Antibody Toolkit to Investigate eEF1A Methylation Dynamics in mRNA Translation Elongation

Authors

Robert Mealey-Farr
Jinho Jeong
Juhyung Park
Shuo Liu
Simone Hausmann
Joel W Francis
Maria Angulo Ibanez
Joonseok Cho
Katrin Chua
Pawel K Mazur
Or Gozani

Publication Date

6-1-2023

Journal

Journal of Biological Chemistry

Abstract

Protein synthesis is a fundamental step in gene expression, with modulation of mRNA translation at the elongation step emerging as an important regulatory node in shaping cellular proteomes. In this context, five distinct lysine methylation events on eukaryotic elongation factor 1A (eEF1A), a fundamental nonribosomal elongation factor, are proposed to influence mRNA translation elongation dynamics. However, a lack of affinity tools has hindered progress in fully understanding how eEF1A lysine methylation impacts protein synthesis. Here we develop and characterize a suite of selective antibodies to investigate eEF1A methylation and provide evidence that methylation levels decline in aged tissue. Determination of the methyl state and stoichiometry on eEF1A in various cell lines by mass spectrometry shows modest cell-to-cell variability. We also find by Western blot analysis that knockdown of individual eEF1A-specific lysine methyltransferases leads to depletion of the cognate lysine methylation event and indicates active crosstalk between different sites. Further, we find that the antibodies are specific in immunohistochemistry applications. Finally, application of the antibody toolkit suggests that several eEF1A methylation events decrease in aged muscle tissue. Together, our study provides a roadmap for leveraging methyl state and sequence-selective antibody reagents to accelerate discovery of eEF1A methylation-related functions and suggests a role for eEF1A methylation, via protein synthesis regulation, in aging biology.

Keywords

Antibodies, Lysine, Methylation, Peptide Chain Elongation, Translational, Peptide Elongation Factor 1, eEF1A, methylation, protein methylation, mRNA transalation, translation elongation, methyltransferase, METTL13, METTL10, eEF1AKMT4, N6AMT2, epigenetic, lysine methylation

Comments

Supplementary Materials

Data Availability Statement

PMID: 37094697