Translational fidelity relies critically on correct aminoacyl-tRNA supply. The trans-editing factor AlaX predominantly hydrolyzes Ser-tRNAAla, functioning as a third sieve of alanyl-tRNA synthetase (AlaRS). Despite extensive studies in bacteria and archaea, the mechanism of trans-editing in mammals remains largely unknown. Here, we show that human AlaX (hAlaX), which is exclusively distributed in the cytoplasm, is an active trans-editing factor with strict Ser-specificity. In vitro, both hAlaX and yeast AlaX (ScAlaX) were capable of hydrolyzing nearly all Ser-mischarged cytoplasmic and mitochondrial tRNAs; and robustly edited cognate Ser-charged cytoplasmic and mitochondrial tRNASers. In vivo or cell-based studies revealed that loss of ScAlaX or hAlaX readily induced Ala- and Thr-to-Ser misincorporation. Overexpression of hAlaX impeded the decoding efficiency of consecutive Ser codons, implying its regulatory role in Ser codon decoding. Remarkably, yeast cells with ScAlaX deletion responded differently to translation inhibitor treatment, with a gain in geneticin resistance, but sensitivity to cycloheximide, both of which were rescued by editing-capable ScAlaX, alanyl- or threonyl-tRNA synthetase. Altogether, our results demonstrated the previously undescribed editing peculiarities of eukaryotic AlaXs, which provide multiple checkpoints to maintain the speed and fidelity of genetic decoding.

中文翻译：

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真核 AlaX 为翻译中氨酰基-tRNA 的质量和数量提供多个检查点


翻译保真度主要依赖于正确的氨酰基-tRNA 供应。反式编辑因子 AlaX 主要水解 Ser-tRNAAla，充当丙氨酰-tRNA 合成酶 (AlaRS) 的第三个筛子。尽管对细菌和古细菌进行了广泛的研究，但哺乳动物中的反式编辑机制仍然很大程度上未知。在这里，我们证明人 AlaX (hAlaX) 专门分布在细胞质中，是一种具有严格 Ser 特异性的活性反式编辑因子。在体外，hAlaX 和酵母 AlaX (ScAlaX) 都能够水解几乎所有 Ser 错误充电的细胞质和线粒体 tRNA；以及经过严格编辑的同源 Ser 电荷细胞质和线粒体 tRNASers。体内或基于细胞的研究表明，ScAlaX 或 hAlaX 的缺失很容易诱导 Ala-和 Thr-to-Ser 错误掺入。 hAlaX 的过度表达阻碍了连续 Ser 密码子的解码效率，暗示其在 Ser 密码子解码中的调节作用。值得注意的是，具有 ScAlaX 缺失的酵母细胞对翻译抑制剂处理的反应不同，遗传霉素耐药性增加，但对放线菌酮敏感性增加，这两种情况都可以通过具有编辑功能的 ScAlaX、丙氨酰或苏氨酰 tRNA 合成酶来挽救。总而言之，我们的结果证明了真核 AlaX 之前未描述的编辑特性，它提供了多个检查点来维持遗传解码的速度和保真度。