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.

Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

“…Therefore, to prevent aggregation and degradation of NAP57 and binding of random RNAs (Walbott et al 2011), it is likely that SHQ1 associates with NAP57 at the site of translation in the cytoplasm, or post-translationally. The dimer may then travel into the nucleus by virtue of the multiple nuclear localization signals of NAP57 (Meier and Blobel 1994;Heiss et al 1999;Youssoufian et al 1999). Only at the site of transcription of H/ACA RNAs is there a need for SHQ1 to be replaced by the specific RNAs.…”

SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs

“…Second, SHQ1, although nuclear at steady state, shuttles between nucleus and cytoplasm but lacks a classical nuclear localization signal. Therefore, it may depend on those of other proteins, such as the multiple nuclear localization signals of NAP57 (Meier and Blobel 1994;Heiss et al 1999;Youssoufian et al 1999). Third, SHQ1 has to act before cotranscriptional association of NAP57 with H/ACA RNA and other core proteins because SHQ1 only binds NAP57 alone and because, once assembled with the core trimer, H/ACA RNAs will no longer exchange (Wang and Meier 2004;Kittur et al 2006).…”

Loss of the Mouse Ortholog of the Shwachman-Diamond Syndrome Gene (Sbds) Results in Early Embryonic Lethality

“…Ablation of dyskerin in male mice leads to apparent abnormality as early as E7.5 with subsequent embryo resorption (11). While dyskerin does localize to the nucleolus as well as the nucleoplasm, consistent with its predicted role in rRNA processing, interpretation of phenotype must also consider dyskerin's apparent role in telomere function (12). Direct comparison to loss of Sbds is limited pending more information on the function or functions of the genes involved in these syndromes.…”