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Biochem Biophys Res Commun
2011 Jul 22;4111:19-24. doi: 10.1016/j.bbrc.2011.06.062.
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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.
Newbery HJ
,
Stancheva I
,
Zimmerman LB
,
Abbott CM
.
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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.
Fig. 1.
Figure 1 shows the results of ClustalW analysis of eEF1A1 and eEF1A2 from X. laevis, X. tropicalis and mouse; the results were coloured using Boxshade. Xt is X. tropicalis, Xl is X. laevis, m is mouse; 1A1 is eEF1A1, 1A2 is eEF1A2. An asterisk denotes the position of the serine residue present in eEF1A2 from all species that is predicted to be a phosphorylation site.
Fig. 2.
Panel A: RT-PCR of eEF1A1 (top panel) and eEF1A2 (bottom panel) in RNA from a range of tissues taken from adult X. laevis. Li = liver, Lu = lung, S = spleen, O = oocytes, G = gall bladder, B = brain, H = heart, M = muscle (two independent tissue samples). No-RT controls were carried out and were negative for all tissues for both genes (data not shown). Panel B: Western blots of eEF1A1 (top) and eEF1A2 (bottom). Gels were run in duplicate using the samples of same protein extract at the same time. Og = optic ganglion, B = brain, Sc = spinal cord, M = muscle, Li = liver, G = gall bladder, Lu = lung, K = kidney, S = spleen. Panel C: Immunohistochemistry for eEF1A2 on spinal cord and cardiac muscle from X. laevis, together with negative controls. eEF1A2 shows widespread expression in cardiac muscle but is expressed in only in neuronal cells in spinal cord.
Fig. 3.
Panel A: RT-PCR of eEF1A1 in X. laevis muscle and liver using primers that amplify full-length mRNA. Lanes labelled with – are no-RT controls. Panel B: Real-time RT-PCR results for eEF1A1 and eEF1A2 in liver (showing expression of eEF1A1 only) and muscle (showing equal levels of expression of eEF1A1 and eEF1A2). Units of expression are relative to the internal control, Rpl8. Panel C: Analysis of expression of eEF1A1 in adult muscle from X. tropicalis. B shows RT-PCR of eEF1A1 and eEF1A2 in muscle from X. tropicalis (Xt) and X. laevis (Xl) together with negative controls (−RT). All PCR products are of the predicted size. Panel D: two Western blots, run in parallel, for eEF1A1 and eEF1A2. M(t) is muscle tissue from X. tropicalis, M(l) is muscle tissue from X. laevis and L(l) is liver from X. laevis.
