The initiation of translation in eukaryotes commences with assembly of a 43S preinitiation complex (PIC) on the small (40S) ribosomal subunit, containing the ternary complex (TC), comprised of GTP-bound eukaryotic initiation factor two and methionyl initiator tRNA (eIF2·GTP·Met-tRNA_i), and additional eIFs (−1, −1A, and −3). The 43S PIC attaches to the 5’ end of mRNA, in a manner stimulated by the eIF4F complex bound to the m⁷G cap, and scans the mRNA 5’-untranslated region (5’UTR) for an AUG start codon, producing a stable 48S PIC on AUG selection by Met-tRNA_i. The GTP bound to eIF2 can be hydrolyzed during scanning, stimulated by GTPase activating protein eIF5, but eIF1 impedes release of inorganic phosphate (P_i) at non-AUG codons. Start codon recognition triggers dissociation of eIF1 from the 40S subunit, enabling P_i release from eIF2·GDP·P_i and subsequent dissociation of eIF2·GDP and other eIFs from the 48S PIC. The eIF5B-catalyzed joining of the large (60S) subunit produces an 80S initiation complex (IC) ready to begin the elongation phase of protein synthesis (Hinnebusch, 2014; Hinnebusch, 2017).

Several different mRNA features influence the rate of translation initiation. The efficiency of 43S PIC attachment to mRNA and subsequent scanning of the 5’UTR are hindered by stable secondary structures, such that long, structure-laden 5’UTRs are generally inhibitory and impose a requirement for RNA helicases associated with eIF4F, including eIF4A and Ded1, to resolve mRNA structures near the cap, within the 5’UTR, and surrounding the start codon (Sen et al., 2015; Sen et al., 2016; Gupta et al., 2018; Gulay et al., 2020). The nucleotide sequence immediately surrounding the start codon—the ‘Kozak context’—particularly at the −3 and +4 positions (numbered from the A of AUG (+1)) also influences the efficiency of start codon selection, with A nucleotides upstream of the AUG codons enhancing initiation on yeast mRNAs (Shabalina et al., 2004; Zur and Tuller, 2013). Binding of eIF4F to the mRNA cap is stimulated by interaction of its eIF4G subunit with the poly(A)-binding protein (PABP) bound to the poly(A) tail, promoting circularization of the mRNA (Hinnebusch, 2011). Formation of this ‘closed-loop structure’ might facilitate reinitiation by ribosomal subunits released from the stop codon of the mRNA following termination of translation and ribosome recycling (Uchida et al., 2002). There is evidence that closed-loop formation is favored for shorter mRNAs (Amrani et al., 2008), which might help account for the negative correlation between mRNA coding sequence length and translation efficiencies observed in yeast and other eukaryotes (Arava et al., 2003; Thompson et al., 2016). However, theoretical modeling indicates that shorter mRNAs are capable of more efficient recycling of ribosomal subunits from the stop codon to the initiation codon without a mechanism for promoting circularization via end-to-end interactions (Fernandes et al., 2017). The abundance of mRNA is also associated with efficient translation, presumably reflecting the need for mRNAs encoding highly expressed proteins to be both abundant—highly transcribed and stable—and optimized for translation.

Translation initiation can also be modulated by altering the activities of general initiation factors. The function of eIF2 in Met-tRNA_i recruitment is inhibited in response to various stresses by phosphorylation of serine-51 of its α-subunit, as phosphorylated eIF2·GDP acts as a competitive inhibitor of eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2. Inhibition of eIF2B reduces assembly of the TC and thereby decreases formation of 43S PICs, attenuating bulk translation initiation in the cell (Pavitt, 2018). Reducing TC levels also stimulates the translation of certain mRNAs by blunting the inhibitory effects of upstream open-reading-frames (uORFs) present in their 5’UTRs (Hinnebusch, 2005; Hinnebusch et al., 2016). Such uORFs are encountered by scanning 43S PICs first, and their translation generally diminishes the number of PICs that reach the main coding sequences (CDS) downstream owing to recycling of ribosomes following termination at the uORF stop codons. Limited reductions in TC by eIF2α phosphorylation allow uORF start codons to be skipped by scanning PICs with attendant increased translation of the main CDS, by various mechanisms. For yeast GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic genes, translation of the first of four uORFs gives rise to 40S subunits that escape recycling at the uORF1 stop codon and resume scanning downstream. At high TC levels, they quickly rebind TC and reinitiate at uORFs 3 or 4, and are efficiently recycled from the mRNA following uORF translation. When TC levels are reduced by eIF2α phosphorylation, catalyzed by protein kinase Gcn2 on amino acid starvation, a fraction of scanning 40S subunits fails to rebind TC until after bypassing all four uORFs, but then bind TC in time to reinitiate at the main GCN4 CDS instead, thereby inducing GCN4 translation (Hinnebusch, 2005).

