title: Dyskerin localizes to the nucleolus and its mislocalization is unlikely to play a role in the pathogenesis of dyskeratosis congenita.
abstract: Mutations in the DKC1 gene are responsible for causing the bone marrow failure syndrome, dyskeratosis congenita (DKC; OMIM 305000). The majority of mutations identified to date are missense mutations and are clustered in exons 3, 4 and 11. It is predicted that the corresponding protein dyskerin is a nucleolar phosphoprotein which functions in both pseudo-uridylation and cleavage of precursor rRNA. Dyskerin contains multiple putative nuclear localization signals (NLSs) at the N-terminus (KKHKKKKERKS) and C-terminus [KRKR(X)(17)KKEKKKSKKDKKAK(X)(17)-KKKKKKKKAKEVELVSE]. By fusing dyskerin with the enhanced green fluorescent protein (EGFP) and by following a time course of expression in mammalian cell lines, we showed that full-length dyskerin initially localizes to the nucleoplasm and subsequently accumulates in the nucleoli. A co-localization to the coiled bodies was observed in some cells where dyskerin-EGFP had translocated to the nucleoli. Analysis of a series of mutant constructs indicated that whereas the most C-terminal lysine-rich clusters [KKEKKKS-KKDKKAK(X)(17)KKKKKKKKAKEVELVSE] influence the rate of nucleoplasmic and nucleolar accumulation, the KRKR sequence is primarily responsible for the nuclear import. Nucleolar localization was maintained when either the N- or C-terminal motifs were mutated, but not when all NLSs were removed. We conclude that the intranuclear localization of dyskerin is accomplished by the synergistic effect of a number of NLSs and that the nucleolar localization signals are contained within the NLSs. Further, examination of dyskerin-EGFP fusions mimicking mutations detected in patients indicated that the intracellular mislocalization of dyskerin is unlikely to cause DKC.
authors.affiliation: Deutsches Krebsforschungszentrum (DKFZ), Department of Molecular Genome Analysis, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
authors.forename: N S
authors.initials: NS
authors.lastname: Heiss
authors.forename: A
authors.initials: A
authors.lastname: Girod
authors.forename: R
authors.initials: R
authors.lastname: Salowsky
authors.forename: S
authors.initials: S
authors.lastname: Wiemann
authors.forename: R
authors.initials: R
authors.lastname: Pepperkok
authors.forename: A
authors.initials: A
authors.lastname: Poustka
mesh_terms: D016475:3T3 Cells; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002466:Cell Nucleolus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019871:Dyskeratosis Congenita / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014709:Vero Cells
chemical_list: D018797:Cell Cycle Proteins; C112148:DKC1 protein, human; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins