E7 mRNA in vitro half-life and nuclease accessibility. (A) In vitro half-life assay of E7 mRNA. RNA transcript was incubated for 0 to 60 min at 30°C in decay reaction mixtures containing rabbit reticulocyte lysate. RNA was phenol extracted and electrophoresed in 4% nondenaturing polyacrylamide gels. c-myc and eIF-4E mRNAs were used as controls. (B) Quantification of mRNA half-life by densitometric analysis. ODu, optical density units. (C) E7 mRNA secondary structure and nuclease accessibility: effect of heat treatment on digestion pattern of HPV16 E7 mRNA by RNase A. Lanes: 1, nontreated E7 mRNA; 2, RNase A-digested E7 mRNA; 3, RNase digest of preheated E7 mRNA.

A p32 component from rabbit reticulocyte lysate binds to E7 mRNA-conjugated Dynabeads. For SDS-PAGE characterization of the E7 mRNA-binding protein purified by magnetic affinity, rabbit reticulocyte lysate was incubated in binding buffer with HPV16 E7 mRNA-activated magnetic beads. Following washings, the reticulocyte component fraction bound to Dynabeads was eluted and electrophoresed in SDS-12.5% PAGE. The gel was silver stained. Lanes: 1, whole rabbit reticulocyte lysate used for magnetic separation; 2, protein eluted with 1 M NaCl from the activated magnetic beads; 3, unbound proteins. Numbers on the left indicate positions of molecular mass standards (in kilodaltons).

Identification of rabbit reticulocyte p32 as α₁-globin. (A) Pir FASTA search for sequence similarity and full-length alignment of two peptide fragment motifs obtained by N-terminal and internal sequencing of p32. HARB, hemoglobin α₁ chain, rabbit. Smith-Waterman score, 155. (B) Addition of BSA to p32-depleted rabbit reticulocyte lysate does not promote E7 mRNA stability. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed by using the reticulocyte unbound proteins remaining in solution after incubation with E7 mRNA-activated magnetic beads plus control protein BSA. (C) Quantification of mRNA half-life as in panel B by densitometric analysis. (D) Addition of rabbit hemoglobin to p32-depleted rabbit reticulocyte lysate promotes E7 mRNA stability. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed with the reticulocyte unbound proteins remaining in solution after incubation with activated magnetic beads plus rabbit hemoglobin (25 μg). (E) Quantification of mRNA half-life as in panel D by densitometric analysis. ODu, optical density units.

HPV16 E7 mRNA-SEQIKA binding: definition of the consensus amino acid sequence. (A) Hemoglobins with double and triple amino acid substitutions inside the α₁-globin_52-57 SEQIKA motif and numbered from 1 to 7 as described in Table 2 were assayed for binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments. In the controls, mRNA was incubated in binding buffer (lane c) and binding buffer plus BSA (25 μg) (lane c₁). (B) Binding assays were supplemented with 50 μg of mouse anti-p32 PAbs and run as in panel A. (C) PEMSA experiments were run by incubating unlabeled HPV16 E7 mRNA with biotinylated control peptide LPGALS, binding peptide SEQIKA, and SEQIKA-derived peptides GEQIKA, SELIKA and SEQILA, each hosting a single amino acid change. The changed residues are underlined. Following electrophoresis, the biotinylated peptide-mRNA complex was visualized by ECL.

HPV16 E7 mRNA-CK7 interaction in squamous cervical cancer SiHa cell lysate. (A) SDS-PAGE characterization of the silver-stained material bound to and eluted from oligonucleotide-activated beads following incubation with 3T3 (lane 1) or SiHa (lane 2) cell lysate. (B) Western blot analysis of reactivity to MAbs raised against human CK4, CK7, CK14, or CK17 of material bound to and eluted from oligonucleotide-activated beads following incubation with 3T3 (lanes 1) or SiHa (lanes 2) cell lysate. (C and D) Analyses as in panels A and B, respectively, for cell lysates that had been treated with 50 μg of anti-SEQIKA PAbs. (E and F) Analyses as in panels A and B, respectively, for cell lysates that had been treated with 50 μg of anti-REWVKFAKPCRE PAbs.

Gel retardation experiments: analysis of interaction between HPV16 E7 mRNA and rabbit reticulocyte lysate. Radiolabeled c-myc (A), eIF-4E (B), and HPV16 E7 (C) mRNAs were incubated in binding buffer (lanes 1), binding buffer plus rabbit reticulocyte lysate (lanes 2), or binding buffer plus rabbit reticulocyte lysate and proteinase K (lanes 3) and electrophoresed in 5% PAGE.

Effect of rabbit reticulocyte p32 on HPV16 E7 mRNA half-life, RNase A digestion, and translation. (A) Depletion of p32 promotes in vitro E7 mRNA decay. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed with p32-depleted rabbit reticulocyte lysate. (B) Quantification of mRNA half-life by densitometric analysis. ODu, optical density units. (C) The p32 component protects E7 mRNA from RNase attack. c-myc, eIF-4E, and HPV16 E7 mRNAs were incubated with (lanes 2) RNase A, (lanes 3) RNase A plus p32 eluted from the magnetic beads, or (lanes 4) BSA (control); (lanes 1) nontreated mRNA. (D) Anti-p32 PAbs promote HPV16 E7 mRNA translation in complete rabbit reticulocyte lysate. Upper part, biotinylated translation pattern obtained following electroblotting onto a polyvinylidene difluoride membrane. Lower part, the same membrane probed with anti-E7 MAb. The positions of prestained molecular size markers are indicated (in kilodaltons).

Rabbit α₁-globin_52-57/human CK7_91-96 SEQIKA binds to, prevents decay of, and blocks translation of HPV16 E7 mRNA. (A) SEQIKA fragment binds to E7 mRNA. PEMSA experiments were run by incubating unlabeled c-myc, eIF-4E, or HPV16 E7 mRNA with biotinylated control peptide LPGALS or SEQIKA. Following electrophoresis, the biotinylated peptide-mRNA complex was visualized by ECL. (B) SEQIKA fragment promotes E7 mRNA stability. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed in vitro with the p32-depleted rabbit reticulocyte lysate obtained following incubation with E7 mRNA-conjugated magnetic beads plus the 6-mer peptide SEQIKA or LPGALS. (C) Quantification of mRNA half-life by densitometric analysis. ODu, optical density units. (D) SEQIKA fragment prevents HPV16 E7 mRNA translation promoted by anti-p32 PAbs in complete rabbit reticulocyte lysate. Translation reaction mixtures were preincubated with anti-p32 PAbs (50 μg) and/or peptide (10 μg) for 10 min prior to mRNA addition. Translation reactions were electrophoresed in SDS-12% PAGE. Following electroblotting onto a polyvinylidene difluoride membrane, E7 oncoprotein expression was analyzed by Western blotting with anti-E7 MAb.

Expression of CK7 in squamous cervical cancer SiHa cell lysate. Equal amounts of 3T3 (lane 1) and SiHa (lane 2) cell lysate (10 μg) were separated by SDS-12% PAGE, and CK7 expression was determined by Western blot analysis with anti-CK7 MAb.