Ribosome recycling step in yeast cytoplasmic protein synthesis is catalyzed by eEF3 and ATP

After each round of protein biosynthesis, the posttermination complex (PoTC) consisting of a ribosome, mRNA, and tRNA must be disassembled into its components for a newround of translation. Here, we show that a Saccharomyces cerevisiae model PoTC was disassembled by ATP and eukaryotic elongation factor 3 (eEF3). GTP or ITP functioned with less efficiency and adenosine 5γ′-(β,γ-imido)triphosphate did not function at all. The kcat of eEF3 was 1.12 min-1, which is comparable to that of the in vitro initiation step. The disassembly reaction was inhibited by aminoglycosides and cycloheximide. The subunits formed from the yeast model PoTC remained separated under ionic conditions close to those existing in vivo, suggesting that they are ready to enter the initiation process. Based onour experimental techniques used in this paper, the release of mRNA and tRNA and ribosome dissociation took place simultaneously. No 40S•mRNA complex was observed, indicating that eEF3 action promotes ribosome recycling, not reinitiation

Modifications on Translation Initiation

Two studies by Meyer et al. and Wang et al. demonstrate a role for m6A modification of mRNA in stimulating translation initiation. These findings add to the growing number of diverse mechanisms for translation initiation in eukaryotes

Identification and characterization of functionally critical, conserved motifs in the internal repeats and n-terminal domain of yeast translation initiation factor 4B (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 wildtype (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

The Stress Granule Transcriptome Reveals Principles of mRNA Accumulation in Stress Granules

Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here, we describe the stress granule transcriptome of yeast and mammalian cells through RNA-sequencing (RNA-seq) analysis of purified stress granule cores and single-molecule fluorescence in situ hybridization (smFISH) validation. While essentially every mRNA, and some noncoding RNAs (ncRNAs), can be targeted to stress granules, the targeting efficiency varies from \u3c1% to \u3e95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-seq analysis by smFISH reveals that only 10% of bulk mRNA molecules accumulate in mammalian stress granules and that only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest that stress granules may not represent a specific biological program of messenger ribonucleoprotein (mRNP) assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes. Transcriptome analysis coupled with single-molecule FISH validation of stress granule cores provides new insight into the mRNAs and ncRNAs that accumulate in stress granules. Ten percent of bulk mRNA molecules accumulate in stress granules, and targeting efficiency correlates with poorer translation efficiency and longer coding and UTR length

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 yeIF4B\u27s 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\u27s 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

The 5\u27-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway

Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5\u27 cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3

Should I stay or should I go? Eukaryotic translation initiation factors 1 and 1A control start codon recognition

Start codon selection is a key step in translation initiation as it sets the reading frame for decoding. Two eukaryotic initiation factors, eIF1 and eIF1A, are key actors in this process. Recent work has elucidated many details of the mechanisms these factors use to control start site selection. eIF1 prevents the irreversible GTP hydrolysis that commits the ribosome to initiation at a particular codon. eIF1A both promotes and inhibits commitment through the competing influences of its two unstructured termini. Both factors perform their tasks through a variety of interactions with other components of the initiation machinery, in many cases mediated by the unstructured regions of the two proteins

A molecular switch created by in vitro recombination of nonhomologous genes

We have created a molecular switch by the in vitro recombination of nonhomologous genes and subjecting the recombined genes to evolutionary pressure. The gene encoding TEM1 β-lactamase was circularly permuted in a random fashion and subsequently randomly inserted into the gene encoding Escherichia coli maltose binding protein. From this library, a switch (RG13) was identified in which its β-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose. Upon removal of maltose, RG13\u27s catalytic activity returned to its premaltose level, illustrating that the switching is reversible. The modularity of RG13 was demonstrated by increasing maltose affinity while preserving switching activity. RG13 gave rise to a novel cellular phenotype, illustrating the potential of molecular switches to rewire the cellular circuitry

Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo

Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2·GTP·Met-tRNA iMet ternary complex (TC) and subsequent Pi release from eIF2·GDP·Pi. It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking P i release at non-AUG codons. We show that Sui? mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui? mutation ISQLG93-97ASQAA (abbreviated 93-97) accelerates eIF1 dissociation and Pi release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui? mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd? mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection

Principles and Properties of Eukaryotic mRNPs

The proper processing, export, localization, translation, and degradation of mRNAs are necessary for regulation of gene expression. These processes are controlled by mRNA-specific regulatory proteins, noncoding RNAs, and core machineries common to most mRNAs. These factors bind the mRNA in large complexes known as messenger ribonucleoprotein particles (mRNPs). Herein, we review the components of mRNPs, how they assemble and rearrange, and how mRNP composition differentially affects mRNA biogenesis, function, and degradation. We also describe how properties of the mRNP interactome lead to emergent principles affecting the control of gene expression