Characterization of the Role of eIF4G in Stimulating Cap- and IRES-Dependent Translation in Aplysia Neurons

John Dyer,Wayne S Sossin

doi:10.1371/journal.pone.0074085

John Dyer, Wayne S Sossin

Open Access

https://doi.org/10.1371/journal.pone.0074085

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Abstract

The rate-limiting step(s) of translation in the nervous system have not been clearly identified. We have been examining this question in the cell body of the Aplysia sensory neuron, where translational regulation is important for the regulation of synaptic strength. In the present study, we examined the role of the adaptor protein eIF4G. We cloned Aplysia eIF4G (Ap4G) and Ap4G contains all the standard metazoan eIF4G protein–protein interaction domains. Overexpressing Ap4G in Aplysia sensory neurons caused an increase in both cap-dependent and internal ribosome entry site (IRES)-dependent translation using a previously characterized bicistronic fluorescent reporter. Unexpectedly, measurement of overall translation using the methionine analog, L-azidohomoalanine, revealed that overexpression of Ap4G did not lead to an increase in overall translation rates. Indeed, the effect of Ap4G on the bicistronic reporter depended on the presence of an upstream open reading frame (uORF) in the 5’ UTR encoded by the vector. We have previously shown that Mnk strongly decreased cap-dependent translation and this depended on a putative 4G binding domain. Here we extend these results showing that even in the absence of the uORF, overexpression of Mnk strongly decreases cap-dependent translation and this depends on the Mnk binding site in eIF4G. Similarly, an increase in cap-dependent translation seen with overexpression of elongation factor 2 kinase did not depend on the uORF. Overall, we show that eIF4G is rate limiting for translation of an mRNA encoding an uORF, but is not generally a rate-limiting step for translation.

Highlights

In many cases, translational control is studied in the context of cell size and cell proliferation, since in most cell lines and in cancerous cells, translational control is critical in determining whether or not the cell decides to double its proteome and divide [1,2]
Aplysia eIF4G (Ap4G) was cloned by a combination of bioinformatics, PCR and 5’ RACE (See Methods). eIF4G is an adaptor that contains a number of identified protein–protein interaction domains and Ap4G contains all domains present in vertebrate eIF4G1 and eIF4G2, including those that bind polyA-binding protein (PABP), eIF4E, eIF4A, eIF3 and Mnk (Figure 1A)
It has been proposed that this decrease in elongation could lead to an increase in specific transcripts that are rate-limited for initiation [5], and our results suggest that the upstream open reading frame (uORF) made translation of enhanced cyan fluorescent protein (eCFP) rate-limiting for eIF4G

Summary

Introduction

Translational control is studied in the context of cell size and cell proliferation, since in most cell lines and in cancerous cells, translational control is critical in determining whether or not the cell decides to double its proteome and divide [1,2]. We have been interested in how extracellular signals alter translational control factors to change the proteome of the neuron during synaptic plasticity In many of these studies, we have been using a bicistronic reporter with enhanced cyan fluorescent protein (eCFP) being driven by cap-dependent translation and enhanced yellow fluorescent protein (eYFP) driven by a verified internal ribosome entry site (IRES) derived from the Aplysia egg-laying hormone mRNA [6]. During these studies it has become clear that more fundamental questions about the regulation of translation in neurons remain open. Given the large number of mRNAs encoding uORFs, this suggests a novel mechanism for translational regulation in neurons

Methods

Results

Conclusion