Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

Abstract

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

eIF4B stimulates eIF4A ATPase and unwinding activities by direct interaction through its 7-repeats region

“…Stable eIF4A/eIF4B complexes have been identified in pulldown experiments for the mammalian counterparts only. 18,19 A direct interaction of yeast eIF4A and eIF4B is supported by the observed stimulation of the eIF4A helicase activity by eIF4B 9 and the promotion of the eIF4A-PIC interaction by eIF4B, 16 and by genetic studies, 20 but has not been shown experimentally. In conventional binding experiments (such as size-exclusion chromatography or pull-down experiments) reactants are mixed at concentrations that favor complex formation, but multiple washing steps and the concomitant dilution may allow dissociation of the binding partners.…”

“…It has been shown previously that yeast eIF4B binds to 40S ribosomal subunits, and mediates recruitment of the PIC to the mRNA, possibly through interactions with eIF4A. 16 PIC recruitment depends on the presence of the r1-7 region of eIF4B, and to a lesser extent on the NTD. 16 Human eIF4A and eIF4B form a stable complex.…”

“…To this end, eIF4B deletion variants lacking one or multiple domains 16 were analyzed for their stimulatory effect on eIF4A ATPase and RNA unwinding activities (Fig. 4, Table 1).…”

“…eIF4B on its own does not affect the eIF4A conformational cycle, but stimulates closing when eIF4G is present. 9,13 Yeast eIF4B consists of an N-terminal domain (NTD), an RNA recognition motif (RRM), a region of seven repeats of moderate homology (r1-7), and a C-terminal domain 16,17 (CTD; Fig. 1).…”

“…16 Human eIF4A and eIF4B form a stable complex. 18,19 An equivalent interaction of yeast eIF4A and eIF4B was deduced from genetic studies, 20,21 the stimulation of the eIF4A helicase activity by eIF4B, 9 and the observation that eIF4B promotes the interaction of eIF4A with the PIC, 16 but the formation of a yeast eIF4A/eIF4B complex has not been observed experimentally. Here we show that yeast eIF4B interacts directly with eIF4A in the presence of RNA and the non-hydrolyzable analog ADPNP, independent of eIF4G.…”