Deciphering a secondary genetic code in neurons: the role of codon bias in regulating neuronal protein levels and implications for Elongator-mediated neurological disease

Familial dysautonomia (FD) results from mutation in IKBKAP/ELP1, a gene encoding the scaffolding protein for the Elongator complex. This highly conserved complex is required for the translation of codon-biased genes in lower organisms. Here we investigate whether Elongator serves a similar function in mammalian peripheral neurons, the population devastated in FD. Using codon-biased eGFP sensors, and multiplexing of codon usage with transcriptome and proteome analyses of over 6,000 genes, we identify two categories of genes, as well as specific gene identities that depend on Elongator for normal expression. Moreover, we show that multiple genes in the DNA damage repair pathway are codon-biased, and that with Elongator loss, their misregulation is correlated with elevated levels of DNA damage. These findings link Elongator's function in the translation of codon-biased genes with both the developmental and neurodegenerative phenotypes of FD, and also clarify the increased risk of cancer associated with the disease. Examining the major genetic pathways that require IKAP are hampered by that Ikbkap null mice are embryonic lethal (E10), but can be examined by genome-wide transcriptome analysis of the dorsal root ganglia from Wnt1-Cre;IkbkapLoxP/LoxP E17.5 fetuses (Ikbkap is deleted from the neural crest). Mice homozygous for a floxed allele of Ikbkap (exon 4) were crossed to mice that are heterozygous for this floxed allele and a Wnt1-Cre transgene. Dorsal root ganglia were removed from E17-18 Wnt1-Cre;IkbkapLoxP/LoxP embryos and stored at -80 until RNA was isolated. Five wildtype and three conditional knock-out animals were analyzed for this experiment.家族性自主神经功能障碍（Familial dysautonomia, FD）由IKBKAP/ELP1基因突变引发，该基因编码延伸体复合物（Elongator complex）的支架蛋白。延伸体复合物是高度保守的蛋白复合体，在低等生物中是密码子偏好性基因翻译过程的必需因子。本研究旨在探究延伸体在哺乳动物外周神经元中是否发挥类似功能——而外周神经元正是FD病程中发生严重毁损的细胞群。研究人员借助密码子偏好性增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）传感器，结合6000余个基因的密码子使用偏好性分析、转录组与蛋白质组多重检测策略，鉴定出两类依赖延伸体实现正常表达的基因类别，以及对应的特异性基因靶点。此外，研究发现DNA损伤修复通路中的多个基因均存在密码子使用偏好性；当延伸体功能缺失时，这些基因的表达失调与DNA损伤水平升高显著相关。本研究结果将延伸体在密码子偏好性基因翻译中的功能，与FD的发育及神经退行性表型建立了直接关联，同时也阐明了该疾病伴随癌症风险升高的分子机制。由于Ikbkap基因全敲除小鼠会在胚胎发育第10天（E10）出现胚胎致死，极大阻碍了对依赖IKAP的核心遗传通路的研究，但可通过对Wnt1-Cre;IkbkapLoxP/LoxP E17.5胎鼠的背根神经节（dorsal root ganglia）进行全基因组转录组分析开展相关研究，该模型中神经嵴来源的细胞内Ikbkap基因被特异性敲除。实验中，将携带Ikbkap第4外显子LoxP位点的纯合小鼠，与该LoxP位点杂合且携带Wnt1-Cre转基因的小鼠进行杂交。收集E17~18周龄的Wnt1-Cre;IkbkapLoxP/LoxP胚胎的背根神经节，于-80℃低温保存直至RNA提取完成。本实验共分析了5只野生型小鼠与3只条件性基因敲除小鼠的样本。