20 research outputs found
Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly
Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs
Characterization of the two smallest core subunits of eIF3 and their roles in translation.
Protein synthesis or mRNA translation is a complex and highly conserved process. Translation consists of initiation, elongation, termination, and ribosome recycling stages. Since most regulation occurs during initiation, its mechanism is being studied intensively to elucidate the molecular basis of every potential control point. The initiation factor eIF3, which in yeast consists of five essential core subunits (eIF3a/TIF32, b/PRT1, c/NIP1, g/TIF35, and i/TIF34) and one transiently associated, non-essential subunit (j/HCR1), is undisputedly one of the key promoters of initiation. In addition, it has also been implicated in playing a critical role during ribosomal recycling, reinitiation, signal transduction, NMD etc. We have focused on determining the molecular mechanism of the roles of eIF3 and its associated eIFs not only in translation initiation but also in termination and in reinitiation. This included the biochemical and genetic mapping of yeast eIF3 binding site on the small ribosomal subunit, among others. We showed that the interaction between the residues 200-400 of a/TIF32-NTD and flexible C-terminal tail RPS0A significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo, thus a/TIF32-NTD together with the recently published PCI (proteasome..
Charakterizace dvou nejmensich podjednotek eIF3 a jejich úloh v translaci.
Protein synthesis or mRNA translation is a complex and highly conserved process. Translation consists of initiation, elongation, termination, and ribosome recycling stages. Since most regulation occurs during initiation, its mechanism is being studied intensively to elucidate the molecular basis of every potential control point. The initiation factor eIF3, which in yeast consists of five essential core subunits (eIF3a/TIF32, b/PRT1, c/NIP1, g/TIF35, and i/TIF34) and one transiently associated, non-essential subunit (j/HCR1), is undisputedly one of the key promoters of initiation. In addition, it has also been implicated in playing a critical role during ribosomal recycling, reinitiation, signal transduction, NMD etc. We have focused on determining the molecular mechanism of the roles of eIF3 and its associated eIFs not only in translation initiation but also in termination and in reinitiation. This included the biochemical and genetic mapping of yeast eIF3 binding site on the small ribosomal subunit, among others. We showed that the interaction between the residues 200-400 of a/TIF32-NTD and flexible C-terminal tail RPS0A significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo, thus a/TIF32-NTD together with the recently published PCI (proteasome..
Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit
The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo
eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA
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
Deletion of <i>hcr1</i> results in accumulation of eRF3 in heavy polysomes.
<p>(<b>A–C</b>) The <i>hcr1Δ</i> strain (H3675) was transformed with either hc <i>HCR1</i> (<b>A</b>), empty vector (<b>B</b>), or hc <i>RLI1</i> (<b>C</b>), and the resulting transformants were grown in SD medium at 30°C to an OD<sub>600</sub> of ∼1 and cross-linked with 0.5% HCHO prior to harvesting. WCEs were prepared, separated on a 5%–45% sucrose gradient by centrifugation at 39,000 rpm for 2.5 h and subjected to Western blot analysis. Several fractions corresponding to the Top, 40S, 60S, and 80S plus polysomal species were pooled, as indicated. Asterisk indicates a non-specific band. (<b>D</b>) Statistical significance of the eRF3 accumulation in heavy polysomes in the <i>hcr1</i> strain and its partial recovery by hc <i>RLI1</i>. Amounts of each individual factor in all fractions were quantified by fluorescence imaging. Thus obtained values for the fractions containing heavy polysomes (14–18) as well as all remaining fractions (1–13) were added up for each of these two groups. Values (mean±SE; n = 4) given in the table then represent relative amounts of factors in heavy polysomes divided by the compound value of the rest of the gradient. Changes in the redistribution of factors between the heavy polysomes and lighter fractions in all three strain were analyzed by the student's <i>t</i>-test and shown to be statistically significant only for eRF3 as shown in the table. (<b>E</b>) Statistical significance of the eIF3 shift from 40S-containing fractions to the top, which is independent of the effect of hc <i>RLI1</i> on eRF3. Essential the same as in panel D, except that the values for the Top fractions (1–4) as well as the 40S fractions (5–6) were added up for each of these two groups. Values (mean±SE; n = 4) given in the table then represent relative amounts of factors in the Top divided by the 40S group. Changes in the redistribution of factors between the 40S and Top fractions in <i>hcr1Δ</i>+EV or +hc <i>RLI1</i> strains vs. wt were analyzed by the student's <i>t</i>-test and shown to be statistically significant only for eIF3 as shown in the table.</p
Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells
Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1? as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation
<i>hcr1Δ</i> and eIF3 mutants genetically interact with release factor mutants.
<p>(<b>A–B</b>) The <i>sup45<sup>Y410S</sup></i> mutation eliminates the negative impact of <i>hcr1Δ</i> on (<b>A</b>) read-through and (<b>B</b>) growth rates. The <i>hcr1Δ</i> strain was crossed with the <i>sup45<sup>Y410S</sup></i> mutant strain and the resulting double mutant was transformed with sc <i>SUP45</i>, hc <i>HCR1</i>, or empty vector (EV), respectively, and (<b>A</b>) processed for stop codon read-through as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003962#pgen-1003962-g001" target="_blank">Figure 1</a> (<i>hcr1Δ</i> read-through values were set to 100%) or (<b>B</b>) subjected to a growth spot assay at indicated temperatures for 2 or 3 days. (<b>C–D</b>) Combining the selected <i>TIF32</i> mutants with <i>sup35<sup>N536T</sup></i> and <i>sup45<sup>Y410S</sup></i> (<b>C</b>) reduces their read-through defects and (<b>D</b>) produces synthetic growth phenotypes. The wt and mutant alleles of <i>TIF32</i> were introduced into <i>tif32Δ</i>, <i>sup35<sup>N536T</sup> tif32Δ</i>, and <i>sup45<sup>Y410S</sup> tif32Δ</i> strains, respectively, by plasmid shuffling. (<b>C</b>) The resulting double mutant strains were grown in SD and processed for the stop codon read-through as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003962#pgen-1003962-g001" target="_blank">Figure 1</a> (the read-through values of both single eRF mutants were set to 100%), or (<b>D</b>) spotted in four serial 10-fold dilutions on SD medium and incubated at indicated temperatures for 4 days. ND; not determined due to severe growth deficiency.</p
Increased gene dosage of ABCE/RLI1 suppresses the slow growth and read-through defects of <i>hcr1Δ</i>.
<p>(<b>A</b>) The <i>hcr1Δ</i> strain was transformed with either empty vector (EV), hc <i>HCR1</i> or hc <i>RLI1</i>. The resulting transformants were subjected to a growth spot assay at 30°C for 2 days. (<b>B</b>) The <i>hcr1Δ</i> strain was transformed with hc vectors carrying either wt or mutant <i>HCR1</i> and <i>RLI1</i> alleles, and <i>SUI1</i> (eIF1) and <i>TIF11</i> (eIF1A). The resulting transformants were grown in SD and analyzed for stop codon read-through as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003962#pgen-1003962-g001" target="_blank">Figure 1</a>. Thus obtained values were normalized to the value obtained with the <i>hcr1Δ</i> strain transformed with wt <i>HCR1</i>, which was set to 100%.</p