Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNP A1) and hnRNP A2 Inhibit Splicing to Human Papillomavirus 16 Splice Site SA409 through a UAG-Containing Sequence in the E7 Coding Region

Figures

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  FIG 1. Alternatively spliced HPV16 mRNAs produced by subgenomic HPV16 expression plasmid pC97ELsL. (A and B) Schematic representation of the HPV16 genome (A) and the HPV16 subgenomic pC97ELsL reporter plasmid (62) (B). Transcription of the HPV16 sequences in the pC97ELsL plasmid is driven by the human cytomegalovirus immediate-early promoter (CMV). HPV16 splice sites are indicated. Numbers refer to the HPV16 reference strain HPV16R (45). Early and late polyadenylation signals pAE and pAL are indicated. p97 and p670 indicate HPV16 early and late promoters, respectively. IRES, internal ribosome entry site; sluc, secreted luciferase. (C, left) Schematic representation of a subset of alternatively spliced mRNAs produced by pC97ELsL and amplified by RT-PCR primers 97S and 880A or 438A. Arrows indicate the location of RT-PCR primers. The names of the first open reading frame (orf) on each alternatively spliced mRNA are indicated to the left. (Right) RT-PCR with primers 97S and 880A on RNA extracted from HeLa cells transfected with pC97ELsL. Control PCR in the absence of reverse transcriptase (RT-minus) is shown. (D, left) Schematic representation of a subset of alternatively spliced mRNAs…

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  FIG 2. hnRNP A1 and A2 affect splicing between HPV16 5′-splice site SD226 and 3′-splice sites SA409 and SA526. (A to E) RT-PCR on RNA extracted from HeLa cells transfected with pC97ELsL (62) in the absence (−) or presence of 2-fold serially diluted plasmid phnRNP A1 or phnRNP A2. RT-PCR primers are indicated below each gel pair. Control PCRs in the absence of reverse transcriptase (RT-minus) are shown to the left. The HPV16 splice sites used to generate the mRNAs amplified by the RT-PCR primers are indicated to the right. (F) RT-PCR on RNA extracted from 293T cells transfected with pC97ELsL in the absence (−) or presence of plasmid phnRNP A1 or phnRNP A2. RT-PCR primers 97S and 880A were used. The HPV16 splice sites used to generate the mRNAs amplified by the RT-PCR primers are indicated to the right. M, molecular size marker; *, primers.

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  FIG 3. Differential effects of hnRNP A1 and A2 on HPV16 E6 and E7 mRNA splicing. (A) RT-PCR on RNA extracted from HeLa cells transfected with pC97ELsL (62) in the absence (−) or presence of 2-fold serially diluted phnRNP A1 or phnRNP A2. RT-PCR primers are indicated to the left of the gel pair. The HPV16 splice sites used to generate the mRNAs amplified by the RT-PCR primers are indicated to the right. Transfections were performed in triplicates, and representative gel pictures are displayed. Control PCRs in the absence of reverse transcriptase (RT-minus) are shown below RT-PCR gels. M, molecular size marker; *, primers. (B) Densitometric quantification of RT-PCR bands was performed on triplicate transfections. Mean values and standard deviations are shown. The quantitations are displayed as fold difference of HPV16 RT-PCR band intensity obtained with RNA extracted from cells transfected with pC97ELsL in the presence of phnRNP A1 or A2 over band intensities in cells transfected with pC97ELsL in the presence of empty pUC plasmid (−). Band intensity ratios are plotted against micrograms of transfected phnRNP A1 or phnRNP A2 plasmid DNA. (C) Western blotting on cell extracts from HeLa…

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  FIG 4. hnRNP A1 and A2 target HPV16 sequences located between HPV16 genomic nucleotide positions 556 and 856. (A) Schematic representation of the HPV16 subgenomic pC97ELsL reporter plasmid (62). Transcription of the HPV16 sequences in the pC97ELsL plasmid is driven by the human cytomegalovirus immediate-early promoter (CMV). HPV16 splice sites are indicated. Numbers refer to the HPV16 reference strain HPV16R (45). Early and late polyadenylation signals pAE and pAL are indicated. IRES, internal ribosome entry site; sluc, secreted luciferase. (B) Schematic representation of HPV16 subgenomic reporter plasmids pX1200, pX1060, pX960, pX856F, and pX556F. Transcription of the HPV16 sequences is driven by the human cytomegalovirus immediate-early promoter (CMV). RT-PCR primers 97S and X525A are indicated. HPV16 sequences in the deletion mutants pX1200, pX1060, pX960, pX856F, and pX556F end at HPV16 nucleotide positions 1200, 1060, 960, 856, and 556. HPV16 splice sites are indicated. Nucleotide positions refer to the nucleotide position of the HPV16 reference genome HPV16R. (C) RT-PCR on RNA extracted from HeLa cells transfected with plasmids pX1200, pX1060, pX960, pX856F, or pX556F in the…

