NLS-dependent and insufficient nuclear localization of XAGE-1 splice variants.

XAGE-1 is a member of the cancer/testis antigen family and it was first identified by searching for the PAGE/ GAGE-related genes. Four transcript variants XAGE-1a, -1b, -1c and XAGE-1d have been discovered and have a broad expression in cancer. As a prominent transcript, XAGE-1b is encoded by 81 amino acids with a molecular weight of 9 kDa. We determined the cellular localization of all four splice variants by confocal microscopy analysis. Among these, XAGE-1a, -1b and -1c showed distinct speckled nuclear localization, while XAGE-1d was distributed evenly both in the cytoplasm and nucleus. By deletion mutagenesis and site directed mutagenesis, we identified the bipartite nuclear localization signal and found that it contributes to the nuclear localization of XAGE-1 variants; but the nuclear localization signal (NLS) only cannot form the characterized distribution of XAGE-1b; amino acids 25-42 also play a role in the formation of nuclear speckles. XAGE-1b, the main transcript of XAGE-1, may act as a partner protein or a member of a protein complex, which plays a role in tumor development and progression.