control siGLO RNA; siSRSF2, cells transfected with an siRNA pool against

590

SRSF2.

591

592

28

593

Figure Legends

594 595

Figure 1: E6 splice isoform expression in epithelial cell transformation.

596

A) Diagram of the E6E7 region of the HPV16 genome. Shaded grey boxes,

597

open reading frames.

598

(nucleotides) refer to the end of the E6 (560) and the beginning of the E7

599

(562) open reading frames. The four E6E7 mRNAs are shown below the

600

genomic map. Numbers mark the nucleotide positions of one splice donor

601

(226) and three possible splice acceptor sites (409, 526, 742). Grey boxes,

602

exons. Black lines, introns. B) qRT-PCR quantification of the expression

603

levels of total E6E7 RNA relative to levels of GAPDH in the four W12 cervical

604

epithelial cell lines (W12E, W12G, W12t, W12ti,). The graph shows the mean

605

and standard error of the mean from three separate experiments. C) Ethidium

606

bromide stained polyacrylamide gel electrophoresis of RT-PCR products

607

amplified from polyadenylated RNA isolated from the above W12 cell lines

608

using primers that amplify all E6 isoforms. The various isoforms are indicated

609

to the right of the gel (E6fl, E6*I, E6*II, E6*X). A second, identical gel to

610

fractionate GAPDH control PCR products from the same RNA/cDNA

611

preparations was electrophoresed at the same time and is shown below. M,

612

marker; RT, reverse transcriptase; “-“, RT-PCR reaction in the absence of

613

reverse transcriptase; “+”, RT-PCR reaction in the presence of reverse

614

transcriptase.

Arrow, P97, HPV16 early promoter. The numbers

615 616

Figure 2: Levels of SRSF proteins 1, 2 and 3 are higher in the W12

617

tumour lines (W12t and W12ti) than in the non-tumour lines (W12E and 29

618

W12G). A) Western blot showing SRSF1 levels in the four W12 lines. B)

619

Western blot showing SRSF2 levels in the four W12 lines. C) Western blot

620

showing SRSF3 levels in the four W12 lines. GAPDH was used as a loading

621

control. Track 1, W12E (non-tumour cell line) proteins extracts. Track 2,

622

W12G (non-tumour cell line) protein extracts. Track 3, W12t (tumour cell line)

623

protein extracts. Track 4, W12ti (tumour cell line) protein extracts.

624 625

Figure 3: The effect of SRSF depletion on E6E7 RNA expression in

626

cervical tumour and non-tumour cells. A) SRSF protein levels before and

627

after depletion by specific siRNA pools in the W12ti cells. B) Ethidium bromide

628

stained polyacrylamide gel electrophoresis of RT-PCR products amplified

629

from polyadenylated RNA isolated from W12ti cells using primers that amplify

630

all E6 isoforms. The effect of depletion of SRSF1 (tracks 5&6), SRSF3 (tracks

631

9&10) and SRSF2 (tracks 11&12) for 48 h on E6 isoform expression in W12ti

632

cervical tumour cells is shown. The E6 RNA isoforms are indicated to the

633

right of the gels. A second, identical gel to fractionate GAPDH control PCR

634

products from the same RNA/cDNA preparations was electrophoresed at the

635

same time and is shown below each gel. RT, reverse transcriptase; “-“, RT-

636

PCR reaction in the absence of reverse transcriptase; “+”, RT-PCR reaction

637

in the presence of reverse transcriptase. “no siRNA”, cells transfected without

638

siRNA; “cntrl”, cells transfected with a control siRNA, siGLO; “SRSF”, cells

639

treated with SRSF siRNA for 48 h. The experiments were repeated at least

640

three times and similar data were obtained in each experiment. C) qPCR

641

quantification of total E6 RNA levels following SRSF depletion. The graph

30

642

shows the mean and standard deviation from the mean of three separate

643

experiments.

644 645

Figure 4. The effect of SRSF depletion on E6E7 RNA expression in CaSki

646

cervical tumour and W12G non-tumour cells.

647

before and after depletion by specific siRNA pools. The antibody used

648

(Mab104) also detects other SR proteins and an adjacent band corresponding

649

to SFSR5 is shown as an internal control for protein levels and to show

650

specificity of SRSF2 depletion. As expected, the starting levels of SRSF2 in

651

W12G cells are much lower than in CaSki cells or W12ti cells compared to the

652

internal SRSF5 control band. B&D) Ethidium bromide stained polyacrylamide

653

gel electrophoresis of RT-PCR products amplified from polyadenylated RNA

654

isolated from cell lines using primers that amplify all E6 isoforms. The E6

655

RNA isoforms are indicated to the right of the gels. A second, identical gel to

656

fractionate GAPDH control PCR products from the same RNA/cDNA

657

preparations was electrophoresed at the same time and is shown below each

658

gel. RT, reverse transcriptase; “-“, RT-PCR reaction in the absence of reverse

659

transcriptase; “+”, RT-PCR reaction in the presence of reverse transcriptase.

660

“no siRNA”, cells transfected without siRNA; “cntrl”, cells transfected with a

661

control siRNA, siGLO; “SRSF”, cells treated with SRSF siRNA for 48 h. The

662

experiments were repeated at least three times and similar data were

663

obtained in each experiment. B) The effect of SRSF2 depletion of E6 isoform

664

expression in CaSki cervical tumour cells. C) qPCR quantification of total E6

665

RNA levels in CaSki cells before and after SRSF2 depletion. The graph

666

shows the mean and standard deviation from the mean of three separate

A) SRSF protein levels

31

667

experiments. D) The effect of SRSF2 depletion on E6 isoform expression in

668

W12G non-tumour cervical cells. E) qPCR quantification of total E6 levels in

669

W12G cells before and after SRSF2 depletion. The graph shows the mean

670

and standard deviation from the mean of three separate experiments.

671 672

Figure 5. W12ti tumour cells exhibit decreased viral oncoprotein

673

expression upon SRSF2 knock down.

674

protein levels with or without SRSF2 knock down in W12ti cells. GAPDH is

675

used as a loading control. SFSR2 depletion is shown with SRSF5 as the

676

internal loading control. “no siRNA“ cells transfected without siRNA; “cntrl”,

677

cells transfected with a control siRNA, “SRSF2”, cells transfected with SRSF2

678

siRNA for 48 h. Below the blots is a quantification of levels of p53 in untreated

679

cells (no siRNA) or treated with the indicated siRNA. The data show the

680

mean and standard deviation from the mean from three separate

681

experiments. The asterisk indicates a significant change in protein expression

682

(p≤0.05) using a student’s “t” test. B) Western blot analysis of E7 and pRb

683

protein levels with or without SRSF2 knock down in W12ti cells. GAPDH is

684

shown as the loading control. The same protein extracts as in (A) were used

685

in this experiment so the same level of SRSF2 depletion was achieved. “no

686

siRNA“ cells transfected without siRNA; “cntrl”, cells transfected with a control

687

siRNA, “SRSF2”, cells transfected with SRSF2 siRNA for 48 h. Below the

688

blots is a quantification of levels of E7 in cells not treated (no siRNA) or

689

treated with the indicated siRNAs. The data show the mean and standard

690

deviation from the mean from three separate experiments. The asterisk

A) Western blot analysis of p53

32

691

indicates a significant change in protein expression (p≤0.05) using a student’s

692

“t” test.

