Regulation of the Elongator and the DPH complex by the Kti11/Kti13 heterodimer

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Post-transcriptional and post-translational modifications of RNA and proteins play an important role in ribosomal protein synthesis. Strikingly, two very different modification reactions, uridine modifications of tRNA anticodons and diphthamide modification of eukaryotic elongation factor EF-2, catalyzed by the Elongator or DPH complex, respectively, share a common component, the Kti11/Kti13 heterodimer. These modifications are of particular importance, as they affect the binding and translocation of tRNAs on the ribosome and thus can influence the translation efficiency and accuracy of a multitude of genes simultaneously. The structure of Elongator and the DPH complex as well as that of Kti11/Kti13 is conserved among eukaryotes, but the molecular functions of these complexes and the way they cooperate are still poorly understood. Elongator is a large and highly conserved macromolecular assembly built up by six distinct proteins that was initially identified in yeast as a RNA polymerase II (Pol II) associated transcription elongation factor from where its name originates. Diverse phenotypes arising from Elongator dysfunction have been described suggesting that it is involved in a whole variety of cellular functions. In humans, Elongator has been associated with neurological disorders (e.g. familial dysautonomia) and carcinogenesis. At the molecular level the protein complex has been shown to be essential for the uridine modification in tRNAs. It cannot be excluded that Elongator fulfills additional cellular and molecular functions and its precise activities are still controversially discussed. However, evidence is accumulating that strongly supports a catalytic role in biosynthesis of the carbamoyl and methoxycarbonyl side chains of uridine. This function requires interaction, perhaps transiently, with the Kti11/Kti13 heterodimeric complex. The DPH complex catalyzes the first step in diphthamide biosynthesis (a modification of a histidine residue) together with Dph4 and is composed of Dph1, Dph2 and Dph3 alias Kti11. DPH1 alias OVCA1 is a candidate tumor suppressor in ovarian cancer and mouse homozygous deletion mutants are embryonic lethal whereas yeast can grow in the absence of diphthamide. We plan to characterize the functional interplay between Kti11/Kti13 with Elongator and the diphthamide biosynthetic (DPH) complex. Hypotheses derived from recent structural insights will guide functional in vivo and in vitro studies to obtain a comprehensive mechanistic understanding of the three protein complexes and to gain further insight in their biological roles. The scientific questions addressed in this proposed research plan will not only enhance the understanding of these central translational control networks, but also facilitate the design of novel therapeutic strategies for several severe human diseases.