Ribosome recycling entails the splitting and release of 80S ribosomes from mRNA at the stop codon following polypeptide chain termination, and occurs in two stages. Dissociation of the 60S subunit from the 80S post-termination complex (post-TerC) occurs first, catalyzed by ABCE1 (Rli1 in yeast), leaving a 40S subunit with the stop codon in the A site and deacylated tRNA in the P site corresponding to the last translated codon of the ORF (Young et al., 2015; Hellen, 2018). The second stage of recycling, dissociation of the tRNA and 40S subunit from the mRNA, is stimulated in vitro by eIF2D, or by the heterodimer MCT-1/DENR, which exhibit homology to separate regions of eIF2D (Skabkin et al., 2010). eIF2D could also stimulate reinitiation of translation by reconstituted mammalian 40S post-TerCs by releasing the deacylated tRNA and making the P site available for recruiting Met-tRNA_i via the TC (Skabkin et al., 2013). Structural analysis shows that human MCT-1/DENR can bind to the 40S decoding center and contact both ends of tRNA_i in a model reinitiation intermediate (Weisser et al., 2017), suggesting their likely positions in a 40S post-TerC. Both DENR and MCT-1 have been shown to promote translation reinitiation downstream of uORFs with strong Kozak consensus sequences in Drosophila and HeLa cells, although the sensitive uORFs in human cells are extremely short, encoding only one amino acid (Schleich et al., 2014; Schleich et al., 2017). Other recent findings implicate DENR in promoting reinitiation following uORF translation on certain mRNAs in mouse cells, including that for the CLOCK gene, although the relevant uORFs were not the single-codon variety (Castelo-Szekely et al., 2019). Interestingly, in reconstituted mammalian systems, eIF2D and MCT-1/DENR were found capable of substituting for eIF2 in GTP-independent recruitment of Met-tRNA_i to the 40S subunit during primary initiation events on certain mRNAs where the AUG codon is placed in the P site without prior scanning, including leaderless mRNAs, mRNAs containing an internal ribosome entry site, or mRNAs with unstructured A-rich 5’UTRs (Dmitriev et al., 2010; Skabkin et al., 2010).

Recently, we showed that yeast mutants lacking the genes for both eIF2D (Tma64) and either MCT-1 (Tma20) or DENR (Tma22) exhibit phenotypes consistent with a broad defect in 40S recycling, including a queuing of translating 80S ribosomes immediately upstream of main CDS stop codons, and increased reinitiation at AUG codons in 3’UTRs. At certain genes, it appeared that unrecycled 40S ribosomes could rejoin with 60S subunits and undergo unconventional 80S reinitiation events in the 3’UTRs (Young et al., 2018), a process observed previously for unrecycled 80S post-TerCs in cells depleted of ABCE1/Rli1 (Young et al., 2015). The ability of Tma64 or Tma20/Tma22 to promote recycling and block reinitiation was also demonstrated in cell extracts with uORF-containing reporter mRNAs (Young et al., 2018). Thus, ostensibly at odds with the findings from mammalian cells where MCT-1/DENR enhance reinititiation following uORF translation, the available evidence suggests that the yeast orthologs of these proteins impede reinitiation by promoting dissociation of 40S post-TerCs from the mRNA. It remains possible, however, that the yeast Tma proteins stimulate, rather than inhibit, reinitiation on particular uORF-containing mRNAs in vivo. It is unknown whether Tma64 or Tma20/Tma22 can substitute for eIF2 in Met-tRNA_i recruitment during primary initiation events on any native mRNAs in yeast cells in the manner described for their mammalian orthologs in vitro (Dmitriev et al., 2010; Skabkin et al., 2010).

