Discovery and characterisation of protein methyltransferases that target translation elongation factor 1A in yeast and human

Abstract

Eukaryotic elongation factor 1A (eEF1A) is an essential protein in the cell that is evolutionarily conserved and abundant. It is involved in numerous cellular processes, including protein synthesis, actin cytoskeleton organisation, protein degradation and nuclear export. As a result, eEF1A has been found to be important in viral replication and is often dysregulated in cancers. It is therefore crucial to understand how this protein is controlled through post-translational modifications. In particular, eEF1A is known to be highly post-translationally methylated. It is therefore important to characterise the methyltransferases that mediate this modification. In this thesis, mass spectrometric techniques were employed, in combination with gene knock outs and recombinant methods, to discover and characterise the methyltransferases of eEF1A. A novel protein methyltransferase, renamed Efm7, was discovered in the budding yeast Saccharomyces cerevisiae that methylates two newly described methylation sites in eEF1A – the N-terminus and the side-chain of the adjacent lysine residue. The N-terminal methylation was shown to be conserved in human, suggesting an important role for this new modification. This thesis also presents the discovery of two new human methyltransferases that methylate lysines 79 and 165 in eEF1A, renamed eEF1A-KMT1 and eEF1A-KMT3, respectively. Both are characterised in vitro, through enzyme assays, and in vivo, through use of CRISPR-mediated knock out of these genes. Proteomic analysis was then used to determine the roles of these enzymes in the human cell. Knock out of EEF1AKMT3 resulted in an upregulation of ribosomal proteins, but this was not observed upon knock out of EEF1AKMT1. Finally, a new technique was developed in order to elucidate the mechanism by which an eEF1A methyltransferase recognises and methylates its substrate residue. This thesis therefore represents significant advances in understanding the methyltransferases that target eEF1A in both yeast and human.