Kti12 - a regulator of the tRNA modification function of Elongator in yeast?

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The conserved eukaryotic protein complex Elongator (Elp1-Elp6) primarily operates in the modification of transfer RNAs (tRNAs). Thus, it ensures proper tRNA functioning required for decoding the genetic information of messenger RNAs (mRNAs) during the processes of mRNA translation and protein biosynthesis. Hence, one might expect that Elongator is permanently active for accurate tRNA decoding at all times. However, data showing that the tRNA modifications oscillate in response to various stimuli suggest Elongator activity is regulatable. In support of this, we have shown Elongator undergoes dynamic phosphorylation dependent on kinase Hrr25 and phosphatase Sit4 and importantly, modulatable by Kti12, an Elongator partner protein related to a tRNA-dependent kinase (PSTK) and other nucleotide (NTP) binding proteins. How Kti12 is precisely involved in Elongator function and whether it operates as a potential Elongator regulator are key questions in need for answers. Therefore, and using yeast as model system to study Elongator regulation by Kti12, we aim to achieve the following goals:- to study (in collaboration with Prof. Stark, Dundee) posttranslational Kti12 modification by mass spectrometric mapping of modification sites followed by site-specific mutagenesis for validation of their significance.- to use a pool of kti12 mutants previously isolated by our collaborator (Prof. Stark, Dundee) and to be complemented by site-specific mutagenesis of the KTI12 gene for identifying protein domains on Kti12 hat are potentially involved in Elongator function and regulation.- to take advantage of bona fide Kti12 properties (Elongator interaction, Hrr25 kinase recruitment to Elongator, Elp1 phosphomodulation, Sit4 antagonism, etc) for examining the requirement of the above identified Kti12 domains for Elongator function and regulation.- to exploit in vitro assays for analysing the proposed tRNA binding capacity of Kti12 and its potential ability to bind NTP cofactors (see above). These studies will involve recombinant Kti12 and mutants identified above for use in tRNA-dependent gel-shift assays and cofactor binding studies. In summary, the proposal centers around in vivo & in vitro methods that aim at identifying structure-function requirements for Kti12 to partner with Elongator and allow Elongator-dependent tRNA modifications to be differentially regulated. Moreover, the project is biomedically relevant with potentials to improve the quality of health and life in the longer term. This lies with a strong body of recent evidence showing that tRNA modifications are linked to human neuropathies and tumour formation Hence, the proposal can contribute to a better understanding of the roles that coordinated formation of Elongator-linked tRNA modifications play in translational control and neurogeneration in higher eukaryal models including our own cells.