Because ribosome recycling is required to provide 40S subunits needed for assembly of 43S PICs to support new initiation events, we wondered whether defective recycling in yeast cells lacking the Tma proteins would reduce the formation of 43S PICs, which in turn, might differentially affect the translation of mRNAs with different inherent rates of initiation. In this view, changes in translation efficiencies (TEs) observed on depletion of the Tma proteins would result from altered competition among mRNAs for limiting PICs rather than loss of mRNA-specific functions of the Tma proteins in either reinitiation or eIF2-independent primary initiation. To explore this possibility, we conducted ribosome profiling on a tma64∆/tma20∆ double mutant, lacking the yeast orthologs of eIF2D and MCT-1, and compared the TEs of all expressed mRNAs in this mutant to their values in isogenic WT cells. The results indicate that eliminating Tma64/Tma20 leads to a reprogramming of translation that generally favors mRNAs that are more efficiently translated in WT cells and disfavors weakly translated mRNAs. Observing the same general reprogramming of TEs in response to eIF2α phosphorylation in WT cells, or to a reduction in WT 40S subunit levels, we propose that competition among mRNAs for limiting 43S PICs is an important driver of changes in TE in cells impaired for 40S recycling.

In this study, we set out to determine whether impairing the second step of ribosome recycling by eliminating Tma64/Tma20, and the predicted sequestration of 40S subunits in either stalled 40S post-TerCs, in 43S PICs scanning 3’UTRs, or in 80S ribosomes translating 3’UTR ORFs, has an impact on global TEs in yeast cells. We also wondered whether eliminating Tma64 would alter the TEs of any mRNAs owing to loss of its possible function as an alternative to eIF2 for recruiting Met-tRNA_i to AUG start codons in the manner demonstrated in vitro for its mammalian counterpart, eIF2D. We found that the tma∆∆ mutant lacking both Tma64 and Tma20 exhibits reduced bulk polysome assembly, consistent with a reduced rate of translation initiation on many mRNAs; and by ribosome profiling we observed altered TEs of specific mRNAs, with sizable groups undergoing increases or decreases in relative TE compared to their values in isogenic WT cells. These groups of upregulated and down-regulated mRNAs were enriched for diametrically opposed properties. The mRNAs translationally upregulated in the tma∆∆ mutant tend to be well-translated in WT cells and to have short CDS and 5’UTRs, strong Kozak AUG contexts, to be unusually abundant and to have longer than average half-lives—all attributes of well-translated ‘strong’ mRNAs. The mRNAs translationally down-regulated in the tma∆∆ mutant tend to be poorly translated in WT cells and to exhibit all of the opposite properties characteristic of ‘weak’ mRNAs. The tma64∆ single mutant, in contrast, displays none of the hallmarks of impaired 40S recycling, shows no reduction in bulk polysome assembly, and exhibits no statistically significant changes in TE for any mRNAs in our profiling experiments. These results suggest that the translation changes in the tma∆∆ mutant are associated with defective ribosome recycling rather than the absence of Tma64/eIF2D per se and any possible role it might play in Met-tRNA_i recruitment in yeast. However, we cannot rule out the possibility that MCT-1/DENR can substitute completely for eIF2D in providing a WT level of eIF2-independent Met-tRNA_i recruitment in the tma64∆ single mutant, and that complete loss of this function in the tma∆∆ mutant contributes to the reprogramming of TEs.

