IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Translational regulation of human papillomavirus type 16 E7 mRNA by the peptide SEQIKA, shared by rabbit alpha(1)-globin and human cytokeratin 7. Translational regulation of human papillomavirus type 16 E7 mRNA by the peptide SEQIKA, shared by rabbit alpha(1)-globin and human cytokeratin 7. J. Virol. (0022-538X) 2002 Kanduc D. In the RNA EMSA (REMSA) the 20-mer e7 mRNA was radiolabelled with 32-P. In the peptide EMSA (PEMSA) the unlabeled 20-mer e7 mRNA was incubated with biotinylated peptide. d.kanduc@biologia.uniba.it 13-Nov-2007: Contacted by JYOTI. in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa tag visualisation tag visualisation Kanduc D. In the RNA EMSA (REMSA) the 20-mer e7 mRNA was radiolabelled with 32-P. In the peptide EMSA (PEMSA) the unlabeled 20-mer e7 mRNA was incubated with biotinylated peptide. d.kanduc@biologia.uniba.it Accepted 2007-DEC-22 by JYOTI. 13-Nov-2007: Contacted by JYOTI. J. Virol. (0022-538X) 2002 Translational regulation of human papillomavirus type 16 E7 mRNA by the peptide SEQIKA, shared by rabbit alpha(1)-globin and human cytokeratin 7. Translational regulation of human papillomavirus type 16 E7 mRNA by the peptide SEQIKA, shared by rabbit alpha(1)-globin and human cytokeratin 7. J. Virol. (0022-538X) 2002 Kanduc D. In the RNA EMSA (REMSA) the 20-mer e7 mRNA was radiolabelled with 32-P. In the peptide EMSA (PEMSA) the unlabeled 20-mer e7 mRNA was incubated with biotinylated peptide. d.kanduc@biologia.uniba.it 13-Nov-2007: Contacted by JYOTI. human-siha Homo sapiens SiHa cervical carcinoma cell line siha SiHa cells huamn cervical carcinoma cells coip coimmunoprecipitation co-immunoprecipitation CoIp immunoprecipitation Co-IP co-immunoprecipitation CoIp immunoprecipitation Co-IP coip predetermined predetermined participant predetermined Kanduc D. In the RNA EMSA (REMSA) the 20-mer e7 mRNA was radiolabelled with 32-P. In the peptide EMSA (PEMSA) the unlabeled 20-mer e7 mRNA was incubated with biotinylated peptide. d.kanduc@biologia.uniba.it Accepted 2007-DEC-22 by JYOTI. 13-Nov-2007: Contacted by JYOTI. J. Virol. (0022-538X) 2002 Translational regulation of human papillomavirus type 16 E7 mRNA by the peptide SEQIKA, shared by rabbit alpha(1)-globin and human cytokeratin 7. Translational regulation of human papillomavirus type 16 E7 mRNA by the peptide SEQIKA, shared by rabbit alpha(1)-globin and human cytokeratin 7. J. Virol. (0022-538X) 2002 Kanduc D. In the RNA EMSA (REMSA) the 20-mer e7 mRNA was radiolabelled with 32-P. In the peptide EMSA (PEMSA) the unlabeled 20-mer e7 mRNA was incubated with biotinylated peptide. d.kanduc@biologia.uniba.it 13-Nov-2007: Contacted by JYOTI. in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa tag visualisation tag visualisation Kanduc D. In the RNA EMSA (REMSA) the 20-mer e7 mRNA was radiolabelled with 32-P. In the peptide EMSA (PEMSA) the unlabeled 20-mer e7 mRNA was incubated with biotinylated peptide. d.kanduc@biologia.uniba.it Accepted 2007-DEC-22 by JYOTI. J. Virol. (0022-538X) 2002 seqika SEQIKA peptide peptide polypeptide oligopeptide oligopeptide polypeptide in vitro In vitro SEQIKA An amino acid sequence which is not represented in UniProt Small peptide present in human KRT7 and rabbit heamaglobin subunit alpha 1/2. 6DD33046DB05D000 hba_sheep Hemoglobin subunit alpha-1/2 Hemoglobin alpha-1/2 chain Alpha-1/2-globin protein protein sheep Ovis aries (Sheep) Sheep MVLSAADKSNVKAAWGKVGGNAGAYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGEKVAAALTKAVGHLDDLPGTLSDLSDLHAHKLRVDPVNFKLLSHSLLVTLACHLPNDFTPAVHASLDKFLANVSTVLTSKYR 1C3CF84DEB3C2176 hba_rat Hemoglobin subunit alpha-1/2 Hba1 Hba-a1 Hemoglobin alpha-1/2 chain Alpha-1/2-globin protein protein rat Rattus norvegicus (Rat) Rat MVLSADDKTNIKNCWGKIGGHGGEYGEEALQRMFAAFPTTKTYFSHIDVSPGSAQVKAHGKKVADALAKAADHVEDLPGALSTLSDLHAHKLRVDPVNFKFLSHCLLVTLACHHPGDFTPAMHASLDKFLASVSTVLTSKYR DEF6857594C42A99 20-mer e7 mrna messenger rna mRNA mrna hpv16 Human papillomavirus type 16 TTATGGTTTCTGAGAACAGA A 20-mer oligonucleotide complementary to the 3' end of HPV16 E7 mRNA 6F22800B3F598BB1 hba_human Hemoglobin subunit alpha Hemoglobin alpha chain Alpha-globin HBA1 HBA2 protein protein human Homo sapiens Human MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR DIP protein P69905 original sequence version: 112 DIP protein P69905 original sequence version: 