Two wobble-splicing events affect ING4 protein subnuclear localization and degradation.
ING4 (inhibitor of growth 4) is a candidate tumor suppressor gene that is implicated as a repressor of cell growth, angiogenesis, cell spreading and cell migration and can suppress loss of contact inhibition in vitro. Another group and we identified four wobble-splicing isoforms of ING4 generated by alternative splicing at two tandem splice sites, GC(N)(7)GT and NAGNAG, which caused canonical (GT-AG) and non-canonical (GC-AG) splice site wobbling selection. Expression of the four ING4 wobble-splicing isoforms did not vary significantly in any of the cell lines examined. Here we show that ING4_v1 is translocated to the nucleolus, indicating that ING4 contains an intrinsic nucleolar localization signal. We further demonstrate that the subcellular localization of ING4 is modulated by two wobble-splicing events at the exon 4-5 boundary, causing displacement from the nucleolus to the nucleus. We also observed that ING4 is degraded through the ubiquitin-proteasome pathway and that it is subjected to N-terminal ubiquitination. We demonstrate that nucleolar accumulation of ING4 prolongs its half-life, but lack of nucleolar targeting potentially increases ING4 degradation. Taken together, our data suggest that the two wobble-splicing events at the exon 4-5 boundary influence subnuclear localization and degradation of ING4.
Tsai
Kuo-Wang
KW
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, ROC.
Tseng
Hsiao-Chun
HC
Lin
Wen-Chang
WC
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D003513:Cycloheximide / Q000378:metabolism; D005091:Exons; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D021381:Protein Transport; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D018797:Cell Cycle Proteins; D018398:Homeodomain Proteins; C483230:ING4 protein, human; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D003513:Cycloheximide; D046988:Proteasome Endopeptidase Complex