Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs

• Published in: Nucleic acids research Vol. 39; no. 14; pp. 6148 - 6160
• Main Authors: Daugeron, Marie-Claire, Lenstra, Tineke L., Frizzarin, Martina, El Yacoubi, Basma, Liu, Xipeng, Baudin-Baillieu, Agnès, Lijnzaad, Philip, Decourty, Laurence, Saveanu, Cosmin, Jacquier, Alain, Holstege, Frank C. P., de Crécy-Lagard, Valérie, van Tilbeurgh, Herman, Libri, Domenico
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.2011
• Subjects: Adenosine - analogs & derivatives; Adenosine - metabolism; Basic-Leucine Zipper Transcription Factors - biosynthesis; Basic-Leucine Zipper Transcription Factors - genetics; Codon, Initiator; Eukaryotic Initiation Factor-5 - genetics; Evolution, Molecular; Gene Expression Profiling; Gene Expression Regulation, Fungal; Life Sciences; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Mutation; Phylogeny; Protein Biosynthesis; RNA; RNA, Transfer - chemistry; RNA, Transfer - metabolism; Saccharomyces cerevisiae Proteins - biosynthesis; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - physiology; Transcription Factors - genetics; Adenosine; Protein Biosynthesis; Fungal; RNA; Gene Expression Regulation; Metalloendopeptidases; Gene Expression Profiling; Phylogeny; Molecular; Eukaryotic Initiation Factor-5; Codon; Initiator; Saccharomyces cerevisiae Proteins; Transfer; Evolution; Transcription Factors; Mutation; Basic-Leucine Zipper Transcription Factors
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkr178

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.