Strikingly, the pattern of TE changes conferred by the tma∆∆ mutations was also evident in the translational reprogramming that accompanies induction of eIF2α phosphorylation by Gcn2 on starvation for amino acids Ile/Val, and also that conferred by depletion of essential 40S subunit protein Rps26. In particular, the group of highly translated ‘SCL’ mRNAs with a heightened propensity for closed-loop formation, owing to their elevated occupancies of eIF4F and PABP, and the group of RPG mRNAs that are strongly enriched in the SCL group (Figure 6—figure supplement 1G), tend to show increased TEs in all three conditions of tma∆∆ mutations, SM treatment of WT cells, and depletion of Rps26. There was also a significant enrichment for ‘weak’ mRNAs among those down-regulated in all three conditions. These same trends were evident on SM treatment of the gcn4∆ mutant, showing that they occur independently of the widespread transcriptional changes conferred by Gcn4 in SM-treated WT cells. Given that eIF2α phosphorylation reduces TC assembly, a key component of 43S PICs, and that both the tma∆∆ mutations and depletion of Rps26 reduce the abundance of free 40S subunits available for PIC assembly, we suggest that the reduced concentration of 43S PICs that should exist in each case is a key factor that skews the competition between strong and weak mRNAs to favor strong mRNAs, and that this altered competition is an important driver of TE changes common to all three conditions. Supporting the idea that the tma∆∆ mutations impair translation initiation by limiting the availability of 43S PICs, they preferentially reduced expression of LUC reporter mRNAs harboring stem-loops inserted close to the mRNA 5’ end, which are expected to impair 43S PIC attachment, compared to reporters bearing cap-distal insertions or the parental reporter lacking a stem-loop insertion.

We note that the tma∆∆ mutant does not exhibit a reduction in the ratio of free 40S to 60S subunits (Figure 1C (ii) versus (i)), ostensibly at odds with our suggestion that the 40S recycling defect in this mutant limits the availability of free 40S subunits for 43S PIC assembly. It is expected that a fraction of the unrecycled 40S subunits will have moved from stop codons and are scanning the 3’UTR to generate the increased 3’UTR translation previously demonstrated in the tma∆∆ mutant (Young et al., 2018) and observed here (Figure 1B). Such 40S post-TerCs, located either at stop codons or scanning in 3’UTRs, may dissociate from mRNA during polysome fractionation by sucrose density gradient centrifugation because they lack base-pairing between a bound tRNA and an mRNA codon—a property which necessitated the use of formaldehyde fixation to capture scanning 40S subunits in 40S profiling experiments (Archer et al., 2016; Sen et al., 2019). If so, this dissociation would prevent us from observing the predicted reduction in free 40S subunits. We have also proposed that sequestration of 40S subunits in the 80S ribosomes engaged in abnormal 3’UTR translation in the tma∆∆ mutant, which occurs on essentially all 3’UTRs (Young et al., 2018), also contributes to the reduced availability of free 40S subunits for PIC assembly in this mutant; and this sequestration also will not reduce the free 40S/60S ratio. Finally, the tma20∆ deletion was shown to confer an elevated free 40S/60S ratio, which may result from a 60S biogenesis defect (Fleischer et al., 2006) and this could offset the depletion of free 40S subunits expected from accumulation of unrecycled 40S subunits at stop codons and in 3’UTRs in the tma∆∆ mutant. Nevertheless, we cannot eliminate the possibility that the impaired ribosome recycling in the tma∆∆ mutant is not the major defect responsible for the reprogramming of translation with features characteristic of a reduction in 43S PIC assembly. As noted above, the complete loss of eIF2-independent Met-tRNA_i recruitment could be involved, or a defect in ribosome biogenesis that results in aberrant 40S subunits in addition to reduced 60S subunit levels might exist, either of which could be exacerbated by defective 40S recycling to confer the reprogramming of TEs observed in the tma∆∆ mutant.

