title: Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1.
abstract: Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish, but little is known about the relationship between its gene structure and nuclear localization of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. auratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poly I:C, or CAB IFN-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NLS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids ("95 KDKSINK 101" and "75 KTWKANFR 82"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K75, K78, R82, K95, and K101) and one non-conserved basic amino acid (K97) are present in this NLS from CaIRF-1. This observation suggests that K97 of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic amino acids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1.
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shi
authors.forename: Yi-Bing
authors.initials: YB
authors.lastname: Zhang
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Jiang
authors.forename: Qi-Ya
authors.initials: QY
authors.lastname: Zhang
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: Gui
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D006054:Goldfish / Q000235:genetics / Q000378:metabolism*; D050856:Interferon Regulatory Factor-1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010802:Phylogeny; D016415:Sequence Alignment
chemical_list: D050856:Interferon Regulatory Factor-1; D019913:Nuclear Localization Signals