Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Discovery and characterisation of a novel human lysine methyltransferase by CRISPR/Cas9 knock out and SILAC proteomics (#79)

Joshua J Hamey 1 , Beeke Wienert 1 , Kate G. R. Quinlan 1 , Marc R Wilkins 1
  1. University of New South Wales, Sydney, NSW, Australia

Lysine methylation is widespread on human proteins, however the enzymes that catalyse its addition remain largely unknown. This limits our capacity to study the function and regulation of this modification. We used the CRISPR/Cas9 system to knock out putative protein methyltransferases METTL21B and METTL23 in K562 cells, to determine if they methylate translation elongation factor 1A (eEF1A). The known eEF1A methyltransferase EEF1AKMT1 was also knocked out as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon knock out of METTL21B, and the expected loss of lysine 79 methylation on knock out of EEF1AKMT1. No loss of eEF1A methylation was seen in the METTL23 knock out. Recombinant METTL21B was shown in vitro to catalyse methylation on lysine 165 in eEF1A, confirming it as the methyltransferase responsible for this methylation site. To gain insight into the specific function of METTL21B-mediated methylation of lysine 165 in eEF1A, we used SILAC followed by LC-MS/MS to analyse the proteomes of two different METTL21B knock outs and compare them to wild-type K562 cells. Two different knock outs of EEF1AKMT1 were also analysed for proteomic changes and were compared to wild-type K562 to determine the effects of loss of lysine 79 methylation. This revealed specific upregulation of large ribosomal subunit proteins upon METTL21B knock out, which was not seen upon EEF1AKMT1 knock out, and changes to further processes related to eEF1A function in knock outs of both METTL21B and EEF1AKMT1. This indicates that the methylation of lysine 165 in human eEF1A has a very specific role in translation.

  1. Hamey, J. J., Wienert, B., Quinlan, K. G., & Wilkins, M. R. (2017). METTL21B is a novel human lysine methyltransferase of translation elongation factor 1A: discovery by CRISPR/Cas9 knock out. Molecular & Cellular Proteomics, mcp-M116.