Mass Spectrometric Analysis of the N Terminus of Translational Initiation Factor eIF4G-1 Reveals Novel Isoforms

Abstract

In eukaryotes, translation initiation factor 4G (eIF4G) acts as the central binding protein for an unusually large number of proteins involved in mRNA metabolism. Several gene products homologous to eIF4G have been described, the most studied being eIF4G-1. By its association with other initiation factors, eIF4G-1 effects mRNA cap and poly(A) recognition, unwinding of secondary structure, and binding to the 43S initiation complex. Multiple electrophoretic isoforms of eIF4G-1 are observed, and multiple cDNAs have been reported, yet the relationship between the two is not known. We report here a new cDNA for eIF4G-1, present as a previously unidentified human expressed sequence tag, that extends the long open reading frame, provides a new in-frame initiation codon, and predicts a longer form of eIF4G-1 than reported previously. eIF4G isoforms from human K562 cells were cleaved with recombinant Coxsackievirus 2A protease and the Nterminal domains purified by m 7 GTP-Sepharose chromatography and polyacrylamide gel electrophoresis. Proteins were digested with proteolytic enzymes and peptides masses determined by matrixassisted laser desorption ionization-time of flight mass spectrometry. In selected cases, peptides were sequenced by electrospray-mass spectrometry fragmentation. This identified the N termini of the three most abundant eIF4G-1 isoforms, two of which had not previously been proposed. These proteins appear to have been initiated from three different AUG codons.The three stages of protein synthesis are catalyzed by three groups of proteins: initiation, elongation, and termination factors (1). Initiation is characterized by formation of a series of initiation complexes, each catalyzed by a different subset of initiation factors. Recruitment of mRNA to the 43 S initiation complex to form the 48 S initiation complex involves eIF3, 1 PABP, and the eIF4 proteins. eIF3 is a 520-kDa multimer that is required for both Met-tRNA i and mRNA binding. PABP is a 70-kDa protein that specifically binds poly(A) and homo-oligomerizes. The eIF4 factors consist of: eIF4A, a 46-kDa RNA helicase; eIF4B, a 70-kDa RNA-binding and -annealing protein that stimulates eIF4A; eIF4E, a 25-kDa cap-binding protein; and eIF4G, a group of proteins of 154 -180 kDa that form specific complexes with all of the other proteins known to be involved in mRNA recruitment.Proteins sharing eIF4G homology represent the products of at least three different genes in mammalian cells (2)(3)(4)

Initiation factor modifications in the preapoptotic phase

“…(b) Isoforms of eIF4GI arise through alternative translation initiation and differ in sequence at the N-terminus. All numbering of eIF4GI protein sequences is based on the N terminally extended sequence described in Bradley et al 53 and Byrd et al…”

Protection of Cap-dependent Protein Synthesisin Vivo and in Vitro with an eIF4G-1 Variant Highly Resistant to Cleavage by Coxsackievirus 2A Protease

“…With improved techniques for producing eIF4G-1 DM at higher levels in intact cells, it should be possible to use the approach demonstrated here to gain further insight into the cytopathic effect of picornaviruses. Furthermore, because there are numerous isoforms of eIF4G-1 (24), this approach may provide a means of testing them for unique activities or preferential recruitment of mRNA populations. FIG.…”

“…The human cDNA initially isolated for eIF4G-1 contained an open reading frame for a protein of 154 kDa (21), referred to as eIF4G-1a (Ref. 24; see Fig. 1).…”

“…Subsequently, two co-linear but longer cDNAs were isolated that diverged at the 5Ј-end (25,26). These extended the open reading frame by an additional 156 codons to produce a theoretical polypeptide of 172 kDa, termed eIF4G-1e (24). Recently, an even longer form, eIF4G-1f, was detected by mass spectrometric analysis (24).…”

“…These extended the open reading frame by an additional 156 codons to produce a theoretical polypeptide of 172 kDa, termed eIF4G-1e (24). Recently, an even longer form, eIF4G-1f, was detected by mass spectrometric analysis (24). eIF4G-1e and eIF4G-1f contain the binding site for PABP, but eIF4G-1a does not (Fig.…”