693 694

Figure 6. W12ti tumour cells undergo apoptosis upon SRSF2 knock

695

down. A) FACS plots of annexin V/propidium iodide staining of W12ti cells

696

transfected with the indicated siRNAs above the plots or with UVB as a

697

positive control for cell death. The percentage cells occupying each quadrant

698

in one experiment are shown. The lower right hand quadrant contains early

699

apoptotic cells while the upper right hand quadrant contains late

700

apoptotic/dead cells. W12ti cells were treated with control siRNA (siCntrl) or

701

siRNA pool against SRSF2 (siSRSF2) or siRNA against HPV16 E6 (siE6) or

702

treated with UVB irradiation (UVB) for 24 h as a positive indicator of

703

apoptosis. Very similar results were obtained in three separate experiments

704

(See Table 1). B) Graph of the percentage of W12ti cells from three

705

independent experiments occupying the live and early apoptotic quadrants of

706

the annexin V and propidium iodide staining plots shown in (A). C) Western

707

blot showing the levels of SRSF2 in mock transfected cells (no siRNA), cells

708

transfected with control siRNA (cntrl) or transfected with siRNA against

709

SRSF2 (SRSF2). SRSF5 is shown as an internal loading control.

710 711

Figure 7. The effect of SRSF2 depletion on W12 cell proliferation and

712

colony formation. Proliferation rates over 72 hours of A) W12ti tumour cells

713

treated with control siRNA (cntrl) or treated with an siRNA pool against

714

SRSF2 (siSRSF2). B) W12G non-tumour cells treated with control siRNA

715

(cntrl) or treated with an siRNA pool against SRSF2 (siSRSF2). The insert

33

716

western blots in A and B show levels of SRSF2 depletion and SRSF5 levels

717

as an internal loading control. C) Colony formation assay of W12ti cells with

718

(SRSF2 siRNA) or without (cntrl siRNA) SRSF2 knock down. The graph

719

shows mean and standard deviation from the mean of numbers of colonies

720

from three independent experiments. Asterisk indicates significant change in

721

colony formation (p≤0.05) using a student’s “t” test.

722 723

Figure 8: SRSF2 does not regulate HPV16 P97 promoter activity but may

724

stabilise E6E7 RNAs. A) Graph of luciferase activity in 293T cells transiently

725

transfected (48 h) with either a luciferase reporter plasmid containing the

726

control SV40 promoter (pGL3promoter, Promega), or the HPV16 long control

727

region encompassing the P97 promoter (HPV16 LCR) (36). Cells transfected

728

with the HPV16 LCR construct were also transfected with either control siRNA

729

(cntrl siRNA) or siRNA against SRSF2 (siSRSF2). The mean and standard

730

deviation from the mean from three separate experiments is shown. B) RT-

731

PCR analysis of E6E7 RNAs in actinomycin D treated W12ti cells 24 hours

732

following transfection with siRNA against SRSF2 or C) control siRNA (cntrl

733

siRNA). The number of hours of actinomycin D treatment is shown above the

734

gels. GAPDH RNA levels are stable over this time period and are shown as a

735

loading control.

34

736

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18. Das, S. and A. R. Krainer. 2014. Emerging functions of SRSF1,

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21. Cohen-Eliav, M., R. Golan-gerstl, Z. Siegfried, C. L. Andersen, K.

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22. Jia, R., C. Li, J. P. McCoy, C. X. Deng, and Z. Zheng. 2010. SRp20 is

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23. Stickeler, E., F. Kittrell, D. Medina, and S. Berget. 1999. Stage-

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27. Karni, R., Y. Hippo, S. W. Lowe, and A. R. Krainer. 2008. The

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28. Anczuków, O., A. Z. Rosenberg, M. Akerman, S. Das, L. Zhan, and

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29. Das, S., O. Anczuków, M. Akerman, and A. R. Krainer. 2012.

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30. He, X., P.L. Ee, J.S. Coon, and W.T. Beck. 2004. Alternative splicing

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36. Hernandez-Lopez, H. R. and S. V. Graham. 2012. Alternative splicing in tumour viruses: a therapeutic target? Biochem J. 445:145-156.

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37. Aasen, T., M. B. Hodgins, M. Edward, and S. V. Graham. 2003. The

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41. Schwarz, E., U. Freese, L. Gissman, W. Mayer, B. Roggenbuck, A.

