Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2α/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Δlsm4ΔC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2α became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2α. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

SUMMARY Integrated Stress Response is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism resulting in partial but not complete translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits “foamy cell” development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met-tRNA i Met ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui ؊ phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui ؊ phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui ؊ phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.Translation initiation is a multistep process culminating in formation of the 80S initiation complex containing methionyl initiator tRNA (Met-tRNA i Met ) base paired with the AUG start codon in the P site of the ribosome. A large number of soluble eukaryotic translation initiation factors (eIFs) have been identified that stimulate the partial reactions of this process (reviewed in reference 12 and 13). A critical step early in the pathway is the binding of Met-tRNA iMet to the 40S ribosomal subunit in a ternary complex (TC) comprised of MettRNA i Met , GTP, and eIF2. The recruitment of TC to 40S subunits is promoted in vitro by eIF1, eIF1A, and the eIF3 complex. The 43S preinitiation complex thus formed interacts with mRNA in a manner stimulated by eIF4F (eIF4A-eIF4E-eIF4G), poly(A)-binding protein, and eIF3, and the 43S complex scans the mRNA until the Met-tRNA i Met base pairs with an AUG triplet. AUG recognition triggers GTP hydrolysis by eIF2 in a reaction stimulated by eIF5, and the eIF2-GDP and other eIFs are ejected from the ribosome. The eIF1, eIF1A, and eIF4G have been implicated in the scanning process in vitro (23, 24). In the final reaction, eIF5B bound to GTP promotes joining of the 60S subunit with the 40S-MettRNA i Met -mRNA complex to produce the 80S initiation complex (15,25). To begin a new round of initiation, the ejected eIF2-GDP complex must be recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B (13).From extensive biochemical analysis of the mammalian initiation factors, it was proposed that eIF3 binds to the 40S ribosome independently of other factors and promotes the recruitment of TC and mRNA in a manner stimulated by eIF1 ...

Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5 of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5 enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.[Keywords: Translation initiation; reinitiation; eIF3; 40S ribosomal subunit; GCN4; short uORF] Supplemental material is available at http://www.genesdev.org.

Translation initiation factor 3 (eIF3) of Saccharomyces cerevisiae forms a multifactor complex (MFC) with eIFs 1, 2, 5 and Met-tRNA i Met . We previously constructed a subunit interaction model for the MFC. Here we incorporated af®nity tags into the three largest eIF3 subunits (eIF3a/TIF32, eIF3b/PRT1 and eIF3c/NIP1) and deleted predicted binding domains in each tagged protein. By characterizing the mutant subcomplexes, we con®rmed all key predictions of our model and uncovered new interactions of NIP1 with PRT1 and of TIF32 with eIF1. In addition to the contact between eIF2 and the N-terminal domain (NTD) of NIP1 bridged by eIF5, the C-terminal domain (CTD) of TIF32 binds eIF2 directly and is required for eIF2±eIF3 association in vivo. Overexpressing a CTD-less form of TIF32 exacerbated the initiation defect of an eIF5 mutation that weakens the NIP1±eIF5±eIF2 connection. Thus, the two independent eIF2±eIF3 contacts have additive effects on translation in vivo. Overexpressing the NIP1-NTD sequestered eIF1±eIF5±eIF2 in a defective subcomplex that derepressed GCN4 translation, providing the ®rst in vivo evidence that association with eIF3 promotes binding of eIF2 and Met-tRNA i Met to 40S ribosomes. Keywords: eukaryotic translation initiation factor/ multifactor complex/protein synthesis/ternary complex/ translational control IntroductionAssembly of the 80S translation initiation complex is a multiple step process involving a large number of soluble eukaryotic initiation factors (eIFs). According to current models, a ternary complex (TC) comprised of eIF2, GTP and Met-tRNA i Met binds to the 40S ribosome with the help of eIFs 1, 1A and 3. The 43S pre-initiation complex thus formed interacts with mRNA in a manner stimulated by eIF4F, poly(A)-binding protein and eIF3, and the resulting 48S complex scans the mRNA until the Met-tRNA i Met base-pairs with the AUG start codon. On AUG recognition, the eIF5 stimulates GTP hydrolysis by eIF2, the eIFs are ejected and the 60S subunit ®nally joins with the 40S±Met-tRNA i Met ±mRNA complex. For a new round of initiation, the ejected eIF2±GDP complex must be recycled to eIF2±GTP by the exchange factor eIF2B (reviewed in Hershey and Merrick, 2000;Hinnebusch, 2000).The eIF3 is intriguing because it can bind directly to the 40S ribosome and stimulates the binding of both TC and mRNA to 40S subunits in vitro (Hershey and Merrick, 2000;Hinnebusch, 2000). While mammalian eIF3 contains 11 different subunits, the yeast factor has only ®ve core subunits (eIF3a/TIF32, eIF3b/PRT1, eIF3c/NIP1, eIF3i/TIF34 and eIF3g/TIF35) and one substoichiometric component (eIF3j/HCR1). The ®ve-subunit complex puri®ed from yeast can restore binding of Met-tRNA i Met (Danaie et al., 1995;Phan et al., 1998) and mRNA to 40S ribosomes in heat-inactivated prt1-1 (eIF3b) mutant extracts. Thus, yeast eIF3 possesses two critical functions ascribed to the more complex mammalian factor. The role of mammalian eIF3 in promoting mRNA binding to the ribosome often is attributed to its interaction with the largest sub...

. These results suggest that eIF3 binds to the solvent side of the 40S subunit in a way that provides access to the interface side for the two eIF3 segments (NIP1-NTD and TIF32-CTD) that interact with eIF1, eIF5, and the eIF2/GTP/Met-tRNA i Met ternary complex.[Keywords: Eukaryotic translation initiation factor (eIF); multifactor complex (MFC); translational control; protein synthesis; 40S ribosome binding; TIF32/NIP1] Supplemental material is available at http://www.genesdev.org.

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.

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5′ and 3′ cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5′ cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5′ sequences making up the 5′ enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5′ enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5′ enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5′ enhancer's stimulatory activity is strictly dependent on and thus follows the 3′ enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5′ leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.