The Translation Initiation Factor eIF3-p48 Subunit Is Encoded byint-6, a Site of Frequent Integration by the Mouse Mammary Tumor Virus Genome

Translation initiation factor eIF3 is a large, multisubunit protein complex that plays a central role in the pathway of initiation by promoting the binding of both methionyl-tRNA i and mRNA to the 40S ribosomal subunit. As part of a broad effort to elucidate the structure of eIF3, we have cloned and sequenced the human cDNA encoding the 48-kDa subunit, eIF3-p48. The recombinant protein comigrates with the authentic p48 subunit in purified eIF3 and coprecipitates with affinity-purified antibodies to the p170 subunit of eIF3. A search of the data base indicates that the mouse gene encoding eIF3-p48 had previously been identified and characterized by others asint-6. The int-6 gene is the site of frequent integration of mouse mammary tumor virus DNA into chromosomes, implicating the gene in the regulation of cell proliferation. In addition, it was shown elsewhere that the homologous humanint-6 gene product binds to the human T-cell leukemia virus type I Tax protein, leading to the translocation of Int-6 to the cytoplasm. We discuss how the cytosolic function of eIF3-p48 (Int-6) in protein synthesis may account for oncogenesis caused by these two viruses. Translation initiation factor eIF3 is a large, multisubunit protein complex that plays a central role in the pathway of initiation by promoting the binding of both methionyl-tRNA i and mRNA to the 40S ribosomal subunit. As part of a broad effort to elucidate the structure of eIF3, we have cloned and sequenced the human cDNA encoding the 48-kDa subunit, eIF3-p48. The recombinant protein comigrates with the authentic p48 subunit in purified eIF3 and coprecipitates with affinity-purified antibodies to the p170 subunit of eIF3. A search of the data base indicates that the mouse gene encoding eIF3-p48 had previously been identified and characterized by others asint-6. The int-6 gene is the site of frequent integration of mouse mammary tumor virus DNA into chromosomes, implicating the gene in the regulation of cell proliferation. In addition, it was shown elsewhere that the homologous humanint-6 gene product binds to the human T-cell leukemia virus type I Tax protein, leading to the translocation of Int-6 to the cytoplasm. We discuss how the cytosolic function of eIF3-p48 (Int-6) in protein synthesis may account for oncogenesis caused by these two viruses. The initiation phase of protein synthesis in eukaryotes is promoted by 10 or more proteins called initiation factors (reviewed in Ref. 1Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). Translation frequently is regulated by phosphorylation of the initiation factors, which causes either stimulation or inhibition of their activities. The levels and specific activities of these proteins are important for determining translation rates and for integrating the process of protein synthesis into the cell's overall metabolism. Abberations in regulating initiation factor activities may result in loss of control of cell proliferation and in malignant transformation of cells (2Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 245-269Google Scholar). To better understand the mechanism of action and regulation of initiation factors, we have sought to determine their primary structures by cloning and sequencing human cDNAs that encode them. One of the mammalian initiation factors, eIF3, is a multisubunit complex of ∼600 kDa that plays a central role in the pathway of initiation (1Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). eIF3 binds to the 40S ribosomal subunit and acts as a ribosomal subunit anti-association factor. It stabilizes the binding of methionyl-tRNA i to 40S ribosomal subunits and is required for mRNA binding. The 10 subunits of human eIF3 possess apparent masses of 170, 116, 110, 66, 48, 47, 44, 40, 36, and 35 kDa (3Hershey J.W.B. Asano K. Naranda T. Vornlocher H.-P. Hanachi P. Merrick W.C. Biochimie. 