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47. Doorbar, J., A. Parton, K. Hartley, L. Banks, T. Crook, M. A.

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48. Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and P.

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49. DeFilippis, R. A., E. C. Goodwin, L. Wu, and D. DiMaio. 2003.

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51. Huang, S. and D. L. Spector. 1996. Dynamic organisation of premRNA splicing factors. J.Cell Biochem. 62:191-197.

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873.

947 948

44

McFarlane et al Figure 1

A

P97 560

E6 E7 562

E6fl

mRNAs

E6*I 409

E6*II 526

E6*X

B

Relative E6 expression

97

226

742

25 20 15 10 5 0

W12E

W12t

W12G

W12ti

RTkb M -

+

-

+

W12ti

W12t

W12E

C

W12G

Cell line

-

+ -

+

1.0

E6fl E6*I E6*II

0.52 0.51 0.40 0.34 0.30 0.22

E6*X

0.52 0.51 0.40 0.34

GAPDH

1 2

3 4 5

6 7 8

9

Figure 1: E6 splice isoform expression in epithelial cell transformation. A) Diagram of the E6E7 region of the HPV16 genome. Shaded grey boxes, open reading frames. Arrow, P97, HPV16 early promoter. The numbers (nucleotides) refer to the end of the E6 (560) and the beginning of the E7 (562) open reading frames. The four E6E7 mRNAs are shown below the genomic map. Numbers mark the nucleotide positions of one splice donor (226) and three possible splice acceptor sites (409, 526, 742). Grey boxes, exons. Black lines, introns. B) qRT-PCR quantification of the expression levels of total E6E7 RNA relative to levels of GAPDH in the four W12 cervical epithelial cell lines (W12E, W12G, W12t, W12ti,). The graph shows the mean and standard error of the mean from three separate experiments. C) Ethidium bromide stained polyacrylamide gel electrophoresis of RT-PCR products amplified from polyadenylated RNA isolated from the above W12 cell lines using primers that amplify all E6 isoforms. The various isoforms are indicated to the right of the gel (E6fl, E6*I, E6*II, E6*X). A second, identical gel to fractionate GAPDH control PCR products from the same RNA/cDNA preparations was electrophoresed at the same time and is shown below. M, marker; RT, reverse transcriptase; “-“, RT-PCR reaction in the absence of reverse transcriptase; “+”, RT-PCR reaction in the presence of reverse transcriptase.

McFarlane et al Figure 1

A kDa

W12E W12G W12t W12ti

McFarlane et al Figure 2

38

SRSF1

38

GAPDH

B 38

SRSF2

38

GAPDH

C 28

SRSF3

17 38

GAPDH 1 2 3 4

Figure 2: Levels of SRSF proteins 1, 2 and 3 are higher in the W12 tumour lines (W12t and W12ti) than in the non-tumour lines (W12E and W12G). A) Western blot showing SRSF1 levels in the four W12 lines. B) Western blot showing SRSF2 levels in the four W12 lines. C) Western blot showing SRSF3 levels in the four W12 lines. GAPDH was used as a loading control. Track 1, W12E (non-tumour cell line) proteins extracts. Track 2, W12G (non-tumour cell line) protein extracts. Track 3, W12t (tumour cell line) protein extracts. Track 4, W12ti (tumour cell line) protein extracts.

