Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations

Re-coding 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 RNAbinding 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 re-coding 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 . Ribosome profiling and RNA-sequencing of cortex samples from 2 Secisbp2 R543Q/fl; Camk-Cre pos mice and 2 control mice.真核硒蛋白中，UGA密码子被重新编码为硒半胱氨酸（selenocysteine, Sec）密码子的过程，依赖于真核硒蛋白mRNA 3'非翻译区（3' untranslated region, 3' UTR）内的硒半胱氨酸插入序列（selenocysteine insertion sequence, SECIS）。硒半胱氨酸插入序列结合蛋白2（SECIS-binding protein 2, SECISBP2）可提升该过程的效率。SECISBP2的致病性突变会降低硒蛋白的表达水平，并引发与人类体内脱碘酶活性及硒蛋白N表达下调相关的表型。SECISBP2被证实具备两项功能：结合硒蛋白mRNA中的SECIS元件，以及协助共翻译过程中的Sec插入。为分别探究这两项功能，本研究构建了两款小鼠模型，其携带此前在患者体内发现的Secisbp2基因两处致病性错义突变。研究发现，RNA结合结构域内的C696R替换会废除SECIS结合能力，且无法使硒蛋白翻译水平高于Secisbp2完全敲除突变体的翻译水平。位于硒半胱氨酸插入结构域的R543Q错义突变仅残留部分活性，并导致硒蛋白翻译水平下调，该结论通过核糖体谱分析得以验证——该分析用于探究突变对单个硒蛋白中UGA密码子重新编码过程的影响。但本研究同时发现，R543Q突变体在体外呈现热不稳定性，且在小鼠肝脏体内会被完全降解，而在大脑中则保留部分功能。神经元内硒蛋白表达的轻度受损，会引发星形胶质细胞增生，并诱导与免疫应答相关的基因转录上调。本研究得出结论：相较于SECISBP2中突变氨基酸所参与的分子相互作用，不同细胞类型中SECISBP2蛋白稳定性的差异，对临床表型的决定作用更为显著。本研究对2只Secisbp2^R543Q/fl; Camk-Cre^+小鼠及2只对照小鼠的皮层样本开展了核糖体谱分析与RNA测序。