The conclusion that conditions expected to reduce the concentration of 43S PICs favor more efficiently translated mRNAs is in full agreement with Lodish’s predictions based on mathematical modeling of the kinetics of protein synthesis (Lodish, 1974). A key prediction of this model is that reducing the concentration of 43S PICs will alter the TEs of particular mRNAs to different degrees depending on their inherent rates of assembling an 80S initiation complex at the start codon from mRNA and the 43S PIC (assigned an overall rate constant of K₁ in the model). On reducing 43S PIC levels, ‘weaker’ mRNAs (with lower K₁ values) will exhibit a proportionally greater reduction in translation compared to ‘stronger’ mRNAs (with higher K₁ values). Considering the relative TE values provided by genome-wide ribosome profiling, the model predicts that weak mRNAs will exhibit a decrease in TE relative to the average mRNA, while strong mRNAs will show an increase in relative TE on reduction of 43S PIC levels, which corresponds to the trends we observed for TE changes in the tma∆∆ mutant, SM treatment of WT, and depletion of Rps26 (Figure 8). The Lodish model is supported by several studies of global translation; and other analyses suggest that reductions in 40S availability can also confer increases in absolute translation rates of very ‘strong’ mRNAs owing to reduced ribosome crowding and collisions during elongation (Mills and Green, 2017).

Figure 8

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It is formally possible that CDS length is a more fundamental driver of TE changes than the basal TE in WT cells under the three mutant/stress conditions analyzed here, but this would beg the question of the molecular mechanism involved. Shorter mRNAs might be more capable of forming the closed-loop intermediate, which could enhance eIF4F association owing to eIF4G-PABP interaction, or more capable of recycling of ribosomes in cis from the stop codon to start codon; but both features would enhance their basal TE and help them compete for limiting 43S PICs at the initiation step. It is also important to note that all the features known to be associated with high basal TE in WT cells are also associated with increased TE in our three mutants/conditions, including elevated propensity for closed-loop formation, higher than average Kozak context scores, higher than average mRNA abundance, and longer than average mRNA half-lives (Costello et al., 2015; Weinberg et al., 2016). Given the highly conserved inverse relationship between CDS length and TE across many different species (Arava et al., 2003; Thompson et al., 2016), it is likely that our finding that a short CDS is a predictor of enhanced TE in all three conditions we examined reflects the fact that mRNAs with short CDSs tend to be inherently stronger mRNAs and thus better able to compete for limiting PICs during initiation in the manner predicted by the Lodish model.

While the same trends in TE reprogramming were evident in all three mutant/stress conditions, there were also extensive condition-specific differences among the particular mRNAs being affected, and in the magnitude of changes observed for the same mRNAs. Thus, it seems clear that particular mRNAs respond to different extents, or even in different directions, to eIF2α phosphorylation induced by SM treatment, to the tma∆∆ mutations, or to the reduction in total 40S abundance in the rps26∆∆ mutant. We envision that these condition-specific responses are superimposed on the reprogramming of TEs imposed by increased competition for limiting 43S PICs to sculpt the overall profile of TE changes across the translatome in different ways.

This condition-specificity of TE changes is exemplified by translational regulation of GCN4. The TE of GCN4 mRNA was strongly induced only by SM treatment, which likely reflects the exquisite sensitivity to TC levels of the delayed reinitiation mechanism via uORFs that governs GCN4 translation. In this mechanism, a slowed rate of TC binding to 40S subunits scanning the GCN4 mRNA leader following translation of uORF1, and not the availability of free 43S PICs per se, leads to the strong induction of GCN4 translation when TC concentrations decline on eIF2α phosphorylation (Hinnebusch, 2005). It is possible that TE increases observed for certain other mRNAs specifically on SM treatment also result from overcoming the inhibitory effects of uORFs on downstream translation via increased eIF2α phosphorylation. This might include mRNAs with single inhibitory uORFs in addition to the more elaborate set of four regulatory uORFs found in GCN4, as reducing TC levels by eIF2α phosphorylation has been shown to mitigate the inhibitory effects of single uORFs (Young and Wek, 2016). In the well-studied case of antizyme inhibitor mRNA, in which ribosome stalling occurs during translation of an inhibitory uORF, evidence indicates that eIF2α phosphorylation decreases the rate of 43S PIC loading at the mRNA 5’ end and thereby suppresses queuing of PICs upstream of the uORF start codon to allow a greater proportion of scanning ribosomes to bypass the uORF and translate the downstream CDS (Ivanov et al., 2018). In addition to the translation of uORF-containing mRNAs being upregulated by eIF2α phosphorylation via increased reinitiation, it is also possible that primary initiation occurs more efficiently at low TC levels on certain mRNAs (Dasso et al., 1990), possibly related to the RNA-binding activity of eIF2β (Laurino et al., 1999) or to the role of eIF2 in promoting the recruitment of other eIFs to the PIC in the multifactor complex (Hinnebusch, 2011), and that such mRNAs would not be similarly upregulated by the diminished free 40S subunit levels predicted for the tma∆∆ or rps26∆∆ mutations.

