tRNAs universally carry a CCA nucleotide triplet at their 3'-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

中文翻译：

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布氏螺旋体的单一添加CCA的酶在胞质溶胶和线粒体中具有独特的功能。

tRNA普遍在其3'末端带有CCA核苷酸三联体。在真核生物中，CCA通过CCA添加酶（CAE）在转录后添加。寄生虫原生动物Trypanosoma brucei的线粒体缺乏tRNA基因，因此从细胞质中导入了所有tRNA。这引起了人们对tRNA修饰及其在该生物中的分布的兴趣，包括如何将CCA添加到tRNA中。在这里，使用BLAST搜索编码布鲁氏杆菌中假定的CAE蛋白的基因，我们确定了一个单一的ORF Tb927.9.8780作为潜在的候选对象。击倒这种假定的蛋白，称为TbCAE，导致截短的tRNA积累，废除翻译，并抑制总的和线粒体CCA添加活性，这表明TbCAE既位于细胞质中又位于线粒体中。然而，线粒体定位的tRNA受TbCAE消融的影响要比其他tRNA小得多。互补分析显示，TbCAE的N末端10个氨基酸因其活性和线粒体定位而不可或缺，另外10个氨基酸的缺失消除了两者。TbCAE敲低引起的生长停滞通过酵母CAE的胞质亚型的表达得以挽救，即使它没有导入线粒体也是如此。这一发现表明，酵母酶通过向初级tRNA转录物中添加CCA来补充TbCAE的基本功能。值得注意的是，可能具有修复功能的线粒体TbCAE活性的消融仅对生长有轻微的影响。互补分析显示，TbCAE的N末端10个氨基酸因其活性和线粒体定位而不可或缺，另外10个氨基酸的缺失消除了两者。TbCAE敲低引起的生长停滞通过酵母CAE的胞质亚型的表达得以挽救，即使它没有导入线粒体也是如此。这一发现表明，酵母酶通过向初级tRNA转录物中添加CCA来补充TbCAE的基本功能。值得注意的是，可能具有修复功能的线粒体TbCAE活性的消融仅对生长有轻微的影响。互补分析显示，TbCAE的N末端10个氨基酸因其活性和线粒体定位而不可或缺，另外10个氨基酸的缺失消除了两者。TbCAE敲低引起的生长停滞通过酵母CAE的胞质亚型的表达得以挽救，即使它没有导入线粒体中也是如此。这一发现表明，酵母酶通过向初级tRNA转录物中添加CCA来补充TbCAE的基本功能。值得注意的是，可能具有修复功能的线粒体TbCAE活性的消融仅对生长有轻微的影响。TbCAE敲低引起的生长停滞通过酵母CAE的胞质亚型的表达得以挽救，即使它没有导入线粒体也是如此。这一发现表明，酵母酶通过向初级tRNA转录物中添加CCA来补充TbCAE的基本功能。值得注意的是，可能具有修复功能的线粒体TbCAE活性的消融仅对生长有轻微的影响。TbCAE敲低引起的生长停滞通过酵母CAE的胞质亚型的表达得以挽救，即使它没有导入线粒体也是如此。这一发现表明，酵母酶通过向初级tRNA转录物中添加CCA来补充TbCAE的基本功能。值得注意的是，可能具有修复功能的线粒体TbCAE活性的消融仅对生长有轻微的影响。