Evolutionary importance of translation elongation factor eEF1A variant switching: eEF1A1 down-regulation in muscle is conserved in Xenopus but is controlled at a post-transcriptional level.

Translation elongation isoform eEF1A1 has a pivotal role in protein synthesis and is almost ubiquitously expressed. In mice and rats that transcription of the gene encoding eEF1A1 is downregulated to undetectable levels in muscle after weaning; eEF1A1 is then replaced by a separately encoded but closely related isoform eEF1A2, which has only previously been described in mammals. We now show that not only is eEF1A2 conserved in non-mammalian vertebrate species, but the down-regulation of eEF1A1 protein in muscle is preserved in Xenopus, with the protein being undetectable by adulthood. Interestingly, though, this down-regulation is controlled post-transcriptionally, and levels of full-length eEF1A1 mRNA remain similar to those of eEF1A2. The switching off of eEF1A1 in muscle is therefore sufficiently important to have evolved through the use of repression operating at different levels in different species. The 3'UTR of eEF1A1 is highly conserved and contains predicted binding sites for several miRNAs, suggesting a possible method for controlling of expression. We suggest that isoform switching may have evolved because of a need for certain cell types to modify the well-established non-canonical functions of eEF1A1.

eEF1A2 and neuronal degeneration.

Translation elongation factor eEF1A (eukaryotic elongation factor 1A) exists as two individually encoded variants in mammals, which are 98% similar and 92% identical at the amino acid level. One variant, eEF1A1, is almost ubiquitously expressed, the other variant, eEF1A2, shows a very restricted pattern of expression. A spontaneous mutation was described in 1972, which gives rise to the wasted phenotype: homozygous wst/wst mice develop normally until shortly after weaning, but then lose muscle bulk, acquire tremors and gait abnormalities and die by 4 weeks. This mutation has been shown to be a deletion of 15 kb that removes the promoter and first exon of the gene encoding eEF1A2. The reciprocal pattern of expression of eEF1A1 and eEF1A2 in muscle fits well with the timing of onset of the phenotype of wasted mice: eEF1A1 declines after birth until it is undetectable by 3 weeks, whereas eEF1A2 expression increases over this time. No other gene is present in the wasted deletion, and transgenic studies have shown that the phenotype is due to loss of eEF1A2. We have shown that eEF1A2, but not eEF1A1, is also expressed at high levels in motor neurons in the spinal cord. Wasted mice develop many pathological features of motor neuron degeneration and may represent a good model for early onset of motor neuron disease. Molecular modelling of the eEF1A1 and eEF1A2 protein structures highlights differences between the two variants that may be critical for functional differences. Interactions between eEF1A2 and ZPR1 (zinc-finger protein 1), which interacts with the SMN (survival motor neuron) protein, may be important in motor neuron biology.

In Vivo Translatome Profiling in Spinal Muscular Atrophy Reveals a Role for SMN Protein in Ribosome Biology.

Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration.

Progressive loss of motor neuron function in wasted mice: effects of a spontaneous null mutation in the gene for the eEF1 A2 translation factor.

Wasted (wst) is a spontaneous autosomal recessive mutation in which the gene encoding translation factor eEF1A2 is deleted. Homozygous mice show tremors and disturbances of gait shortly after weaning, followed by motor neuron degeneration, paralysis, and death by about 28 days. We have now conducted a more detailed analysis of neuromuscular pathology in these animals. Reactive gliosis was observed at 19 days postnatal in wst/wst cervical spinal cord, showing a rostrocaudal gradient. This was followed a few days later by motor neuron vacuolation and neurofilament accumulation, again with a rostrocaudal progression. Thoracic/abdominal muscles from wst/wst mice aged 17 days showed evidence of progressive denervation of motor endplates, including weak synaptic transmission and retraction of motor nerve terminals. Similar abnormalities appeared in distal, lumbrical muscles from about 25 days of age. We conclude that spontaneous failure of eEF1A2 expression in the wasted mutant first triggers gliosis in spinal cord and retraction of motor nerve terminals in muscle, and then motor neuron pathology and death. The early initiation and rapid progression of motor unit degeneration in wst/wst mice suggest that they should be considered an important and accessible model of early-onset motor neuron degeneration in humans.

Of mice, men and motor neurons.

The use of mouse models has been of particular importance in studying the pathogenesis of amyotrophic lateral sclerosis. Here, we describe both transgenic and classical mutants for which the genetic lesion is known. We draw attention, wherever possible, to pathological factors common to multiple models.

Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours.

BACKGROUND: The tissue-specific translation elongation factor eEF1A2 was recently shown to be a potential oncogene that is overexpressed in ovarian cancer. Although there is no direct evidence for an involvement of eEF1A2 in breast cancer, the genomic region to which EEF1A2 maps, 20q13, is frequently amplified in breast tumours. We therefore sought to establish whether eEF1A2 expression might be upregulated in breast cancer. METHODS: eEF1A2 is highly similar (98%) to the near-ubiquitously expressed eEF1A1 (formerly known as EF1-alpha) making analysis with commercial antibodies difficult. We have developed specific anti-eEF1A2 antibodies and used them in immunohistochemical analyses of tumour samples. We report the novel finding that although eEF1A2 is barely detectable in normal breast it is moderately to strongly expressed in two-thirds of breast tumours. This overexpression is strongly associated with estrogen receptor positivity. CONCLUSION: eEF1A2 should be considered as a putative oncogene in breast cancer that may be a useful diagnostic marker and therapeutic target for a high proportion of breast tumours. The oncogenicity of eEF1A2 may be related to its role in protein synthesis or to its potential non-canonical functions in cytoskeletal remodelling or apoptosis.

