Identifying regulatory elements and their RNA-binding proteins in the 3' untranslated regions of papillomavirus late mRNAs

Abstract

Human papillomaviruses (HPVs) infect cutaneous and mucosal epithelia to cause benign (warts) and malignant lesions (e.g. cervical cancer). Bovine papillomaviruses (BPVs) infect fibroblasts to cause fibropapillomas but can also infect cutaneous epithelial cells. For HPV-1, -16, -31 and BPV-1, cis-acting RNA elements in the late 3' untranslated region (3'UTR) control expression of virus proteins by binding host cell proteins. The present study compared the effects on gene expression of the cis-acting elements of seven PV late 3'UTRs (HPV-6b, -11, -16, -31 and BPV-1, -3 and -4) representing a range of different genera and species and pathological properties. pSV-beta-galactosidase reporter plasmids containing the late 3'UTRs from seven PVs were transiently transfected into cervical adenocarcinoma HeLa cells, and reporter gene expression quantified by reverse transcription-quantitative PCR and a beta-galactosidase assay. All elements inhibited gene expression in keratinocytes. Cancer-related types HPV-16 and -31, had the greatest inhibitory activity whereas the lowest inhibition was found in the non-cancer related types, BPV-3 and HPV-11. Using RBPmap version 1.1, bioinformatics predictions of factors binding the elements identified proteins which function mainly in mRNA splicing. Markedly, in terms of protein binding motifs, BPV late 3'UTR elements were similar to those of HPV-1a but not to other HPVs. Using HPV-1a as a model and siRNA depletion, the bioinformatics predictions were tested and it was found that PABPC4 was responsible for some of the 3'UTR repressive activity. The data revealed candidate proteins that could control PV late gene expression.

Keywords: 3'untranslated region; RNA regulatory element; RNA-binding protein motifs; late gene expression; papillomavirus.

Figure 1

Relatedness of the 3'UTRs of selected PVs. (A) Diagram of the region of cloned fragments, starting from the 3' end of last exon and expanding into late 3' UTR, of eight PVs analyzed in this study. Numbers indicate genomic nucleotide positions. Regulatory elements have been functionally defined for HPV-1a, -16, 31 and BPV-1 (7-11) but not for the other PVs. (B) Phylogenetic tree of eight PV late 3'UTRs, the PV late 3'UTR sequences were retrieved from Genbank. Accession numbers appear in parentheses. Bootstrap (1,000 replicates) values in percentage are shown. Bar at 0.1 substitutions per nucleotide. 3'UTRs, 3' untranslated region; PV, papillomaviruses; HPV, human papillomavirus; BPV, bovine papillomavirus.

Figure 2

Inhibitory activity on gene expression of the late 3'UTRs of seven PVs compared with pSV (A) PCR products separated by 1% agarose gel electrophoresis, (B) average band intensity of PCR products obtained from pSV containing late 3'UTR of each PVs transiently transfected to HeLa cells; (C) Quantitative PCR products from pSV containing late 3'UTR of each PV transiently transfected to HaCaT cells (D) average absorbance of HeLa cell lysates from beta-galactosidase activity assays. Bar charts show the mean and standard error of the mean from three separate experiments. ^*P<0.05. 3' untranslated region; PV, papillomavirus; pSV, pSV-beta-galactosidase positive control; (-)RT, no reverse transcriptase added; M, size marker.

Figure 3

Percentages of the predicted binding sites of RNA binding proteins for PV late 3'UTR fragments using the computational tool RBPmap version 1.1(31). PV, papillomavirus; 3'UTR, 3' untranslated region; EJC, exon junction complex.

Figure 4

Predicted RNA binding proteins, by using RBPmap version 1.1 (http://rbpmap.technion.ac.il/) (31), at high stringency level [P-value (significant) < 0.001 and P-value (suboptimal) < 0.01] and binding site numbers that have functions related to (A) mRNA splicing, (B) mRNA stability, (C) mRNA transport and (D) mRNA degradation. HPV, human papillomavirus; BPV, bovine papillomavirus.

Figure 5

Comparisons of predicted RNA binding protein sites in HPV-1a late 3'UTR and the 7 PVs (A) Percentage of RNA binding protein binding sites found in eight PV 3'UTRs categorised into 11 mRNA metabolic pathways/events analyzed using RBPmap version 1.1(31) at high stringency level, P-value (significant) <0.001 and P-value (suboptimal) <0.01, (B) Graphs showing relative levels of siRNA knockdown of PABPC1, PABPC4 and HuR with siRNA control. (C) Western blot analysis of HeLa cell lysate after siRNA depletion using primary antibodies against PABPC1, PABPC4, HuR and GAPDH. The GADPH control for PABPC4 and HuR shows faint bands above the main band. This is due to the X-ray film moving during exposure. (D) Average absorbance from beta-galactosidase activity assays measured in HeLa cell lysates transiently co-transfected with pSV-HPV-1a late 3'UTR and siRNA for PABPC1, PABPC4 and HuR depletion. Bar charts show the mean and standard error of the mean from three separate experiments. ^*P<0.05. HPV, human papillomavirus; 3'UTR, 3' untranslated region; PV, papillomavirus; si, small interfering; PABPC, poly(A) binding protein; HuR, human antigen R; pSV, pSV-beta-galactosidase positive control.