Fujun Zhou

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

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 yeIF4Bs 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 ribosomes 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 translation of long mRNAs with structured 5′ UTRs and low closed-loop potential but weak dependence on eIF4G

Significance Protein synthesis initiates in eukaryotes when the 40S ribosomal subunit, loaded with initiator tRNA, attaches to the 5′ end of the mRNA, scans the 5′ UTR, and selects the AUG start codon. Ribosome attachment and scanning are impeded by structures in the 5′ UTR that can be resolved by RNA helicases Ded1 and eukaryotic translation initiation factor 4A (eIF4A), with cofactors eIF4B and eIF4G. We show that eIF4B can stimulate translation independently of eIF4A and that eIF4B, eIF4A, and Ded1 are preferentially required for translating long mRNAs, burdened with 5′ UTR structures, that inefficiently form the closed-loop intermediate with the mRNA ends joined by eIF4G. In contrast, eIF4G appears to be most crucial for closed-loop assembly on short, highly translated, and unstructured mRNAs. DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5′ untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F–poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5′ UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.

Yeast Eukaryotic Initiation Factor 4B (eIF4B) Enhances Complex Assembly between eIF4A and eIF4G in Vivo

Background: Mammalian eIF4B stimulates eIF4A helicase activity, but its function in promoting translation initiation in yeast is unclear. Results: Yeast eIF4B enhances eIF4G·eIF4A association in vivo and in vitro. Conclusion: yeIF4B stimulates eIF4F assembly by promoting an eIF4G HEAT domain conformation conducive for binding eIF4A. Significance: A new function is established for eIF4B of supporting eIF4F assembly for mRNA activation. Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m7G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts− mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001

Identification and characterization of functionally critical, conserved motifs in the internal repeats and N-terminal domain of yeast translation initiation factor 4B (yeIF4B).

Background: The internal repeats and NTD of yeIF4B stimulate translation initiation. Results: The minimal number of repeats and conserved motifs in the repeat and NTD necessary for yeIF4B function was determined. Conclusion: Two repeats provide appreciable function, except when the NTD is missing or eIF4F function is limiting or compromised. Significance: The results provide a comprehensive description of functionally critical sequence elements in yeIF4B. eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

eIF4A is stimulated by the pre-initiation complex and enhances recruitment of mRNAs regardless of structural complexity

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5′-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that the PIC stimulates the ATPase of eIF4A, indicating that the factor acts in association with initiating ribosomal complexes rather than exclusively on isolated mRNAs. ATP hydrolysis by eIF4A accelerates the rate of recruitment for all mRNAs tested, regardless of their degree of secondary structure, indicating that the factor plays important roles beyond unwinding mRNA structure. Structures in the 5′-UTR and 3′ of the start codon synergistically inhibit mRNA recruitment, in a manner relieved by eIF4A, suggesting that the factor resolves global mRNA structure rather than just secondary structures in the 5′-UTR. We suggest that eIF4A breaks the many weak interactions formed within an mRNA that occlude the 5′-UTR and facilitates engagement of the 5′-UTR with the PIC.

eIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast

The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor context. Ribosome profiling of NTT substitution R13P reveals heightened discrimination against unfavorable AUG context genome-wide. Both R13P and K16D substitutions destabilize the closed complex at UUG codons in reconstituted PICs. Thus, electrostatic interactions involving the eIF1A NTT stabilize the closed conformation and promote utilization of suboptimal start codons. We predict UM-associated mutations alter human gene expression by increasing discrimination against poor initiation sites.

eIF3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2·GTP·Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncover a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.