1997; 78: 903-907Crossref Scopus (61) Google Scholar). We have previously reported the cloning and characterization of cDNAs encoding the p110 and p36 subunits (4Asano K. Kinzy T.G. Merrick W.C. Hershey J.W.B. J. Biol. Chem. 1997; 272: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and others have reported on the p170 and p116 subunits (5Méthot N. Rom E. Olsen H. Sonenberg N. J. Biol. Chem. 1997; 272: 1110-1116Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 6Johnson K.R. Merrick W.C. Zoll W.L. Zhu Y. J. Biol. Chem. 1997; 272: 7016-7113Google Scholar). This report focuses on the p48 subunit of eIF3 and provides evidence that eIF3-p48 is identical to the product of the murine int-6 gene, where the mouse mammary tumor virus (MMTV) 1The abbreviations used are: MMTV, mouse mammary tumor virus; kb, kilobase pair; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag; bp, base pair(s). genome frequently integrates. Peptides from rabbit eIF3 subunits were prepared by proteolysis and sequenced as described previously (7Merrick W.C. Methods Enzymol. 1979; 60: 108-123Crossref PubMed Scopus (79) Google Scholar). Internal peptide sequencing of purified HeLa eIF3 (8Brown-Luedi M. Meyer L.J. Milburn S.C. Yau P.M.-P. Corbett S. Hershey J.W.B. Biochemistry. 1982; 21: 4202-4206Crossref PubMed Scopus (48) Google Scholar) was conducted in the Protein Structure Laboratory of the University of California, Davis. Clones 142907 (GenBank™ accession number R71564), 47908 (H11132 and H11044), and 71140 (T47475 andT47476) were kindly provided by the Lawrence Livermore National Laboratory (Livermore, CA) and were sequenced on both strands. Plasmid pTZp48 was constructed by subcloning the following PCR DNA fragment into the BamHI and HindIII sites of pTZ19R (9Mead D.A. Szczesna-Skorupa E. Kemper B. Protein Eng. 1986; 1: 67-74Crossref PubMed Scopus (569) Google Scholar); the 1.3-kb DNA fragment was generated by PCR amplification of a heat-treated human liver cDNA library (Stratagene) with the following primers: oligo-1 (5′-CCCAAGCTTAAGATGGCGGAGTACGACTTGAC-3′, corresponding to nucleotides 4–26 in the DNA sequence with accession number U54562, tagged with a HindIII site) and oligo-2 (5′-CCCGGATCCTCAGTAGAAGCCAGAATCTT-3′, corresponding to nucleotides 1344–1325, tagged with a BamHI site), and was digested withBamHI and HindIII. Poly(A)+ mRNA purified from mouse FM3A cells was employed for an reverse transcription-PCR reaction (Gene Amp RNA PCR kit, Perkin-Elmer) using the primers 5′-GCGGAATTCCCCGGCAAGATGGCGG-3′ (corresponding to nucleotides 1–17 in accession number U54563, tagged with an EcoRI site) and 5′-CCCAAGCTTCCCATGTTTGTCTGCCAGG-3′ (corresponding to nucleotides 376–358, tagged with a HindIII site). The resulting 0.4-kb DNA fragment was sequenced and subcloned into pBluescript SKII (Stratagene), followed by sequencing again. The human and corrected mouse sequences are deposited in GenBank™ as U54562 and U54563, respectively. PCR reactions were conducted with 16 human libraries from a QUICK-screen cDNA library panel (CLONTECH) and Taq DNA polymerase (Stratagene). For the first amplification, the primers used were oligo 3 (5′-CCCGAATTCATGACTCCAGAAGAAGCTGA-3′, corresponding to nucleotides 1090–1109 in U54563, tagged with EcoRI) and oligo 2 (described above). For the second amplification, oligo 3 and oligo 7 (5′-GACCCTAAAAGGAAACACAGGGAAATAA-3′, corresponding to nucleotides 1168–1174, with the antisense sequence of the insertion underlined) were used with 0.2% of the first amplification reaction mixture as template. The conditions were 35 cycles (30 s at 94 °C, 30 s at 53 °C, and 1 min at 72 °C) for the first amplification and 20 cycles (30 s at 94 °C, 30 s at 68 °C, and 1 min at 72 °C) for the second. One-tenth of each reaction was subjected to electrophoresis on a 3% NuSieve GTG agarose gel containing ethidium bromide, which was photographed under UV light. In vitro translation of eIF3-p48 mRNA employed the TnT translation kit (Promega) with CsCl-purified covalently closed circular pTZp48 as template. Immunoprecipitation was conducted essentially as described (4Asano K. Kinzy T.G. Merrick W.C. Hershey J.W.B. J. Biol. Chem. 1997; 272: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The immune complexes were isolated with GammaBind G Sepharose beads (Pharmacia Biotech Inc.), washed, and eluted with SDS. Only a small fraction (<5%) of thede novo synthesized p48 was precipitated with anti-eIF3 (or with anti-p170 antiserum), because p48 alone is not recognized by these antibodies. eIF3 purified from both rabbit reticulocytes and human HeLa cells was fractionated by SDS-PAGE (see Fig. 3 a), and the p48 band was excised. After proteolysis and high pressure liquid chromatography fractionation as described under “Experimental Procedures,” the following peptide sequences were obtained: LGHVVMGNNAVSPYXQX(VIEK) from the rabbit protein, and VIQQESSYTYK and NQN(S)R(P/I)(EAPN) from the human factor (uncertain residues are enclosed in parentheses). The rabbit peptide sequence was used to search the nonredundant protein and expressed sequence tag (EST) data bases, and a perfect match of the first 14 amino acid residues was found to the mouse int-6 gene product (10Marchetti A. Buttitta F. Gallahan M.S.D. Smith G.H. Callahan R. J. Virol. 1995; 69: 1932-1938Crossref PubMed Google Scholar). This surprising result suggests that eIF3-p48 corresponds to the int-6 gene product and therefore may be involved in oncogenesis, as discussed in greater detail below. The rabbit peptide sequence also was found in the derived amino acid sequences of 5 human ESTs homologous to murineint-6. Furthermore, the two human peptide sequences match portions of mouse Int-6 and the human homolog. The entireint-6 DNA sequence was used to search the EST data base, and 50 related ESTs were identified. Three overlapping clones (142907, 47098, and 71140; shown in Fig. 1) of human cDNA were selected and kindly provided by the Lawrence Livermore National Laboratory (Livermore, CA). These were sequenced on both strands and then edited to generate a 1507-bp sequence (accession number U54562) that encodes the human Int-6 homolog, eIF3-p48. One of the human ESTs encoding eIF3-p48 (Int-6), N02633, has been mapped to chromosome 6 with an approximate cytological range of 6q15–21 (from the Whitehead Institute for Biological Research).Figure 1Structures of eIF3-p48 cDNAs. Lines and empty boxes indicate the untranslated and the p48 coding regions, respectively. The source of cDNA is given on the right, and EST clone names are given on theleft. Filled rectangles in the p48 coding region denote parts that match the three partial peptide sequences. Thedashed line in EST clone 142907 represents noncoding DNA possibly derived from fusion with foreign DNA or from an unspliced intron. The open triangle in EST clone 47098 indicates the 21-bp insertion, which causes premature termination of the open reading frame. For comparison, the murine int-6 cDNA is shown below, with the site of the missing C indicated by a solid triangle and the resulting N-terminal extension shown by thedashed box. The human int-6 cDNA sequence is identical to the one already reported (11Desbois C. Rousset R. Bantignies F. Jalinot P. Science. 1996; 273: 951-953Crossref PubMed Scopus (130) Google Scholar) as U62962, except that the latter lacks 28 bp at the 5′ terminus that includes the correct initiation codon.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our identification of the initiator codon in the eIF3-p48 cDNA differs from that presumed in the reported mouse Int-6 cDNA (10Marchetti A. Buttitta F. Gallahan M.S.D. Smith G.H. Callahan R. J. Virol. 1995; 69: 1932-1938Crossref PubMed Google Scholar). The open reading frame actually begins at an in-frame upstream AUG that results in a protein with 49 additional N-terminal amino acid residues. The reported murine int-6 sequence (10Marchetti A. Buttitta F. Gallahan M.S.D. Smith G.H. Callahan R. J. Virol. 1995; 69: 1932-1938Crossref PubMed Google Scholar) lacks a C residue following nucleotide 85, which when corrected places in-frame the AUG at nucleotides 62–64 (numbered as in accession number L35556). This C residue is present in our cDNA (U54562) and in the humanint-6 cDNA sequence (U62962) reported previously (11Desbois C. Rousset R. Bantignies F. Jalinot P. Science. 