title: Characterization of the role of eIF4G in stimulating cap- and IRES-dependent translation in aplysia neurons.
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.
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Dyer
authors.forename: Wayne S
authors.initials: WS
authors.lastname: Sossin
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001048:Aplysia; D002478:Cells, Cultured; D003001:Cloning, Molecular; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000502:physiology*; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012315:RNA Caps; D012270:Ribosomes / Q000378:metabolism*; D011984:Sensory Receptor Cells / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D039603:Eukaryotic Initiation Factor-4G; D012315:RNA Caps
references.citation: Curr Opin Neurobiol. 2010 Aug;20(4):450-6
references.pmid: 20430610
references.citation: J Neurochem. 2006 Apr;97(1):79-91
references.pmid: 16515558
references.citation: Neuron. 2009 Jan 15;61(1):10-26
references.pmid: 19146809
references.citation: Nat Cell Biol. 2009 Jul;11(7):903-8
references.pmid: 19525934
references.citation: Neurosci Lett. 1996 Dec 27;221(1):29-32
references.pmid: 9014173
references.citation: J Neurochem. 2011 Jun;117(5):841-55
references.pmid: 21426346
references.citation: Nucleic Acids Res. 2001 Dec 15;29(24):5226-32
references.pmid: 11812856
references.citation: J Neurosci. 2013 Jan 30;33(5):1872-86
references.pmid: 23365227
references.citation: J Biol Chem. 2010 Jul 16;285(29):21922-33
references.pmid: 20463023
references.citation: Nat Neurosci. 2010 Jul;13(7):897-905
references.pmid: 20543841
references.citation: EMBO J. 2000 Feb 1;19(3):434-44
references.pmid: 10654941
references.citation: Cell. 2009 Feb 20;136(4):731-45
references.pmid: 19239892
references.citation: Mol Cell Biol. 2013 Mar;33(5):937-46
references.pmid: 23263986
references.citation: J Neurochem. 2000 Aug;75(2):872-81
references.pmid: 10899966
references.citation: Mol Cell Biol. 2006 Nov;26(22):8586-98
references.pmid: 16982686
references.citation: Oncogene. 2006 Oct 16;25(48):6416-22
references.pmid: 17041626
references.citation: Nature. 2013 Jan 17;493(7432):371-7
references.pmid: 23172145
references.citation: Nucleic Acids Res. 2005 Nov 28;33(20):6593-602
references.pmid: 16314320
references.citation: Phys Biol. 2010 May 12;7(2):021001
references.pmid: 20463379
references.citation: Nat Neurosci. 2003 Mar;6(3):219-20
references.pmid: 12592407
references.citation: Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7029-36
references.pmid: 11416183
references.citation: Gene. 2002 Oct 16;299(1-2):1-34
references.pmid: 12459250
references.citation: EMBO J. 2005 Dec 7;24(23):4094-105
references.pmid: 16281055