Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations
Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations
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DOI：
10.1074/jbc.ra119.009369
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发表时间：
2019-07
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通讯作者：
Wenchao Zhao;Simon Bohleber;Henrik Schmidt;Sandra Seeher;M. Howard;Doreen Braun;S. Arndt;Uschi Reuter;H. Wende;C. Birchmeier;Noelia Fradejas-Villar;U. Schweizer
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作者：
Wenchao Zhao;Simon Bohleber;Henrik Schmidt;Sandra Seeher;M. Howard;Doreen Braun;S. Arndt;Uschi Reuter;H. Wende;C. Birchmeier;Noelia Fradejas-Villar;U. Schweizer
硒蛋白中UGA密码子作为硒代半胱氨酸（Sec）密码子的取代依赖于真核生物硒蛋白mRNA的3′-UTR中的硒代半胱氨酸插入序列（SECIS）。SECIS结合蛋白2（SECISBP 2）增加了这一过程的效率。SECISBP 2的致病性突变降低硒蛋白的表达，并导致与人类脱碘酶活性和硒蛋白N表达降低相关的表型。SECISBP 2具有两种功能：结合硒蛋白mRNA中的SECIS元件和促进共同翻译的Sec插入。为了分别探测这两种功能，我们在这里建立了两个小鼠模型，它们携带先前在患者中鉴定的Secisbp 2中的两个致病性错义突变。我们发现，C696 R取代的RNA结合结构域废除SECIS结合，并不支持硒蛋白的翻译水平以上的一个完整的Secisbp 2无效突变。位于硒代半胱氨酸插入结构域的R543 Q错义取代导致残留活性并导致硒蛋白翻译减少，如通过核糖体分析以确定对单个硒蛋白中UGA重编码的影响所示。然而，我们发现R543 Q变体在体外是热不稳定的，在体内小鼠肝脏中完全降解，而在大脑中部分发挥作用。神经元中硒蛋白表达的中度损伤导致星形胶质细胞增生和与免疫反应相关的基因的转录诱导。我们的结论是，差异SECISBP 2蛋白在个别细胞类型的稳定性可能会决定临床表型，在更大程度上比分子相互作用，涉及突变的氨基酸SECISBP 2。
Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3′-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2.