132 15E13666573BBBAE hba_colli Hemoglobin subunit alpha-A HBAA Hemoglobin alpha-A chain Alpha-A-globin protein protein colli Columba livia (Domestic pigeon) MVLSANDKSNVKAVFAKIGGQAGDLGGEALERLFITYPQTKTYFPHFDLSHGSAQIKGHGKKVAEALVEAANHIDDIAGALSKLSDLHAQKLRVDPVNFKLLGHCFLVVVAVHFPSLLTPEVHASLDKFVLAVGTVLTAKYR A4CE77962BBE6EFB hba_rabit Hemoglobin subunit alpha-1/2 Hemoglobin alpha-1/2 chain Alpha-1/2-globin protein protein rabit Oryctolagus cuniculus (Rabbit) Rabbit MVLSPADKTNIKTAWEKIGSHGGEYGAEAVERMFLGFPTTKTYFPHFDFTHGSEQIKAHGKKVSEALTKAVGHLDDLPGALSTLSDLHAHKLRVDPVNFKLLSHCLLVTLANHHPSEFTPAVHASLDKFLANVSTVLTSKYR D117DDF66C13DD2B hba_pig Hemoglobin subunit alpha HBA Hemoglobin alpha chain Alpha-globin protein protein pig Sus scrofa (Pig) Pig VLSAADKANVKAAWGKVGGQAGAHGAEALERMFLGFPTTKTYFPHFNLSHGSDQVKAHGQKVADALTKAVGHLDDLPGALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHHPDDFNPSVHASLDKFLANVSTVLTSKYR DIP protein P01965 original sequence version: 87 95869BABB4C4EDB8 k2c7_human Keratin, type II cytoskeletal 7 KRT7 SCL Cytokeratin-7 Sarcolectin Keratin-7 Type-II keratin Kb7 protein protein human Homo sapiens Human MSIHFSSPVFTSRSAAFSGRGAQVRLSSARPGGLGSSSLYGLGASRPRVAVRSAYGGPVGAGIREVTINQSLLAPLRLDADPSLQRVRQEESEQIKTLNNKFASFIDKVRFLEQQNKLLETKWTLLQEQKSAKSSRLPDIFEAQIAGLRGQLEALQVDGGRLEAELRSMQDVVEDFKNKYEDEINHRTAAENEFVVLKKDVDAAYMSKVELEAKVDALNDEINFLRTLNETELTELQSQISDTSVVLSMDNSRSLDLDGIIAEVKAQYEEMAKCSRAEAEAWYQTKFETLQAQAGKHGDDLRNTRNEISEMNRAIQRLQAEIDNIKNQRAKLEAAIAEAEERGELALKDARAKQEELEAALQRGKQDMARQLREYQELMSVKLALDIEIATYRKLLEGEESRLAGDGVGAVNISVMNSTGGSSSGGGIGLTLGGTMGSNALSFSSSAGPGLLKAYSIRTASASRRSARD 070CBE8F66A62497 hba_bovin Hemoglobin subunit alpha HBA Hemoglobin alpha chain Alpha-globin protein protein bovin Bos taurus (Bovine) Bovine MVLSAADKGNVKAAWGKVGGHAAEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGAKVAAALTKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLLVTLASHLPSDFTPAVHASLDKFLANVSTVLTSKYR 6D20CADDA05C0DC5 hpv16e7-hba 3 10 unspecified role unspecified role prey prey rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes 7 unspecified role unspecified role bait bait physical association physical association Figure 2, 3, 4 In this approach, the REMSA was performed on the 32P-labeled viral transcript following incubation with rabbit reticulocyte lysate. A parallel sample containing proteinase K was run as a control. Figure 2C, lane 2, shows the presence of an mRNA-protein complex that could be detected following 5% PAGE. No gel shift signal was evident when the reaction mixture was supplemented with proteinase K (Fig. 2C, lane 3), documenting the protein nature of the complex. No protein complex was evident when c-myc or eIF-4E mRNA was used (Fig. 2A and B, respectively, lane 2). Figure 3, lane 2, shows that the component eluted from E7 mRNA-coated beads was positive to silver staining and migrated as a 32-kDa polypeptide (p32). The p32 component was absent in the depleted reticulocyte lysate preparation (Fig. 3, lane 3). A 32 kDa protein component within rabbit reticulocytes was shown to bind to hpv16 e7. The binding of E7 mRNA to a p32 component from rabbit reticulocyte lysate controls HPV16 E7 expression by preventing both degradation and translation of the viral transcript. The 32 kDa protein component within rabbit reticulocytes was shown to inhibit hpv16 e7 mRNA degradation and translation. The effect of p32 on HPV16 E7 mRNA degradation was analyzed with the p32-depleted reticulocyte lysate preparation. This showed that E7 mRNA stability was markedly reduced when assayed in the p32-depleted reticulocyte lysate. Also p32 addition protected E7 mRNA (but not c-myc or eIF-4E mRNAs) from RNase A digestion. There was also a striking translational signal obtained by carrying out translation in the presence of anti-p32 Abs hpv16e7-seqika 3 7 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait region biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined lys5leu binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro physical association physical association Figure 6A-D, 7C Rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA binds to, prevents decay of, and blocks translation of HPV16 E7 mRNA. Mutation of K (lys) at position 5 to L (leu) prevented binding suggesting that this residue is essential for peptide binding to HPV16 E7 mRNA. PEMSA results are shown in Fig. 6A. It is clearly evident that the rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA fragment did interact with HPV16 E7 mRNA. The interaction was peptide and mRNA specific, since no interaction was found with the cytoskeletal paxillin LPGALS peptide or the control c-myc or eIF-4E mRNA. In addition, Fig. 6B and C show that the 6-mer peptide SEQIKA stabilized HPV16 E7 mRNA when incubated in the p32-depleted reticulocyte lysate. Likewise, the SEQIKA fragment restored the translational inhibition removed by anti-p32 PAbs in the in vitro translational assay carried out with complete rabbit reticulocyte lysate (Fig. 6D). The peptide binding sequence was further investigated by synthesizing three biotinylated 6-mer peptides changed at the three invariant positions S52, Q54, and K56, i.e., GEQIKA, SELIKA, and SEQILA, with the changed residues in italics. The three substituted 6-mer peptides were then used in the PEMSAs reported in Fig. 7C. It can be seen that the K amino acid residue was essential for peptide binding to HPV16 E7 mRNA. hpv16e7-sxqxkx 1 7 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro physical association physical association Table 2, Figure 7A, 7B Rabbit {alpha}1-globin 53-58 SEQIKA motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-sxqxkx-1 1 8 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 7 unspecified role unspecified role bait bait physical association physical association Table 2, Figure 7A, 7B Human {alpha}1-globin 53-58 SAQIKG motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-sxqxkx-2 1 13 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 7 unspecified role unspecified role bait bait physical association physical association Table 2, Figure 7A, 7B Bovine {alpha}1-globin 53-58 SAQVKG motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-sxqxkx-3 1 6 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 7 unspecified role unspecified role bait bait physical association physical association Table 2, Figure 7A, 7B Rat {alpha}1-globin 53-58 SAQVKA motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-sxqxkx-4 1 7 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro physical association physical association Table 2, Figure 7A, 7B Sheep {alpha}1-globin 53-58 SAQVKG motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-sxqxkx-5 1 7 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro physical association physical association Table 2, Figure 7A, 7B Sheep {alpha}1-globin 52-57 SDQVKA motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-sxqxkx-6 1 9 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 7 unspecified role unspecified role bait bait physical association physical association Table 2, Figure 7A, 7B Pigeon {alpha}1-globin 53-58 SAQIKG motif showed binding to radiolabeled HPV16 E7 mRNA by gel retardation experiments and this binding was suppressed by anti-p32Pabs. To gain some preliminary insights into the mode of rabbit {alpha}1-globin52-57/human CK791-96 SEQIKA action, I tested the mRNA-binding ability of hemoglobins having double and triple amino acid substitutions inside the {alpha}1-globin52-57 motif. Table 2 describes the hemoglobins that were assayed. It can be seen that the common {alpha}1-globin52-57 motif is the sequence SXQXKX, where S52, Q54, and K56 are invariant amino acid residues in the hemoglobins analyzed. Then, the hemoglobins described in Table 1 were tested by REMSA performed on the 32P-labeled viral transcript. Figure 7A illustrates that all of the hemoglobins listed in Table 1 were able to bind HPV16 E7 mRNA, indicating that the conserved globin motif S52-Q54-K56 was sufficient for the interaction with the transcript. Anti-p32 PAbs suppressed hemoglobin binding to HPV16 E7 mRNA (Fig. 7B). hpv16e7-krt7 2 7 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey human-siha Homo sapiens SiHa cervical carcinoma cell line siha SiHa cells huamn cervical carcinoma cells physical association physical association Figure 9 FIG. 9. HPV16 E7 mRNA-CK7 interaction in squamous cervical cancer SiHa cell lysate. The in-vivo interaction assay showed that there was binding of the 20-mer oligonucleotide with krt7. The assay was carried out using magnetic beads conjugated with the biotinylated 20-mer oligonucleotide 5'-TTATGGTTTCTGAGAACAGA, complementary to the 3' end of E7 mRNA, as bait. Oligonucleotide-coated beads were incubated with SiHa cell lysate and then eluted with the high-salt elution buffer.