Dyskerin localizes to the nucleolus and its mislocalization is unlikely toplay a role in the pathogenesis of dyskeratosis congenita

Mutations in the DKC1 gene are responsible for causing the bone marrow fail ure syndrome, dyskeratosis congenita (DKC; OMIM 305000), The majority of mu tations identified to date are missense mutations and are clustered in exon s 3, 4 and 11, It is predicted that the corresponding protein dyskerin is a nucleolar phosphoprotein which functions in both pseudouridylation and cle avage of precursor rRNA, Dyskerin contains multiple putative nuclear locali zation 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 o f expression in mammalian cell lines, we showed that full-length dyskerin i nitially localizes to the nucleoplasm and subsequently accumulates in the n ucleoli, 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 [KKEKKKSKKDKKAK(X)(17)KKKKKKKKAKEVELVSE] influence the rate of nu cleoplasmic and nucleolar accumulation, the KRKR sequence is primarily resp onsible 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 acc omplished by the synergistic effect of a number of NLSs and that the nucleo lar localization signals are contained within the NLSs, Further, examinatio n of dyskerin-EGFP fusions mimicking mutations detected in patients indicat ed that the intracellular mislocalization of dyskerin is unlikely to cause DKC.