Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously we reported tissue-specific regulation of readthrough of the first kelch stop codon. Here, we characterize major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3′ of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3′ of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, we show that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.

翻译终止密码子通读发生在从病毒到哺乳动物的生物体中，并且在解码果蝇和病毒 mRNA 时尤为普遍。重新编码 UGA、UAG 或 UAA 以指定氨基酸允许由单个基因编码的蛋白质的一部分在 C 端延伸。果蝇 kelch mRNA的延伸产物为 160 kDa，而未延伸的 Kelch 蛋白（Cullin3-RING 泛素连接酶的一个亚基）为 76 kDa。以前我们报告了第一个kelch通读的组织特异性调节终止密码子。在这里，我们描述了各种细胞类型的主要效率差异。免疫印迹显示，在马氏小管、卵巢和睾丸中的通读水平较低，但在幼虫和成体中枢神经系统 (CNS) 组织的裂解物中有丰富的通读产物。通读的记者在成人大脑中证明了超过 30% 的通读，并且在幼虫和成人大脑中的成像显示通读发生在神经元中，而不是神经胶质细胞中。在转基因果蝇中评估了通读终止密码子两侧的通读刺激序列的程度以及发生低效通读的人体组织培养细胞中。有可能形成通读终止密码子的 mRNA 茎环 3' 的 99 个核苷酸序列刺激了通读效率。然而，即使只有 6 个核苷酸的kelch mRNA 序列 3' 终止密码子，成人神经元的通读效率也只下降到 6%。最后，我们发现果蝇中枢神经系统中的高效通读很常见。对于许多神经元蛋白质来说，单个蛋白质的 C 末端扩展形式可能相对丰富。