McFarlane et al Figure 2

no siRNA

B

SR siRNA

cntrl siRNA

A

nosiRNA

McFarlane et al. Figure 3

siRNA SRSF3

kb

GAPDH

1.02

M

1

W12ti cells

2

cntrl

SRSF1

cntrl

3

5

7

4

6

8

SRSF2

SRSF3 9

10 11

12 E6fl E6*I E6*II

0.52

SRSF1 0.40 0.34 0.30

GAPDH

E6*X

0.22

SRSF2

0.52

GAPDH

0.40

GAPDH

C

Relative expression levels

RT

1 .2

-

+

-

+

-

+

-

+

-

+

-

+

Total E6 RNA levels in W12ti cells

1 0 .8 0 .6 0 .4 0 .2 0

Cntrl

SRSF1

SRSF3

SRSF2

Figure 3: The effect of SRSF depletion on E6E7 RNA expression in W12ti cervical tumour cells. A) SRSF protein levels before and after depletion by specific siRNA pools in the W12ti cells. B) Ethidium bromide stained polyacrylamide gel electrophoresis of RT-PCR products amplified from polyadenylated RNA isolated from W12ti cells using primers that amplify all E6 isoforms. The effect of depletion of SRSF1 (tracks 5&6), SRSF3 (tracks 9&10) and SRSF2 (tracks 11&12) for 48 h on E6 isoform expression in W12ti cervical tumour cells is shown. The E6 RNA isoforms are indicated to the right of the gels. A second, identical gel to fractionate GAPDH control PCR products from the same RNA/cDNA preparations was electrophoresed at the same time and is shown below each gel. RT, reverse transcriptase; “-“, RT-PCR reaction in the absence of reverse transcriptase; “+”, RT-PCR reaction in the presence of reverse transcriptase. “no siRNA”, cells transfected without siRNA; “cntrl”, cells transfected with a control siRNA, siGLO; “SRSF”, cells treated with SRSF siRNA for 48 h. The experiments were repeated at least three times and similar data were obtained in each experiment. C) qPCR quantification of total E6 RNA levels following SRSF depletion.

McFarlane et al. Figure 3

siSRSF2

cntrl

cntrl

A

siSRSF2

McFarlane et al. Figure 4

SRSF5 SRSF2 GAPDH

no siRNA

CaSki

siRNA M

kb 1.02

1

C

CaSki cells cntrl 2

SRSF2

3

4

5

6 E6fl E6*I

0.52

E6*II

0.40 0.34 0.30 0.22 0.52

1.2

Total E6 RNA in CaSki cells

1 0.8 0.6 0.4 0.2 0 Cntrl

GAPDH

0.40 0.34

D

siRNA

M

+

1

-

+

-

E

W12G cells cntrl

2

3

4

SRSF2

5

6

1.02

E6fl E6*I

0.52

E6*II

0.40 0.34 0.30 0.22 0.52

GAPDH

0.40

RT

-

+

-

+

-

SRSF2

+

Relative expression levels

-

no siRNA

RT

kb

Relative expression levels

B

W12G

Total E6 RNA in W12G cells

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2

0

Cntrl

SRSF2

+

Figure 4: The effect of SRSF depletion on E6E7 RNA expression in CaSki cervical tumour and W12G non-tumour cells. A) SRSF protein levels before and after depletion by specific siRNA pools. The antibody used (Mab104) also detects other SR proteins and an adjacent band corresponding to SFSR5 is shown as an internal control for protein levels and to show specificity of SRSF2 depletion. As expected, the starting levels of SRSF2 in W12G cells are much lower than in CaSki cells or W12ti cells compared to the internal SRSF5 control band. B&D) Ethidium bromide stained polyacrylamide gel electrophoresis of RT-PCR products amplified from polyadenylated RNA isolated from cell lines using primers that amplify all E6 isoforms. The E6 RNA isoforms are indicated to the right of the gels. A second, identical gel to fractionate GAPDH control PCR products from the same RNA/cDNA preparations was electrophoresed at the same time and is shown below each gel. RT, reverse transcriptase; “-“, RT-PCR reaction in the absence of reverse transcriptase; “+”, RT-PCR reaction in the presence of reverse transcriptase. “no siRNA”, cells transfected without siRNA; “cntrl”, cells transfected with a control siRNA, siGLO; “SRSF”, cells treated with SRSF siRNA for 48 h. The experiments were repeated at least three times and similar data were obtained in each experiment. B) The effect of SRSF2 depletion of E6 isoform expression in CaSki cervical tumour cells. C) qPCR quantification of total E6 RNA levels in CaSki cells before and after SRSF2 depletion. D) The effect of SRSF2 depletion on E6 isoform expression in W12G non-tumour cervical cells. E) qPCR quantification of total E6 levels in W12G cells before and after SRSF2 depletion.

