Dyskerin localizes to the nucleolus and its mislocalization is unlikely to play a role in the pathogenesis of dyskeratosis congenita
Dyskerin localizes to the nucleolus and its mislocalization is unlikely to play a role in the pathogenesis of dyskeratosis congenita
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DOI：
10.1093/hmg/8.13.2515
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发表时间：
1999-12-01
影响因子：
3.5
通讯作者：
Poustka, A
中科院分区：
文献类型：
--
作者：
Heiss, NS;Girod, A;Poustka, A
DKc1基因突变是导致骨髓衰竭综合征，先天性角化不良(DKC；OMIM 305000)的原因，迄今发现的突变大多是错义突变，聚集在外显子3，4和11上，预测相应的蛋白质Dyskerin是一种核仁磷酸蛋白，在前体rRNA假尿苷酸化和裂解中发挥作用，Dyskerin在N端(KKHKKKERKS)和C端[KRKR(X)(17)KKEKKKSKKDKKAK(X)(17)-KKKKKKKKAKEVELVSE].含有多个可能的核定位信号(NLSS)通过将dyskerin与增强型绿色荧光蛋白(EGFP)融合并在哺乳动物细胞系中表达一段时间，我们发现全长dyskerin最初定位于核质中，随后积聚在核仁中，在一些dyskerin-EGFP移位到核仁的细胞中观察到与卷曲小体的共定位，对一系列突变结构的分析表明，尽管最C端富含赖氨酸的簇[KKKKKKKKKKKAKEVELVSE]影响核浆和核仁积累的速度，但KRKR序列主要负责核输入。当N-末端或C-末端基序突变时，核仁定位保持不变，但当所有NLSS被移除时，核仁定位不变。我们的结论是，Dyskerin的核内定位是由多个NLSS协同作用完成的，核仁定位信号包含在NLSS中。此外，对患者中检测到的突变的Dyskerin-EGFP融合的检测表明，Dyskerin在细胞内的错误定位不太可能导致DKC。
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 pseudouridylation 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 [KKEKKKSKKDKKAK(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.