Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

Abstract

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex.

The role of translation elongation factor eEF1 subunits in neurodevelopmental disorders

“…Each isoform appears to function in the canonical role of translation elongation with similar protein synthesis kinetics, but with different affinities for GTP/GDP, with eEF1A1 favoring GTP and eEF1A2 showing greater affinity for GDP (Kahns et al., ). Despite this difference, both eEF1A1 (Mansilla et al., ) and eEF1A2 (Y. Cao, Portela, Janikiewicz, Doig, & Abbott, ) have been shown to bind to the eEF1B complex. Although Mansilla et al.…”

“…EEF1B1 has been shown to be a processed pseudogene, whereas EEF1B3 is a human‐specific intronless gene transcribed only in brain and muscle (Chambers, Peters, & Abbott, ). Although eEF1Bα is essential for cell survival in yeast, with knockout strains showing growth defects (Hiraga, Suzuki, Tsuchiya, & Miyakawa, ), it is dispensable for short‐term viability, at least, of human cells in culture (see below) (Y. Cao et al., ). The C‐terminal region of the protein encompasses the residues necessary for guanine exchange activity (Janssen & Möller, ), whereas the N‐terminal binds to eEF1Bγ, linking eEF1Bα to the rest of the eEF1 complex (Mansilla et al., ).…”

“…Three splice forms encoded by a single gene, EEF1D, each giving rise to a protein of predicted size of about 38 kDa, have been reported. The same locus also encodes a more recently described brain, spinal cord, and testis specific isoform called eEF1BδL, which is expressed throughout brain development in the mouse (Kaitsuka & Matsushita, ; Cao et al., ). This longer isoform includes an additional 367 amino acids at the N‐terminus, a domain containing a nuclear localization signal.…”

“…It is as yet unclear whether the eEF1BδL longer isoform binds to the other subunits of eEF1B in the same way as the canonical isoform, and is thus able to act as a guanine exchange factor. The smaller isoforms of eEF1D are ubiquitously expressed in mouse, but are more highly expressed in brain and liver at fetal and neonatal stages than from p10 onwards (Y. Cao et al., ). In contrast, eEF1BδL is expressed at in brain at all developmental stages tested, increasing slightly with age (Y. Cao et al., ).…”

New evidence that biallelic loss of function in EEF1B2 gene leads to intellectual disability

“…EEF1B2 is located on 2q33 and is ubiquitously expressed. Although eEF1Bα is essential for cell survival in yeast, it seems dispensable for short‐term viability, at least, of human cells in culture …”