McFarlane et al. Figure 4

p53

SRSF2

B

siRNA

cntrl

no siRNA

SRSF2

kDa 62 49

cntrl

A

no siRNA

McFarlane et al Figure 5

siRNA

kDa 38

GAPDH

GAPDH

38

SRSF5 SRSF2

pRb

97

100

* p=0.021

80 60 40 20 0

E7

120

Relative E7 levels

Relative p53 levels

17

*

100

p=0.033

80 60 40 20 0

no siRNA

cntrl siRNA

SRSF2 siRNA

no siRNA

cntrl siRNA

SRSF2 siRNA

Figure 5. W12ti tumour cells exhibit decreased viral oncoprotein expression upon SRSF2 knock down . A) Western blot analysis of p53 protein levels with or without SRSF2 knock down in W12ti cells. GAPDH is used as a loading control. SFSR2 depletion is shown with SRSF5 as the internal loading control. “no siRNA“ cells transfected without siRNA; “cntrl”, cells transfected with a control siRNA, “SRSF2”, cells transfected with SRSF2 siRNA for 48 h. Below the blots is a quantification of levels of p53 in untreated cells (no siRNA) or treated with the indicated siRNA. The data show the mean and standard deviation from the mean from three separate experiments. The asterisk indicates a significant change in protein expression (p≤0.05) using a student’s “t” test. B) Western blot analysis of E7 and pRb protein levels with or without SRSF2 knock down in W12ti cells. GAPDH is shown as the loading control. The same protein extracts as in (A) were used in this experiment so the same level of SRSF2 depletion was achieved. “no siRNA“ cells transfected without siRNA; “cntrl”, cells transfected with a control siRNA, “SRSF2”, cells transfected with SRSF2 siRNA for 48 h. Below the blots is a quantification of levels of E7 in cells not treated (no siRNA) or treated with the indicated siRNAs. The data show the mean and standard deviation from the mean from three separate experiments. The asterisk indicates a significant change in protein expression (p≤0.05) using a student’s “t” test.

McFarlane et al Figure 5

McFarlane et al Figure 6

siSRSF2

siE6

2.1

34.3

29.9

18.3

5.5

58.1

B

4.4

3.0

43.4

5.5

31.5

4.8

48.8

C

70

Relative percentage cells

UVB 58.6

siRNA

60

SRSF2

30.4

21.4

cntrl

siCntrl

no siRNA

A

SRSF5 SRSF2

50 40 Live 30

Apoptotic

20 10 0

cntrl

siSRSF2

siE6

UVB

Cell treatment

Figure 6. W12ti tumour cells undergo apoptosis upon SRSF2 knock down. A) FACS plots of annexin V/propidium iodide staining of W12ti cells transfected with the indicated siRNAs above the plots or with UVB as a positive control for cell death. The percentage cells occupying each quadrant in one experiment are shown. The lower right hand quadrant contains early apoptotic cells while the upper right hand quadrant contains late apoptotic/dead cells. W12ti cells were treated with control siRNA (siCntrl) or siRNA pool against SRSF2 (siSRSF2) or siRNA against HPV16 E6 (siE6) or treated with UVB irradiation (UVB) for 24 h as a positive indicator of apoptosis. Very similar results were obtained in three separate experiments (See able 1). B) Graph of the percentage of W12ti cells from three independent experiments occupying the live and early apoptotic quadrants of the annexin V and propidium iodide staining plots shown in (A). C) Western blot showing the levels of SRSF2 in mock transfected cells (no siRNA), cells transfected with control siRNA (cntrl) or transfected with siRNA against SRSF2 (SRSF2). SRSF5 is shown as an internal loading control.