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  FIG 5. Dose-dependent, differential effects of hnRNP A1 and A on HPV16 E6 and E7 mRNA alternative splicing. (A) RT-PCR on RNA extracted from HeLa cells transfected with pX856F in the absence (−) or presence of 2-fold serially diluted phnRNP A1 or phnRNP A2 (2, 1, 0.5, and 0.25 μg). RT-PCR primers were 97S and 856A. The splicing pattern each RT-PCR band represents is indicated to the right. M, molecular size marker. (B) Western blotting on cell extracts from HeLa cells transfected with serially diluted phnRNP A1 or phnRNP A2 plasmid. Blots were stained with antibodies specific for hnRNP A1 or A2 as described in Materials and Methods. MW, molecular weight marker. (C) Densitometric quantification of the RT-PCR bands in panel A. The quantitations are displayed as fold difference of HPV16 RT-PCR band intensity obtained with RNA extracted from HeLa cells transfected with phnRNP A1 or phnRNP A2 plasmid over band intensities in cells transfected with empty pUC plasmid. Fold difference is plotted against the amount of transfected phnRNP A1 or phnRNP A2 plasmid.

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  FIG 6. Mapping of the hnRNP A1 target site in the HPV16 E7 coding region to a splicing-inhibitory HPV16 RNA sequence located between HPV16 genomic nucleotide positions 579 and 604. (A) RT-PCR on RNA extracted from HeLa cells transfected with plasmid pX556F, pX579, pX581, pX604, pX616, or pX644 in the absence (pUC) or presence of phnRNP A1. The bands representing unspliced HPV16 E6 mRNAs and HPV16 mRNAs spliced from SD226 to SA409 are indicated to the right. cDNAs were amplified by RT-PCR primers 97S and X556AF. (B) Quantitation of results in panel A. The two lines represent percentage unspliced or spliced HPV16 mRNA calculated for each HPV16 plasmid transfected with either pUC plasmid or hnRNP A1 expression plasmid and plotted against each transfected HPV16 expression plasmid. (C, upper) The wild-type HPV16 sequence from HPV16 nucleotide positions 564 to 604 is displayed. The region between HPV16 nucleotide positions 581 and 604 that mediates splicing inhibition is marked in red. The UAG-containing sequence that constitutes a potential target site for hnRNP A1 and A2 is underlined. This sequence represents the sequence in wild-type plasmid pX604. (Lower) The mutant HPV16 sequence in…

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  FIG 7. hnRNP A1 and A2 bind to an HPV16 RNA sequence located between HPV16 genomic nucleotide positions 594 and 604. (A) Schematic representation of the 5′ end of the HPV16 genome encoding E6, E7, and part of E1. HPV16 early (p97) and late (p670) promoters and HPV16 splice sites SD226, SA409, SA526, and SA742 are indicated. Names and nucleotide positions of overlapping, biotinylated RNA oligonucleotides are shown below. Nucleotide positions of the 5′ and 3′ ends of the RNA oligonucleotides refer to the HPV16 reference genome HPV16R (45). The region in the HPV16 genome covered by the RNA oligonucleotides is indicated as a gray box. (B) Sequences of selected biotinylated HPV16 RNA oligonucleotides used in RNA-mediated protein pulldowns of proteins from HeLa cell nuclear extracts. The sequence to which hnRNP A1 and A2 mapped is indicated in red. (C and D) Western blotting of factors pulled down by Streptavidin-coated magnetic beads carrying either of the indicated biotinylated HPV16 RNA oligonucleotides. Filters were stained with antibody to hnRNP A1 (C) or hnRNP A2 (D) as described in Materials and Methods. (E) Coomassie staining of purified GST- and GST-hnRNP A1 protein. (F) Western…

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  FIG 8. Nucleotide substitutions abolish pulldown of hnRNP A1 and A2 by HPV16 RNA oligonucleotides. (A) Sequences of biotinylated HPV16 RNA oligonucleotides used in RNA-mediated protein pulldowns of proteins from HeLa cell nuclear extracts. The sequence to which hnRNP A1 and A2 pulldown was mapped in Fig. 7 is indicated in red. Nucleotide substitutions are marked in dark blue. Numbers refer to nucleotide positions in HPV16R. Names of RNA oligonucleotides are listed to the left. (B and C) Western blotting of factors pulled down by Streptavidin-coated magnetic beads carrying either of the indicated biotinylated HPV16 RNA oligonucleotides. Filters were stained with antibody to either hnRNP A1 (B) or hnRNP A2 (C). (D) The sequence of the wild-type and mutant splicing silencer sequence in pC97ELsL and pC97ELsLM1, respectively, is shown. (E) RT-PCR on RNA extracted from HeLa cells transfected with plasmid pC97ELsL or pC97ELsLM1. RT-PCR primers 97S and 880A were used. (F) Quantitation of the triplicate samples. Mean values and standard deviations are shown. (G) RT-minus control. (H) HeLa cells were transfected with pC97ELsL in the presence of hnRNP A1 or hnRNP A2 as described in Materials and…

