ABSTRACT

In kinetoplastid protists, all mitochondrial tRNAs are encoded in the nucleus and imported from the cytoplasm to maintain organellar translation. This also applies to the tryptophanyl tRNA (tRNA^Trp) encoded by a single-copy nuclear gene, with a CCA anticodon to read UGG codon used in the cytosolic translation. Yet, in the mitochondrion it is unable to decode the UGA codon specifying tryptophan. Following mitochondrial import of tRNA^Trp, this problem is solved at the RNA level by a single C34 to U34 editing event that creates the UCA anticodon, recognizing UGA. To identify the enzyme responsible for this critical editing activity, we scrutinized the genome of Trypanosoma brucei for putative cytidine deaminases as the most likely candidates. Using RNAi silencing and poisoned primer extension, we have identified a novel deaminase enzyme, named here TbmCDAT for mitochondrial Cytidine Deaminase Acting on tRNA, which is responsible for this organelle-specific activity in T. brucei. The ablation of TbmCDAT led to the downregulation of mitochondrial protein synthesis, supporting its role in decoding the UGA tryptophan codon.

摘要

在动质体原生生物中，所有线粒体 tRNA 都在细胞核中编码并从细胞质输入以维持细胞器翻译。这也适用于由单拷贝核基因编码的色氨酸 tRNA (tRNA ^{Trp )，具有 CCA 反密码子以读取用于胞质翻译的 UGG 密码子。}然而，在线粒体中，它无法解码指定色氨酸的 UGA 密码子。在线粒体输入 tRNA ^Trp后，这个问题在 RNA 水平上通过一个 C34 到 U34 编辑事件得到解决，该事件创建 UCA 反密码子，识别 UGA。为了确定负责这一关键编辑活动的酶，我们仔细检查了布氏锥虫的基因组推定的胞苷脱氨酶是最有可能的候选者。使用 RNAi 沉默和中毒引物延伸，我们鉴定了一种新的脱氨酶，在此命名为 TbmCDAT，用于作用于 tRNA 的线粒体胞苷脱氨酶，它负责T. brucei中的这种细胞器特异性活性。TbmCDAT 的消融导致线粒体蛋白质合成的下调，支持其在解码 UGA 色氨酸密码子中的作用。