Identification of cDNAs encoding two novel nuclear proteins, IMUP-1 and IMUP-2, upregulated in SV40-immortalized human fibroblasts.
Using a model system of young, senescent and SV40-immortalized WI-38 fibroblasts, we identified two mRNAs upregulated in immortalized cells (imup-1, immortalization-upregulated protein 1, and imup-2). Compared to normal tissues, both genes were more frequently expressed in cancer cells. The open reading frame of imup-1 spans 321bp, coding for a 10.9 kDa protein of 106 amino acids, while an insertion of 59bp in the otherwise identical mRNA of imup-2 leads to a frameshift, resulting in an 8.5 kDa protein of 85 amino acids. Database searches identified these genes on chromosome 19, which could account for the cloned imup-1 and imup-2 transcripts by alternative splicing. Southern blot analysis of digested genomic DNA confirmed that both transcripts are derived from a single locus. The expressed proteins IMUP-1 and IMUP-2 share 46 identical N-terminal amino acids, whereas the C-termini are unrelated. Green fluorescent protein-fusions of both IMUP-1 and IMUP-2 accumulated in the nucleus of HeLa cells. The C-terminus of IMUP-1 contains a bipartite nuclear localization signal, the deletion of which impaired nuclear translocation. In-vitro translated proteins bound to poly(rG), but did not interact with single-stranded DNA or double-stranded DNA. The nuclear localization of IMUP-1 and IMUP-2 as well as the upregulation of both underlying mRNAs in immortalized cells suggest a function in immortalization.
Kim
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Kato Cytoprotein Network Project, ERATO, Japan Science and Technology Corporation (JST), c/o Sagami Chemical Research Center, Nishi-Ohnuma 4-4-1, Sagamihara, 229-0012, Kanagawa, Japan.
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Ishizuka
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Kato
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D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005796:Genes / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009696:Nucleic Acids / Q000378:metabolism; D011068:Poly G / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013539:Simian virus 40 / Q000235:genetics; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D009696:Nucleic Acids; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011068:Poly G