In vivo characterization of the role of tissue-specific translation elongation factor 1A2 in protein synthesis reveals insights into muscle atrophy.

Translation elongation factor 1A2 (eEF1A2), uniquely among translation factors, is expressed specifically in neurons and muscle. eEF1A2-null mutant wasted mice develop an aggressive, early-onset form of neurodegeneration, but it is unknown whether the wasting results from denervation of the muscles, or whether the mice have a primary myopathy resulting from loss of translation activity in muscle. We set out to establish the relative contributions of loss of eEF1A2 in the different tissues to this postnatal lethal phenotype. We used tissue-specific transgenesis to show that correction of eEF1A2 levels in muscle fails to ameliorate the overt phenotypic abnormalities or time of death of wasted mice. Molecular markers of muscle atrophy such as Fbxo32 were dramatically upregulated at the RNA level in wasted mice, both in the presence and in the absence of muscle-specific expression of eEF1A2, but the degree of upregulation at the protein level was significantly lower in those wasted mice without transgene-derived expression of eEF1A2 in muscle. This provides the first in vivo confirmation that eEF1A2 plays an important role in translation. In spite of the inability of the nontransgenic wasted mice to upregulate key atrogenes at the protein level in response to denervation to the same degree as their transgenic counterparts, there were no measurable differences between transgenic and nontransgenic wasted mice in terms of weight loss, grip strength, or muscle pathology. This suggests that a compromised ability fully to execute the atrogene pathway in denervated muscle does not affect the process of muscle atrophy in the short term.

Haploinsufficiency for translation elongation factor eEF1A2 in aged mouse muscle and neurons is compatible with normal function.

Translation elongation factor isoform eEF1A2 is expressed in muscle and neurons. Deletion of eEF1A2 in mice gives rise to the neurodegenerative phenotype "wasted" (wst). Mice homozygous for the wasted mutation die of muscle wasting and neurodegeneration at four weeks post-natal. Although the mutation is said to be recessive, aged heterozygous mice have never been examined in detail; a number of other mouse models of motor neuron degeneration have recently been shown to have similar, albeit less severe, phenotypic abnormalities in the heterozygous state. We therefore examined the effects of ageing on a cohort of heterozygous +/wst mice and control mice, in order to establish whether a presumed 50% reduction in eEF1A2 expression was compatible with normal function. We evaluated the grip strength assay as a way of distinguishing between wasted and wild-type mice at 3-4 weeks, and then performed the same assay in older +/wst and wild-type mice. We also used rotarod performance and immunohistochemistry of spinal cord sections to evaluate the phenotype of aged heterozygous mice. Heterozygous mutant mice showed no deficit in neuromuscular function or signs of spinal cord pathology, in spite of the low levels of eEF1A2.

Eef1a2 promotes cell growth, inhibits apoptosis and activates JAK/STAT and AKT signaling in mouse plasmacytomas.

BACKGROUND: The canonical function of EEF1A2, normally expressed only in muscle, brain, and heart, is in translational elongation, but recent studies suggest a non-canonical function as a proto-oncogene that is overexpressed in a variety of solid tumors including breast and ovary. Transcriptional profiling of a spectrum of primary mouse B cell lineage neoplasms showed that transcripts encoding EEF1A2 were uniquely overexpressed in plasmacytomas (PCT), tumors of mature plasma cells. Cases of human multiple myeloma expressed significantly higher levels of EEF1A2 transcripts than normal bone marrow plasma cells. High-level expression was also a feature of a subset of cell lines developed from mouse PCT and from the human MM. METHODOLOGY/PRINCIPAL FINDINGS: Heightened expression of EEF1A2 was not associated with increased copy number or coding sequence mutations. shRNA-mediated knockdown of Eef1a2 transcripts and protein was associated with growth inhibition due to delayed G1-S progression, and effects on apoptosis that were seen only under serum-starved conditions. Transcriptional profiles and western blot analyses of knockdown cells revealed impaired JAK/STAT and PI3K/AKT signaling suggesting their contributions to EEF1A2-mediated effects on PCT induction or progression. CONCLUSIONS/SIGNIFICANCE: EEF1A2 may play contribute to the induction or progression of some PCT and a small percentage of MM. Eef1a2 could also prove to be a useful new marker for a subset of MM and, ultimately, a possible target for therapy.

Structural models of human eEF1A1 and eEF1A2 reveal two distinct surface clusters of sequence variation and potential differences in phosphorylation.

BACKGROUND: Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Balpha and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins. CONCLUSIONS: The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis.