The alternative splicing factor hnRNP A1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element

Abstract

Human papillomavirus type 16 (HPV16) infects anogenital epithelia and is the etiological agent of cervical cancer. We showed previously that HPV16 infection regulates the key splicing/alternative splicing factor SF2/ASF and that virus late transcripts are extensively alternatively spliced. hnRNP A1 is the antagonistic counterpart of SF2/ASF in alternative splicing. We show here that hnRNP A1 is also up-regulated during differentiation of virus-infected epithelial cells in monolayer and organotypic raft culture. Taken together with our previous data on SF2/ASF, this comprises the first report of HPV-mediated regulation of expression of two functionally related cellular proteins during epithelial differentiation. Further, using electrophoretic mobility shift assays and UV crosslinking we demonstrate that hnRNP A1 binds the HPV16 late regulatory element (LRE) in differentiated HPV16 infected cells. The LRE has been shown to be important in temporally controlling virus late gene expression during epithelial differentiation. We suggest that increased levels of these cellular RNA processing factors facilitate appropriate alternative splicing necessary for production of virus late transcripts in differentiated epithelial cells.

Fig. 1

RNA sequence of the HPV16 LRE at the junction of the L1 open reading frame and the long control region (LCR). The L1 UAA stop codon is in bold type. The four weak 5′ splice sites (Cumming et al., 2003) are underlined and the 3′ GU-rich region is in italic type. A putative hnRNP A1 binding site is boxed. Truncated LRE probes used in experiments described in Fig. 5 are indicated beneath the sequence.

Fig. 2

hnRNP A1 is up-regulated during differentiation of W12E cells. Cell lysates were prepared from undifferentiated (U) and differentiated (D) W12E and HaCaT cells. 10 μg of each extract was fractionated on SDS-PAGE and Western blotted with antibodies against the proteins indicated beside the graphs associated with each set of Western blots. (A) SF2/ASF (B) hnRNP A1 (C) U2AF⁶⁵ (D) Sm protein (E) U1A (F) CstF-64 (G) HuR. Graphs show quantification of the Western blot data by densitometry scanning and show the mean and standard deviation from the mean of three to five separate experiments. The scale on the Y-axis in (A) and (B) is different to the scale for (C–G). (H) Western blots of undifferentiated and differentiated W12 and HaCaT cells probed with antibodies against involucrin, keratin 10 and filaggrin to demonstrate extent of differentiation of the cells. W12 cell extracts were probed with an anti-L1 antibody to demonstrate production of virus late proteins, associated with terminally differentiated, virus-infected epithelial cells. Blots were reprobed with an antibody against GAPDH as a loading control.

Fig. 3

Immunostaining of organotypic raft culture sections demonstrating epithelial differentiation stage-specific and HPV16-mediated regulation of hnRNP A1. 4 μm raft tissue sections stained with (A) anti-involucrin antibody or (B) anti-hnRNP A1 antibody. Nuclei are stained with DAPI. The basal and suprabasal layers of the epithelia are indicated. The dotted red line indicates the junction of the raft tissue and the collagen layer. (C) Immunohistochemistry staining for hnRNP A1 (brown colour) of a 4 μm section of normal human foreskin keratinocytes. Nuclei are counterstained with eosin (blue colour). The basal and suprabasal layers of the epithelium are indicated.

Fig. 4

hnRNP A1 binds the HPV16 LRE. (A) Electrophoretic mobility shift assay showing interaction of hnRNP A1 and LRE RNA in HeLa and W12 cells. Free probe and the RNA/protein complexes formed are indicated. Asterisks and an arrowhead indicate RNA/protein complexes that have been supershifted due to binding of the anti-hnRNP A1 antibody in tracks 3 and 5. Track 1, [α-³²P] rUTP-labelled LRE RNA probe alone (the LRE RNA is a stem loop structure (Cumming et al., 2003); the upper band is due to secondary structure formation); track 2, LRE RNA probe incubated with HeLa nuclear extract; track 3, as in track 2 but nuclear extract preincubated with anti-hnRNP A1 antibody; track 4, LRE RNA probe incubated with W12E nuclear extract; track 5, as in track 4 but nuclear extract preincubated with anti-hnRNP A1 antibody. (B) Western blot of nuclear (tracks 2 and 4) and cytoplasmic (tracks 1 and 3) extracts from undifferentiated (tracks 1 and 2) and differentiated (tracks 3 and 4) W12E cells showing fractionation of U2AF⁶⁵ and hnRNP A1 mainly with the nuclear fraction and involucrin mainly with the cytoplasmic fraction as expected. The increase in levels of involucrin in tracks 3 and 4 confirms that the W12 cells have differentiated. (C) Autoradiogram of an SDS-PAGE fractionation of UV crosslinking of [α-³²P] rUTP-labelled LRE RNA probe with nuclear (NE) and cytoplasmic (CE) fractions of HeLa and undifferentiated (U) and differentiated (D) W12E cells. (D) Western blot of the SDS-PAGE in (C) probed with anti-hnRNP A1 antibody.

Fig. 5

Nuclear hnRNP A1 binds the LRE directly. (A) Electrophoretic mobility shift analysis of binding of GST (500 nM) and GST-hnRNP A1 (45, 90, 180 nM) to [α-³²P] rUTP-labelled LRE or truncated LRE RNA probes. The probes used are shown in Fig. 1 and indicated below the autoradiograph. Arrows indicate free probe. A bracket indicates RNA/protein complexes. (B) Competition electrophoretic mobility shift experiment using a non-specific competitor, unlabelled in vitro-transcribed pBluescript polylinker RNA (70 nts) (pBS) (lanes 1–5) or a specific competitor, unlabelled in vitro transcribed LRE RNA (79 nts) (lanes 6–10). P; [α-³²P] rUTP-labelled LRE probe alone, N; probe plus nuclear extract. Both competitors were added to reactions at 1, 2, 4, 8 and 16-fold molar excess. Arrows indicate free probe. A bracket indicates protein/RNA complexes.