One way to account for TE changes conferred specifically by elimination of the Tma factors might be to propose that mRNAs differ in their dependence on the recycling of 40S post-TerCs from the stop codon to the initiation codon of the same mRNA for reinitiation. Indeed, there are theoretical predictions that short mRNAs achieve their relatively higher TEs compared to long mRNAs by this feedback mechanism (Fernandes et al., 2017). In this view, however, shorter mRNAs would be preferentially impaired by a reduction in 40S recycling, whereas we observed that mRNAs down-regulated in the tma∆∆ mutant tend to be longer than average. In fact, if the predicted deleterious effect of reduced 40S recycling from stop codon to start codon on short mRNAs acts to dampen the stimulatory effect of limiting 43S PICs on their translation, then this could help explain why the tma∆∆ mutations confer an overall weaker reprogramming of TEs compared to SM treatment, given that eIF2α phosphorylation reduces 43S PICs without affecting recycling.

We also considered the possibility that TE changes conferred specifically by elimination of the Tma factors could arise from altered reinitiation following uORF translation, as mammalian MCT-1/DENR were shown to stimulate reinitiation in a reconstituted system, and depletion of these factors in animal cells reduces reinitiation on certain mRNAs. In yeast, by contrast, elimination of these proteins in combination with eIF2D (Tma64) had the opposite effect of enhancing reinitiation following termination at uORFs of reporter mRNAs in cell extracts (Young et al., 2018). However, based on our analysis of GCN4 translational control, and from quantifying the ratios of ribosome densities in uORFs versus downstream CDSs (RRO values), we uncovered no evidence that eliminating the Tma proteins significantly perturbs reinitiation following uORF translation in yeast cells. Additional experiments using other indicators of reinitiation should be undertaken to examine this issue further. Recently, it was shown that elimination of DENR (Tma22) and eIF2D (Tma64) impairs translation of ATF4 mRNA both in human cells (Bohlen et al., 2020) and in Drosophila (Vasudevan et al., 2020), which is regulated by the delayed reinitiation mechanism established for yeast GCN4 mRNA. It appears that loss of DENR/eIF2D reduces reinitiation following translation of ATF4 uORFs 1-2 owing to a failure to dissociate tRNAs from 40S post-TCs at the penultimate codons of these uORFs (Bohlen et al., 2020). Presumably, the penultimate codons in GCN4 uORFs 1-2 do not require this activity of the yeast recycling factors.

There were many mRNAs whose TEs were affected specifically by depletion of Rps26 and 40S subunit levels. One possible contribution to these specific changes might be the elevated ratio of free 60S to free 40S subunits present only in this condition. This could have the effect of increasing rates of 60S subunit joining to 48S PICs at start codons by mass action, and thereby increase the relative TEs of a subset of mRNAs in which subunit joining is a rate-limiting step of initiation. Some support for this idea comes from our findings here, and from previous results (Mittal et al., 2017), that depleting 40S subunits can impair GCN4 translation, which might result from enhanced subunit joining at the inhibitory uORFs that, in turn, reduces the amount of reinitiation downstream at the GCN4 CDS. Another possibility is that the aberrant-free ribosomal subunit ratios activate a signaling pathway that alters the translation of particular mRNAs in a manner distinct from the influence of limiting 43S PICs. Clearly, more work is required to achieve a full understanding of the numerous condition-specific TE changes.

