Identification of nuclear and cytoplasmic proteins that interact specifically with an AU-rich, cis-acting inhibitory sequence in the 3' untranslated region of human papillomavirus type 1 late mRNAs

Abstract

Expression of human papillomavirus late genes encoding L1 and L2 capsid proteins is restricted to terminally differentiated epithelial cells. We have previously identified and characterized an AU-rich, cis-acting negative regulatory element in the 3 untranslated region of human papillomavirus type 1 late mRNAs. This element acts posttranscriptionally to reduce mRNA levels and the translation efficiency of mRNAs. The experiments reported here are a continuation of our previous work. We have used RNA gel shifts and UV cross-linking assays to identify cellular proteins that interact with the inhibitory RNA sequence of human papillomavirus type 1. RNA gel shift assays established that cellular proteins interact with the AU-rich sequence. The binding of nuclear proteins was inhibited by competition with poly(U), whereas the binding of cytoplasmic proteins was inhibited by competition with poly(U) and also by competition with poly(A) and poly(G). Two nuclear proteins and two cytoplasmic proteins that bind specifically to the AU-rich RNA sequence were identified by UV cross-linking. These proteins did not bind to the 3 untranslated region of human papillomavirus type 1 early mRNAs, which does not show inhibitory activity. The cellular proteins identified in our experiments may therefore be involved in the inhibition of human papillomavirus type 1 late gene expression in nondifferentiated epithelial cells.

The Inhibitory Activity of the AU-Rich RNA Element in the Human Papillomavirus Type 1 Late 3′ Untranslated Region Correlates with Its Affinity for the elav-Like HuR Protein

“…Plasmid constructions. The following plasmids have been described previously: pCCKH1 and p⌬XKb (53); pKSXB, pKSAUM, pKSUM, pKSAUM/UM, pKSCUC, pKSB2, pKSC1, pKSC2, pKSC3, and pKSFOS (44); and pGEX-HuR (33). To generate pT7HuR, a PCR fragment was amplified from pGEX-HuR (33) with oligonucleotides HURSTART (5Ј-GTCGACACAATGTCTAATGG TTATGGAG-3Ј) and HURSTOP (5Ј-GGTACCTTATTTGTGGGACTTGTT GG-3Ј) and ligated to pBluescript (Stratagene) digested with EcoRV and treated with calf intestinal alkaline phosphatase.…”

“…In vitro transcription, RNA gel shift assay, and UV cross-linking. Following linearization of pKSXB, pKSAUM, pKSUM, pKSAUM/UM, pKSCUC, pKSB2, pKSC1, pKSC2, pKSC3, or pKSFOS, in vitro transcription was performed as described previously (53), except that the RNAs were labeled with [␣-32 P]CTP. The molarities of the probes were calculated after determination of the specific activity by using Cerenkov ␤-counting.…”

“…Competitor RNAs were synthesized in the absence of radiolabeled nucleotide. RNA gel shift assays were performed essentially as described previously (53). Briefly, 25 to 40 fmol (or 4 fmol in the K d determination experiments) of radiolabeled RNA probe was incubated with 1 g of GST-HuR protein (or dilutions thereof) in a total volume of 20 l of binding buffer (60 mM KCl, 10 mM HEPES [pH 7.6], 3 mM MgCl 2 , 1 mM dithiothreitol (DTT), 5% glycerol, 5 g of heparin per l) for 15 min at room temperature.…”

“…For K d determinations, both bound and free RNAs were quantified (free RNA was defined as the signal position in the gel where RNA probe alone migrated). UV cross-linking was performed as described previously (53). Probes used for UV cross-linking were labeled with [␣-32 P]UTP.…”

“…Replacing uracil (U) with cytidine (C) in these motifs inactivated the h1ARE (44). The wild-type and mutant h1AREs were then used as substrates in a UV crosslinking assay to identify cellular proteins that bind preferentially to the wild-type sequence (44,52,53), since such factors could potentially be mediating the inhibitory activity of the h1ARE. Three nuclear proteins bound to the wild-type but not to the mutant inactive sequence (44).…”