Characterization of hCINAP, a Novel Coilin-interacting Protein Encoded by a Transcript from the Transcription Factor TAFIID32 Locus

Niovi Santama,Stephen C Ogg,Anna Malekkou,Spyros E Zographos,Karsten Weis,Angus I Lamond

doi:10.1074/jbc.m501982200

Abstract

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.

Highlights

Cajal bodies are subnuclear domains that contain newly imported snRNP3 and snoRNP complexes, as well as certain transcription factors
In the case of the survival of motor neurons (SMN) protein, its binding to coilin is dependent on the presence in coilin of symmetrical dimethyl arginine residues, a modification shared with the Sm family of snRNP proteins, which localize in Cajal bodies [11]
In the case of NOPP140, it has been shown that deletion mutants removing the amino-terminal portion of NOPP140, which no longer bind to coilin, have a dominant negative effect on Cajal body formation when exogenously expressed in mammalian cells [9]

Summary

Introduction

Cajal bodies are subnuclear domains that contain newly imported snRNP3 and snoRNP complexes, as well as certain transcription factors. In the case of NOPP140, it has been shown that deletion mutants removing the amino-terminal portion of NOPP140, which no longer bind to coilin, have a dominant negative effect on Cajal body formation when exogenously expressed in mammalian cells [9]. These data support the important role of coilin in Cajal body formation and further indicate that the mechanisms involved likely require the interaction of coilin with other partner proteins.

Methods

Results

Conclusion