Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.
Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.
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DOI：
10.1093/nar/gkr178
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发表时间：
2011-08
影响因子：
14.9
通讯作者：
Libri D
中科院分区：
文献类型：
--
作者：
Daugeron MC;Lenstra TL;Frizzarin M;El Yacoubi B;Liu X;Baudin-Baillieu A;Lijnzaad P;Decourty L;Saveanu C;Jacquier A;Holstege FC;de Crécy-Lagard V;van Tilbeurgh H;Libri D
EKC/KEOPS复合物在真核生物和微生物中普遍保守，并参与了几个细胞过程，包括转录，端粒稳态和基因组不稳定性。然而，到目前为止，该复合物的分子功能仍然难以捉摸。我们分析了EKC/KEOPS突变体的转录组，并观察到高度富集Gcn 4p转录激活因子的靶点的特异性谱。发现GCN 4表达在突变体中通过其前导序列中存在的抑制性上游ORF（uORF）的缺陷识别在翻译水平上被激活。我们发现EKC/KEOPS突变体在解码ANN密码子的tRNA的位置37（t6 A37）处的N6-苏氨酰氨基甲酰基腺苷修饰是有缺陷的，这影响了抑制性uORF的起始并引起Gcn 4去阻遏。结构建模揭示了Kae 1和细菌酶之间的相似性，这些酶参与类似于t6 A37形成的氨甲酰化反应，支持EKC在tRNA修饰中的直接作用。EKC突变体与翻译起始因子和苏氨酸生物合成基因的强烈遗传相互作用进一步支持了这些发现。总的来说，我们的数据提供了一个新的转折，以了解EKC/KEOPS的主要功能及其对几个基本的细胞功能，如转录和端粒稳态的影响。
The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.
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