Use of a Yeast tRNase Killer Toxin to Diagnose Kti12 Motifs Required for tRNA Modification by Elongator

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life

Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm5U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm5U to (S)-mchm5U in tRNAGlyUCC, and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm5U, and also of mcm5Um, its 2′-O-ribose methylated derivative. This suggests that accumulated mcm5U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm5U- and mcm5Um-containing forms of the selenocysteine-specific tRNASec in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines

Das Kluyveromyces lactis Killertoxin: Regulatoren des Zellimports und der intrazellulären Wirkung in Saccharomyces cerevisiae

Killerstämme der Milchhefe Kluyveromyces lactis sekretieren einen heterotrimeren (αßγ) Toxinkomplex (Zymocin), welcher das Wachstum sensitiver Hefen wie z.B. Saccharomyces cerevisiae inhibiert. Die Zytotoxizität des Zymocins resultiert aus der Endonucleaseaktivität der γ-Untereinheit. Diese spaltet im Zytosol sensitiver Hefezellen tRNAGlu, tRNALys und tRNAGln auf der 3‘-Seite Elongator-abhängig modifizierter Wobble-Uridine (U34) im Anticodonbereich. Im ersten Teil der Arbeit (Publikation 1, 2 und 3) konnten vier neue Binde- bzw Importfaktoren (PMA1, IPT1, UGP1 und ISR1) identifiziert werden, die vermutlich in Ereignisse nach der Zymocinbindung an den Zellwandrezeptor Chitin involviert sind. Im zweiten Teil (Manuskript 1 und 2) steht der evolutionär hochkonservierte Elongator im Mittelpunkt. Der Komplex hat Acetyltransferase-Aktivität und ist essentiell für die Bildung von modifizierten Seitenketten im Anticodon (U34) einiger tRNAs. Die Aktivität des Elongators ist dabei nicht nur von einer vollständigen Assemblierung, sondern auch vom Phosphorylierungsstatus der Elp1-Untereinheit, abhängig. Es konnte gezeigt werden, dass neben der Typ2A-Proteinphosphatase Sit4, auch die Caseinkinase Hrr25 den Elp1-Phosphorylierungsstatus beeinflusst.von Constance Mehlgarte