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  FIG 9. C terminus of hnRNP A1 is required for efficient inhibition of splicing between HPV16 5′-splice site SD226 and 3′-splice site SA409 in the E6 coding region. (A) Schematic representation of the hnRNP A1 and A2 proteins and the A1A2 and A2A1 hybrids. RNA recognition motifs 1 and 2 (RRM1 and RRM2), RGG, and pyrimidine-rich nuclear/cytoplasmic localization sequence (PY-NLS) (also termed M9) are indicated. hnRNP A1 consists of 320 amino acids and hnRNP A2 of 341 amino acids. The break points of the A1A2 and A2A1 hybrids are indicated. H, HA tag. (B) Western blotting on extracts from HeLa cells transfected with plasmids expressing the hnRNP A1A2 and hnRNP A2A1 hybrids. Blots are stained with anti-HA tag antibodies that detect the HA-tagged hnRNP A1A2 and hnRNP A2A1 hybrids but not the untagged hnRNP A1 and hnRNP A2 proteins. MW, molecular weight marker. (C) RT-PCR on RNA extracted from HeLa cells transfected with pC97ELsL (62) in the absence (−) or presence of phnRNP A1, phnRNP A2, or plasmids expressing the hnRNP A1A2 or hnRNP A2A1 hybrids (pA1NA2C and pA2NA1C). RT-PCR primers 97S and X765A were used (for the location of these RT-PCR primers, see Fig. 1C). The mRNA splicing each…

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  FIG 10. hnRNP A1 and A2 inhibit splicing of HPV16 E6 and E7 mRNAs produced from the episomal HPV16 DNA genome. (A, left) Schematic representation of genomic HPV16 plasmid pHPV16AN (40). LoxP sites and HPV16 early (p97) and late (p670) promoters and early (pAE) and late (pAL) poly(A) signals are indicated. Positions of PCR primers 16S and 16A are indicated. (Right) The effect of the cre recombinase on pHPV16AN in transfected cells is illustrated. Schematic representation of the 5′ end of the unspliced HPV16 E6 mRNAs and the mRNAs spliced between SD226 and SA409 are shown. (B) PCR with primers 16S and 16A on Hirt DNA extracted from HeLa cells transfected with the indicated HPV16 plasmids in the presence or absence (−) of the cre-expressing plasmid pCAGGS-nlscre. Primers 16S and 16A are located on each side of the LoxP sites in the HPV-16 plasmids, and the PCR reaction yields a 366-nucleotide PCR fragment that is diagnostic for recombination at the LoxP sites. A larger band is amplified from plasmid DNA that has not recombined (a parallel transfection performed in the absence of pCAGGS-nlscre [−cre]). (C) RT-PCR on RNA extracted from HeLa cells transfected with pHPV16AN and pCAGGS-nlscre in…

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  FIG 11. Knockdown of hnRNP A1 enhances splicing of HPV16 E6 and E7 mRNAs produced from the episomal HPV16 DNA genome. (A) pHPV16AN (40) and pCAGGS-nlscre were cotransfected in duplicates with scrambled (scr) siRNAs or siRNAs to hnRNP A1 (siA1) or hnRNP A2 (siA2), RNA was extracted, and RT-PCR was performed with primers 97S and X525A. Control PCRs in the absence of reverse transcriptase (RT-minus) are shown. (B) Densitometric quantification of the RT-PCR bands was performed in triplicates. Mean values are shown and standard deviations are indicated. The quantitations are displayed as fold difference of HPV16 RT-PCR band intensity obtained with RNA extracted from cells transfected with pHPV16AN and pCAGGS-nlscre in the presence of hnRNP A1 or hnRNP A2 siRNAs over band intensities in cells transfected with pHPV16AN and pCAGGS-nlscre in the presence of scrambled siRNAs (scr). (C) Knockdown of hnRNP A1 and hnRNP A2 was confirmed by Western blotting of hnRNP A1 or hnRNP A2.

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  FIG 12. Knockdown of hnRNP A1 in HPV16-driven cervical cancer cell line SiHa promotes splicing of the HPV16 E6 and E7 mRNAs and enhances production of HPV16 E7 protein. (A) HPV16-positive cervical cancer cell line SiHa was transfected in duplicates with scrambled (scr) siRNAs or siRNAs to hnRNP A1 (siA1) or hnRNP A2 (siA2), RNA was extracted, and RT-PCR was performed with primers 97S and X525A. (B) Densitometric quantification of the RT-PCR bands was performed in triplicates. Mean values are shown, and standard deviations are indicated. The quantitations are displayed as fold difference of HPV16 RT-PCR band intensity obtained with RNA extracted from SiHa cells transfected with siRNAs to hnRNP A1 or hnRNP A2 (siA1 and siA2) siRNAs over band intensities in cells transfected with scrambled siRNAs (scr). (C) Knockdown of hnRNP A1 and hnRNP A2 was confirmed by Western blotting of hnRNP A1 or hnRNP A2. (D) HPV16-positive cervical cancer cell line SiHa was either untransfected (−) or transfected in duplicates with scrambled (scr) siRNAs or siRNAs to hnRNP A1 (siA1) or hnRNP A2 (siA2), and protein was extracted with RIPA buffer and analyzed by Western blotting with antibodies to HPV16 E7 or…