McFarlane et al Figure 6

cntrl

A 5 0 0 ,0 0 0 4 5 0 ,0 0 0 4 0 0 ,0 0 0

siSRSF2

McFarlane et al Figure 7

W12ti cells

SRSF5 SRSF2

Total cell numbers

3 5 0 ,0 0 0

3 0 0 ,0 0 0

Cntrl

2 5 0 ,0 0 0

siSRSF2

2 0 0 ,0 0 0 1 5 0 ,0 0 0 1 0 0 ,0 0 0 5 0 ,0 0 0 0

cntrl

B 1 8 0 ,0 0 0

Total cell numbers

1 6 0 ,0 0 0

72

48 Hours after SRSF2 depletion

siSRSF2

24

W12G cells

SRSF5 SRSF2

1 4 0 ,0 0 0 1 2 0 ,0 0 0 1 0 0 ,0 0 0

Cntrl

8 0 ,0 0 0

siSRSF2

6 0 ,0 0 0 4 0 ,0 0 0 2 0 ,0 0 0 0

24

48

72

Hours after SRSF2 depletion

C

W12ti colony formation upon SRSF2 depletion

45

No. of colonies

40

p=0.025

35 30

*

25 20 15 10 5 0

cntrl siRNA

SRSF2 siRNA

Figure 7. The effect of SRSF2 depletion on W12 cell proliferation and colony formation. Proliferation rates over 72 hours of A) W12ti tumour cells treated with control siRNA (cntrl) or treated with an siRNA pool against SRSF2 (siSRSF2). B) W12G non-tumour cells treated with control siRNA (cntrl) or treated with an siRNA pool against SRSF2 (siSRSF2). The insert western blots in A and B show levels of SRSF2 depletion and SRSF5 levels as an internal loading control. C) Colony formation assay of W12ti cells with (SRSF2 siRNA) or without (cntrl siRNA) SRSF2 knock down. The graph shows mean and standard deviation from the mean of numbers of colonies from three independent experiments. Asterisk indicates significant change in colony formation (p≤0.05) using a student’s “t” test.

McFarlane et al Figure 7

A

R elative lu ciferase un its

McFarlane et al. Figure 8

100

10

1

0.1

0.01 p G L 3 promoter

H P V 16 L C R +cntrl siRNA

SRSF2 siRNA

B

C

ActD 10 mg/ml

0h 1h

4h

cntrl siRNA ActD 10 mg/ml

0h 1h

4h

kb

kb

1.0

1.0

E6fl E6*I E6*II

0.52 0.51

E6fl E6*I E6*II

0.52 0.51 0.40 0.34

0.40 0.34 0.52 0.51 0.40 0.34

H P V 16 L C R + siS R S F 2

GAPDH M

1

2

3

0.52 0.51 0.40 0.34

GAPDH M

1

2

3

Figure 8: SRSF2 does not regulate HPV16 P97 promoter activity but may stabilise E6E7 RNAs. A) Graph of luciferase activity in 293T cells transiently transfected (48 h) with either a luciferase reporter plasmid containing the control SV40 promoter (pGL3promoter, Promega), or the HPV16 long control region encompassing the P97 promoter (HPV16 LCR) (39). Cells transfected with the HPV16 LCR construct were also transfected with either control siRNA (cntrl siRNA) or siRNA against SRSF2 (siSRSF2). The mean and standard deviation from the mean from three separate experiments is shown. B) RT-PCR analysis of E6E7 RNAs in actinomycin D treated W12ti cells 24 hours following transfection with siRNA against SRSF2 or C) control siRNA (cntrl siRNA). The number of hours of actinomycin D treatment is shown above the gels. GAPDH RNA levels are stable over this time period and are shown as a loading control.

McFarlane et al. Figure 8