Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPsin vivo

Abstract

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F K mRNA K PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP-eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4GKmRNAK PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G-mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP-eIF4G association is only one of several interactions that stabilize eIF4FKmRNA complexes, and emphasize that closed-loop mRNP formation via PABP-eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F K mRNA assembly.

The Role of the Poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants

“…These findings support the earlier observation that the interaction between eIF4G and PABP in yeast is RNA-dependent 33 as the RNA-binding domain RNA1 assists the interaction between PABP and eIF4G. 37 Whether functionally analogous sequence elements to RNA1, Box1, and Box2 identified in yeast eIF4G1 are present in plant or animal eIF4G is unknown although no obvious sequences exhibiting conservation with these yeast elements are apparent.…”

“…37 These data suggest that the interaction of PABP with eIF4G is only one of several interactions that stabilizes eIF4G binding to an mRNA, at least in yeast. These findings support the earlier observation that the interaction between eIF4G and PABP in yeast is RNA-dependent 33 as the RNA-binding domain RNA1 assists the interaction between PABP and eIF4G.…”

“…Indeed, as eIFiso4G lacks any sequence analogous to the N-terminal region of eIF4G of plant, animal, or yeast eIF4G proteins, there is no possibility of even functionally analogous sequences for the RNA1, Box1, and Box2 elements of yeast eIF4G1. 37 Instead, PABP binds eIFiso4G within the HEAT-1 domain at 2 contiguous sites that overlap extensively with the eIF4A binding site in this same domain (Fig. 1A).…”

“…Domains for yeast eIF4G are defined as described previously. 37 (B) Below the N-terminal PABP binding site of wheat eIF4G is a sequence logo for the region corresponding to the PABP interaction site in human eIF4G. For this analysis, a sequence alignment from 6 monocot and 7 dicot eIF4G homologs was generated using EBI MUS-CLE.…”

“…Although the factors providing the necessary specificity for binding mRNA are eIF4E and PABP, the RNA binding domains in plant eIF4G, eIFiso4G, eIF4A, and eIF4B suggest that these likely contribute to stabilizing the complex to an mRNA as observed for animal and yeast eIF4G, 37,116,117 while the presence of protein interaction domains in each factor serves to bring together the activities required to promote the binding of 40 S subunits and their subsequent scanning. The interaction of PABP with the cap-associated factors not only increases these activities and stabilizes the binding of the complex to the 5 0 -cap, but may promote the re-recruitment of 40 S subunits following translation termination, thereby increasing the yield of protein from a given mRNA before its eventual destruction.…”