Deciphering the underlying mechanisms of de novo missense mutations in eukaryotic translation elongation factor eEF1A2

Centre for Genomics & Experimental Medicine, Institute for Genetics & Molecualr Medicine, University of Edinburgh

Eukaryotic translation elongation factor eEF1A2 is a neuronal specific isoform, essential for the delivery of aminoacylated tRNAs to the ribosome, in a GTP dependent manner. 36 heterozygous de novo missense mutations in eEF1A2 have been identified in individuals suffering from epilepsy, autism and intellectual disability. In order to determine possible therapeutic strategies, the molecular basis to these mutations must first be researched. My work examines the effect of the mutations in vitro and in vivo, to identify a gain or loss of function. Changes to the binding partners of eEF1A2 were examined, to identify loss or gain in binding might elucidate the molecular basis to these mutations. WT eEF1A2 was compared with two mutant conditions eEF1A2D252H and eEF1A2G70S. The proteins were expressed in neurons, subjected to IP pulldown and label free mass spectrometry to identify interacting components and any changes. Results were validated by co-immunoprecipitation, both in vitro and in vivo. Compared to WT, eEF1A2D252H showed a loss of binding to its cognate guanine exchange factor eEF1B, suggesting it would no longer be able to recycle GDP and function canonically. However no loss of binding could be identified in eEF1A2G70S. Findings suggest that eEF1A2D252H may operate through a loss of function mechanism, but are as yet unclear for other mutations. It appears as though mutations may work in different manners, depending on their location in the protein, culminating in the same phenotype.

References

1. LeSourd et al, 2006 - eEF1B: At the dawn of the 21st century
2. Jiang et al, 2005 - Three-dimensional reconstruction of the valyl-tRNA synthetase/elongation factor-1H complex and localization of the delta subunit
3. Cao et al, 2014 - Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

Funded by: MRC

* entered into the PhD student poster competition