Characterization of hCINAP, a novel coilin-interacting protein encoded by a transcript from the transcription factor TAFIID32 locus.
Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.
Santama
Niovi
N
Department of Biological Sciences, University of Cyprus and Cyprus Institute of Neurology and Genetics, P.O. Box 20537, 1678 Nicosia, Cyprus. santama@ucy.ac.cy
Ogg
Stephen C
SC
Malekkou
Anna
A
Zographos
Spyros E
SE
Weis
Karsten
K
Lamond
Angus I
AI
D000255:Adenosine Triphosphate / Q000737:chemistry; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D020541:Coiled Bodies / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D035381:TATA-Binding Protein Associated Factors / Q000737:chemistry / Q000502:physiology*; D035362:Transcription Factor TFIID / Q000737:chemistry / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C106849:TAF9 protein, human; D035381:TATA-Binding Protein Associated Factors; C432963:TSPYL2 protein, human; D035362:Transcription Factor TFIID; C054562:yellow fluorescent protein, Bacteria; C068921:p80-coilin; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D005982:Glutathione Transferase