1996; 273: 951-953Crossref PubMed Scopus (130) Google Scholar); it is found as well in a mouse cDNA obtained by reverse transcription-PCR amplification of mRNA isolated from mouse FM3A cells as described under “Experimental Procedures” (results not shown). That the upstream AUG is the correct initiation site is suggested by the size of the protein product expressed in the reticulocyte lysate (see Fig. 3 b). The derived amino acid sequence of human eIF3-p48 is identical to the corrected mouse Int-6 sequence; both proteins contain 445 amino acid residues with a calculated mass of 52,187 Da. Northern blot analysis of HeLa poly(A)+ mRNAs gave a single 1.6-kb band when probed with DNA from the coding region of p48 (results not shown). Therefore, the 1507-bp DNA reported here as U54562 represents nearly full-length cDNA. eIF3-p48 (Int-6) sequences are found with a frequency of about 0.02% in the human EST data base, with essentially all tissues represented, suggesting that p48 is a ubiquitous, moderately abundant protein, as expected for a subunit of eIF3. Although homologs in many animal and plant species were identified in the data base (results not shown), no homolog of eIF3-p48 was found in the complete Saccharomyces cerevisiae genome data base. A second, more minor difference between the human and mouse cDNAs is the presence in one of the human int-6 clones (47098) of a 21-nucleotide insertion following nucleotide 1170, which lies between the Leu388 and Gly389 codons (Fig. 1). The insertion occurs at the junction of exons 11 and 12, conforms to the 3′ intron consensus sequence (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.7-16.8Google Scholar), and therefore appears to be due to alternative splicing. The insertion encodes FPCVSF-stop; thus the mRNA is predicted to generate a C-terminal truncation. Given the possible oncogenic properties of truncated eIF3-p48 (Int-6) (see below), the frequency of occurrence of this insertion was determined. The 21-bp insertion was found in only 1 of 26 ESTs encoding that region of human eIF3-p48 and therefore may be a unique or very rare event. To investigate the occurrence of the alternative splice/insertion event in human cells, two independent cDNA libraries from 8 different tissues were analyzed by PCR as described under “Experimental Procedures.” Whereas no larger PCR product indicative of the 21-bp insertion was detected in any of the primary amplification reactions (Fig. 2, upper panel), when a nested PCR analysis was performed by using a primer corresponding to the insert sequence, 7 of the 16 libraries representing 5 different tissues generated a band indicative of the insert (Fig. 2, lower panel). Thus the putative alternative splice/insertion event occurs widely but at such a low frequency that the resulting truncated protein likely would not affect cell physiology. To demonstrate that the cloned human int-6 DNA sequence actually encodes the p48 subunit of eIF3, a full-length cDNA was generated by PCR amplification of a human liver cDNA library (Stratagene), and the product was cloned under control of the T7 promoter to generate pTZp48, as described under “Experimental Procedures.” The pTZp48 insert was sequenced to confirm the sequence reported here (accession numberU54562), and eIF3-p48 was expressed in an in vitro coupled transcription/translation system (Promega). The largest and most abundant of the three 35S-labeled products comigrates precisely with the p48 subunit of purified eIF3 when analyzed by SDS-PAGE (Fig. 3 b). The minor, lower molecular weight products presumably are partially degraded forms of p48. Further evidence that pTZp48 encodes a subunit of eIF3 was obtained by immunoprecipitation of the eIF3 complex present in thein vitro translation reaction. Labeled p48 and its degraded forms are detected in precipitates obtained with crude anti-eIF3 antiserum (Fig. 3 c), even though eIF3-p48 is not directly recognized by this antiserum (13Meyer L.J. Milburn S.C. Hershey J.W.B. Biochemistry. 1982; 21: 4206-4212Crossref PubMed Scopus (36) Google Scholar, 14Milburn S.C. Duncan R.F. Hershey J.W.B. Arch. Biochem. Biophys. 1990; 276: 6-11Crossref PubMed Scopus (14) Google Scholar). Immunoprecipitates formed with affinity-purified antibodies specific for the p170 subunit of eIF3 also contain the recombinant eIF3-p48, but that with preimmune serum does not (Fig. 3 c). Apparently, newly synthesized p48 exchanges inefficiently into endogenous eIF3 complexes, leading to its coprecipitation. Evidence that the cDNA described here encodes eIF3-p48 includes matches to three peptide sequences derived from a 48-kDa protein in purified eIF3, co-migration of the recombinant protein with the 48-kDa subunit, and co-immunoprecipitation with anti-eIF3 and anti-p170 antibodies. eIF3-p48 itself appears not to be very antigenic, because the eIF3 antiserum does not recognize the p48 subunit. This lack of antigenicity and the comigration of the p48 and p47 subunits of eIF3 during routine SDS-PAGE resulted in a failure in earlier to the two which were called (13Meyer L.J. Milburn S.C. Hershey J.W.B. Biochemistry. 1982; 21: 4206-4212Crossref PubMed Scopus (36) Google S.C. Duncan R.F. Hershey J.W.B. Arch. Biochem. Biophys. 1990; 276: 6-11Crossref PubMed Scopus (14) Google Scholar). the p48 and p47 subunits are different as shown by the cloning of the p48 cDNA described as well as the cloning and sequencing of a cDNA encoding H.-P. N. J. C. A. and J. B. for The is to that of the p116 and p110 which also were not during SDS-PAGE and were identified earlier as a single band S.C. Duncan R.F. Hershey J.W.B. Arch. Biochem. Biophys. 1990; 276: 6-11Crossref PubMed Scopus (14) Google Scholar). Thus the cloning and sequencing of cDNAs for eIF3 subunits has to a of our of the of this complex factor. That eIF3-p48 is by the int-6 gene is and into mouse at a number of different of the of in mammary 1982; 1: Scholar). One of these called was identified in a mammary cell line obtained from a mouse as well as in two independent mammary (10Marchetti A. Buttitta F. Gallahan M.S.D. Smith G.H. Callahan R. J. Virol. 1995; 69: 1932-1938Crossref PubMed Google Scholar). exons that encode a protein to be and of the genome may result in the of a truncated protein that either is or as a (10Marchetti A. Buttitta F. Gallahan M.S.D. Smith G.H. Callahan R. J. Virol. 1995; 69: 1932-1938Crossref PubMed Google Scholar). Thus truncated eIF3-p48 (Int-6) may be a a truncated Int-6 protein has not been in the tumor cells, and possible of are such as of to Int-6 also has been identified as binding to the human T-cell leukemia virus type I Tax protein (11Desbois C. Rousset R. Bantignies F. Jalinot P. Science. 1996; 273: 951-953Crossref PubMed Scopus (130) Google Scholar). It is that binding of Tax Int-6 from the to the truncated eIF3-p48 is generated and as an how it be involved in A number of of the have been in regulation of cell proliferation (2Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 245-269Google Scholar). of the cDNA encoding causes malignant transformation of and cells A. Sonenberg N. 1990; PubMed Scopus Google Scholar) and in HeLa cells A. S. A. 1990; PubMed Scopus Google Scholar). The of the of the that are for the of this initiation resulting in of protein synthesis (2Sonenberg N. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 245-269Google Scholar). of a of the also causes malignant transformation S. Sonenberg N. Science. PubMed Scopus Google Scholar), presumably by the of protein synthesis phosphorylation of This is by the that of the of which be by also cell R. Hershey J.W.B. Sonenberg N. J. 1995; PubMed Scopus Google Scholar). In each of these loss of proliferation control appears to the cell's failure to It is therefore how truncated forms of eIF3-p48 to of cell Large C-terminal to loss of which in the of an eIF3 subunit would be expected to result in an inhibition of protein it is possible that p48 is not part of the of mammalian eIF3, as no homolog is found in eIF3. we that it function as a subunit involved in eIF3 Therefore, of eIF3-p48 result in loss of of eIF3 and thus of protein Tax in the of human T-cell leukemia virus type cells (11Desbois C. Rousset R. Bantignies F. Jalinot P. Science. 1996; 273: 951-953Crossref PubMed Scopus (130) Google Scholar) to eIF3-p48 (Int-6) and it from eIF3, resulting in of protein A second is that eIF3-p48 plays two in cells, one as a subunit of eIF3 and as a of cell with the leukemia protein as previously (11Desbois C. Rousset R. Bantignies F. Jalinot P. Science. 1996; 273: 951-953Crossref PubMed Scopus (130) Google Scholar). this is the the of a cell be by the of the protein, either or cell The of eIF3-p48 (Int-6) is surprising (11Desbois C. Rousset R. Bantignies F. Jalinot P. Science. 1996; 273: 951-953Crossref PubMed Scopus (130) Google Scholar). of an initiation factor has a in antibodies the F. C. A. S. R. Sonenberg N. S. A. PubMed Scopus Google Scholar). are in to investigate the function of the truncated or p48 subunit of eIF3 in protein synthesis and cell We for purified FM3A for in peptide sequencing and and for reading of the