Molecular Cloning of a Zinc Finger Autoantigen Transiently Associated with Interphase Nucleolus and Mitotic Centromeres and Midbodies

Abstract

We have cloned a novel human autoimmune antigen in a patient suffering from rheumatoid arthritis with high levels of antibodies to the nucleolus organizer regions. Initially the human autoimmune serum was used to select a cDNA of 317 amino acids from a hamster expression library. Using the hamster DNA as a probe, we isolated the human homologous cDNA of 320 amino acids. Human and hamster polypeptides share a 95% amino acid homology. The deduced 36-kDa protein contains a putative amino-terminal NLS signal, nine cysteine-X-X-cysteine motifs highly conserved, and a carboxyl-terminal poly acidic region. Several homologous expressed sequence tags have been identified in data bases suggesting that orthologous proteins are present throughout evolution 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 protein is widely distributed in human tissues with significantly higher expression levels in heart and skeletal muscle. Specific antibodies to the recombinant protein and transfection experiments demonstrated by immunofluorescence the localization of the protein predominantly but not exclusively to the nucleolus of interphase mammalian cells. In actinomycin D-treated cells the protein remains associated with the nucleolus but is not segregated, like 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 conserved zinc finger nucleolar autoantigen to the mitotic centromeres may provide the means for several aspects of cell cycle control and transcriptional regulation.

Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii

“…Eukaryotic proteins with these motifs are thought to be involved in RNA binding or single-stranded DNA binding (25,38). CCHC zinc finger genes can also have diverse roles in transcriptional modulation, translational control, protein degradation pathways, and growth regulation (6,(27)(28)(29)35). In T. gondii, transcriptional and translational control mechanisms play important roles in stage differentiation.…”

Genomic structure and chromosome location of the human gene encoding the zinc finger autoantigen ZNF330

“…These include among other examples: anti-centromere sera against kinetochore and centromere associated antigens (Moroi et al, 1980), nucleolus components (Ochs et al, 1985;Savino et al, 1999), the upstream binding factor (UBF) for polymerase I (Rodríguez-Sá nchez et al, 1987;Chan et al, 1991;Bolívar et al, 1996), and autoantibodies directed against FXR1, the autosomal gene related to the fragile X syndrome (Bolívar et al, 1998). Recently, we have used a human autoimmune anti-NOR serum to identify a novel nucleolar zinc finger protein (Bolívar et al, 1999). The new autoantigen, originally named NOA36 (from now on it will be noted as ZNF330 according to the HUGO Gene Nomenclature Committee) was described as being highly conserved from nematodes to humans as shown by comparison of the cloned hamster and human cDNA with sequences from several ESTs databases.…”

“…This new gene gives rise to a 1.8-kb mRNA whose open reading frame encodes a protein evolutionarily conserved with nine CXXC motifs plus other highly conserved cysteine and histidine residues compatible with a structural organization of a putative transcription factor of the zinc finger type (Miller et al, 1985;Klug and Rhodes, 1987). Previous immunolocalization of ZNF330 in culture cells, demonstrated a nucleolar and a cytoplasm association during interphase, and a transition to the centromeres and midbodies in mitosis (Bolívar et al,1999). With intent to further unravel functional aspects of ZNF330, we now describe that the autoantigen is released from nucleoli in RNAse treated culture cells, although surprisingly it remains associated with the cytoplasm after nuclease treatment.…”

“…This serum was produced against the rodent antigen although it reacts positively by IF and blots with the antigen from several cell lines tested, including human (Bolívar et al, 1999). For this study CHO culture cells were grown on coverslips, washed briefly in PBS and fixed in cold methanol:acetone for 7 min at -20°C.…”

“…The human ZNF330 cDNA sequence (Bolívar et al, 1999) was used to screen a human placenta genomic library cloned in the lambda Fix II vector (Stratagene, La Jolla, CA). The recombinant phage-containing filters were hybridized with the human probe, washed and subjected to autoradiography according to standard procedures (Sambrook et al, 1989).…”