Santama Niovi, Ogg Stephen C, Malekkou Anna, Zographos Spyros E, Weis Karsten, Lamond Angus I
Department of Biological Sciences, University of Cyprus and Cyprus Institute of Neurology and Genetics, P.O. Box 20537, 1678 Nicosia, Cyprus.
J Biol Chem. 2005 Oct 28;280(43):36429-41. doi: 10.1074/jbc.M501982200. Epub 2005 Aug 2.
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.
卷曲螺旋蛋白是卡哈尔体的一种标记蛋白，卡哈尔体是一种核内亚结构域，作为核RNA-蛋白复合物组装和成熟的场所。利用酵母双杂交筛选来鉴定与卷曲螺旋蛋白相互作用的蛋白，我们鉴定出了hCINAP（人类卷曲螺旋蛋白相互作用核ATP酶蛋白），这是一种含有P环核苷酸结合基序和ATP酶活性的172个氨基酸的核因子。hCINAP蛋白序列在从人类到植物和酵母的全长范围内高度保守，并且在所测试的所有人类组织和细胞系中普遍表达。CINAP的酵母同源物是一个单拷贝的必需基因。在哺乳动物细胞中，带有标签的hCINAP存在于含有卷曲螺旋蛋白的复合物中，并且重组的、在大肠杆菌中表达的hCINAP在体外直接与卷曲螺旋蛋白结合。卷曲螺旋蛋白的214个羧基末端残基似乎对与hCINAP的相互作用至关重要。免疫荧光和荧光蛋白标记均表明，hCINAP特异性定位于细胞核，以广泛、弥散的核质模式分布，排除核仁，在卡哈尔体中也有一定程度的富集。在HeLa细胞中过表达hCINAP导致每个细胞核中卡哈尔体的平均数量减少，这与其影响卡哈尔体的稳定性和/或其组装速率一致。hCINAP mRNA是来自TAF9基因座的可变剪接转录本，该基因座编码基础转录因子亚基TAFIID32。然而，hCINAP和TAFIID32 mRNA从不同的ATG密码子翻译而来，并使用不同的阅读框，导致它们在各自的蛋白质序列中没有同一性。
