Mutations in EFL1, an SBDS partner, are associated with infantile pancytopenia, exocrine pancreatic insufficiency and skeletal anomalies in a Shwachman-Diamond like syndrome
Mutations in EFL1, an SBDS partner, are associated with infantile pancytopenia, exocrine pancreatic insufficiency and skeletal anomalies in a Shwachman-Diamond like syndrome
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DOI：
10.1136/jmedgenet-2016-104366
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发表时间：
2017-08-01
影响因子：
4
通讯作者：
Elpeleg, Orly
中科院分区：
文献类型：
--
作者：
Stepensky, Polina;Chacon-Flores, Montserrat;Elpeleg, Orly
背景 对于核糖体成熟的最后一步，新生的 40S 和 60S 亚基从细胞核输出到细胞质。为了防止这些核糖体亚基过早结合，真核起始因子 6 (eIF6) 与细胞核内的 60S 亚基结合。它在细胞质中的释放需要 EFL1 和 SDBS 蛋白的相互作用。在Shwachman-Diamond综合征（SDS）中，缺陷的SDBS蛋白阻止eIF6被驱逐，抑制其再循环到细胞核以及随后活性80S核糖体的形成。目的本研究旨在确定来自三个不相关家族的6名患者的SDS样疾病的分子基础，该疾病表现为全血细胞减少、胰腺外分泌功能不全和骨骼异常。方法采用全外显子组分析进行突变鉴定。荧光显微镜研究评估了 Tif6-GFP（酵母 eIF6 同源物）在酵母 WT 和突变细胞中的定位。人类和酵母 EFL1 蛋白（WT 和突变体）在酿酒酵母 BCY123 菌株中表达，并使用圆二色性和小角 X 射线散射来评估这些蛋白的折叠和灵活性。采用绿孔雀石比色法测定WT和Efl1突变体的GTPase活性。结果4名患者的EFL1中p.R1095Q变异纯合，2名患者p.M882K变异纯合。残基 R1095 和 M882 在物种间保守。突变蛋白的 GTP 酶活性以及 SDBD 蛋白或 60S 核糖体亚基对其的激活均不受影响。 efl1 Delta 酵母细胞与 EFL1 突变体的互补挽救了缓慢生长的表型。尽管如此，与WT细胞中的核定位相比，Tif6-GFP在突变酵母细胞中重新定位到细胞质。结论EFL1中的突变在临床上表现为SDS样表型。与 SDS 的分子病理学类似，突变的 EFL1 蛋白不会促进细胞质 Tif6 从 60S 亚基的释放，可能会阻止成熟核糖体的形成。
Background For the final step of the maturation of the ribosome, the nascent 40S and 60S subunits are exported from the nucleus to the cell cytoplasm. To prevent premature association of these ribosomal subunits, eukaryotic initiation factor 6 (eIF6) binds the 60S subunit within the nucleus. Its release in the cytoplasm requires the interaction of EFL1 and SDBS proteins. In Shwachman-Diamond syndrome (SDS), a defective SDBS protein prevents eIF6 eviction, inhibiting its recycle to the nucleus and subsequent formation of the active 80S ribosome.Objective This study aims to identify the molecular basis of an SDS-like disease, manifested by pancytopenia, exocrine pancreatic insufficiency and skeletal abnormalities in six patients from three unrelated families.Methods Whole exome analysis was used for mutation identification. Fluorescence microscopy studies assessed the localisation of Tif6-GFP, the yeast eIF6 homologue, in yeast WT and mutant cells. Human and yeast EFL1 proteins, WT and mutants, were expressed in Saccharomyces cerevisiae BCY123 strain, and circular dichroism and small-angle X-ray scattering were used to assess the folding and flexibility of these proteins. Green malachite colorimetric assay was performed to determine the GTPase activity of WT and Efl1 mutants.Results Four patients were homozygous for p.R1095Q variant and two patients were homozygous for p.M882K variant in EFL1. Residue R1095 and M882 are conserved across species. Neither the GTPase activity of the mutant proteins nor its activation by the SDBD protein or the 60S ribosomal subunit were affected. Complementation of efl1 Delta yeast cells with the EFL1 mutants rescued the slow growth phenotype. Nonetheless, Tif6-GFP was relocalised to the cytoplasm in mutant yeast cells in contrast to its nuclear localisation in WT cells.Conclusions Mutations in EFL1 clinically manifest as SDS-like phenotype. Similar to the molecular pathology of SDS, mutant EFL1 proteins do not promote the release of cytoplasmic Tif6 from the 60S subunit, likely preventing the formation of mature ribosomes.