Molecular cloning of a zinc finger autoantigen transiently associated withinterphase nucleolus and mitotic centromeres and midbodies - Orthologous proteins with nine cxxc motifs highly conserved from nematodes to humans

We have cloned a novel human autoimmune antigen in a patient suffering from rheumatoid arthritis with high levels of antibodies to the nucleolus organ izer regions. Initially the human autoimmune serum was used to select a cDN A of 317 amino acids from a hamster expression library. Using the hamster D NA as a probe, we isolated the human homologous cDNA of 320 amino acids. Hu man and hamster polypeptides share a 95% amino acid homology, The deduced 3 6-kDa protein contains a putative amino-terminal NLS signal, nine cysteine- X-X-cysteine motifs highly conserved, and a carboxyl-terminal poly acidic r egion. Several homologous expressed sequence tags have been identified in d ata bases suggesting that orthologous proteins are present throughout evolu tion from worms to humans. A Drosophila expressed sequence tag was further completely sequenced for a full-length protein with 60% amino acid identity to the human homologue. Northern blot analysis revealed that this novel pr otein is widely distributed in human tissues with significantly higher expr ession levels in heart and skeletal muscle, Specific antibodies to the reco mbinant protein and transfection experiments demonstrated by immunofluoresc ence the localization of the protein predominantly but not exclusively to t he nucleolus of interphase mammalian cells, In actinomycin D-treated cells the protein remains associated with the nucleolus but is not segregated, li ke other ribosomal factors such as upstream binding factor. In mitosis the protein was found to be associated with centromeres and concentrated at the midbody in cytokinesis. Transient distribution of this evolutionarily cons erved zinc finger nucleolar autoantigen to the mitotic centromeres may prov ide the means for several aspects of cell cycle control and transcriptional regulation.