Our finding that depletion of Rps26 increases the relative TEs of the group of highly-translated SCL mRNAs was also observed in profiling data from Gilbert et al. (Thompson et al., 2016) for two mutants lacking a different ribosomal protein belonging to the large or small subunit, in accordance with the Lodish model. However, findings ostensibly at odds with the model were presented recently (Ferretti et al., 2017) involving two groups of yeast mRNAs found to bind preferentially to 40S subunits either containing Rps26 (868 "+Rps26" mRNAs) or lacking Rps26 (743 "ΔRps26" mRNAs). The "+Rps26" mRNAs tend to be more highly translated in WT cells versus the "ΔRps26" mRNAs, and thus are enriched in ‘strong’ mRNAs; however, five representative mRNAs of the "+Rps26" group showed a greater reduction in translation on Rps26 depletion in comparison to eight representative mRNAs of the "ΔRps26" group. This last finding departs from the prediction of the Lodish model that strong mRNAs should be translated proportionally better than weak mRNAs on reduction of 40S subunit levels by depletion of Rps26. However, by interrogating our own ΔTE_rps26∆∆ values for the "+Rps26" and "ΔRps26" groups of mRNAs, we found an increase in median TE for the "+Rps26" group, and a decrease in median TE for the "ΔRps26" group, (Figure 5—figure supplement 4), in keeping with the Lodish model. It is unclear whether the different conclusions regarding the effects of Rps26 depletion on translation of the "+Rps26" and "ΔRps26" mRNAs in our two studies reflect differences in the strains or culture conditions (undefined rich medium there versus defined medium here), the use of polysome association (there) versus ribosome density (here) to assess TE changes, or the particular subsets of representative "+Rps26" and "ΔRps26" mRNAs interrogated for TE changes by Ferretti et al., 2017.

A recent study by Khajuria et al., 2018 on the consequences of reducing the levels of ribosomal subunits in human hematopoietic stem and progenitor cells (HSPCs), regarded as a model of Diamond-Blackfan anemia (DBA), also revealed a widespread reprogramming of translation quite different than observed here in the yeast rps26∆∆ mutant. The mRNAs showing TE reductions in that study tended to be highly translated in untreated HSPCs, and to exhibit short CDSs and short 5’UTRs lacking uORFs, which seems at odds with the predictions of the Lodish model. Although we cannot account for this discrepancy, we note that the global TE values in the untreated HSPCs did not show the marked inverse correlation with CDS lengths that has been observed previously in yeast (including our study) (Arava et al., 2003; Thompson et al., 2016), and highly conserved throughout Eukarya (Fernandes et al., 2017), including mouse and HeLa cells (Thompson et al., 2016). We also note that examining the profiling data of Khajuria et al. indicates that the TEs of RPG mRNAs in untreated HSPCs are well below average, but were found to be above average in a study of HeLa cells (Guo et al., 2010; Guo et al., 2015), just as they are in yeast. The lower than average TEs of RPG mRNAs in HSPC cells places the reductions in their TEs conferred by reduced ribosome levels in line with the Lodish model; however, the fact that the entire cohort of translationally down-regulated mRNAs have a greater than average median TE in untreated HSPCs seems to suggest that other changes in translation factors or regulatory proteins occur in response to ribosome depletion in HSPCs that override the predicted broad outcomes of the Lodish model. As noted above, other factors also appear to modulate the response to limiting 43S PICs in our rps26∆∆ mutant to produce extensive condition-specific TE changes.

At odds with our results on the rps26ΔΔ mutant, a study examining the effects of deleting many different RPGs in yeast reached the conclusion that genes for which translation was consistently lower in ribosome-deficient yeast cells had moderately shorter than average CDS lengths, whereas genes for which translation was consistently higher had dramatically longer CDS lengths (Cheng et al., 2019). However, using the Ribo-seq and RNA-seq data from this study to calculate TE changes, we observed no clear relationship between TE changes and CDS lengths conferred by the two 40S RPG deletions with the strongest reductions in free 40S levels (rps29b∆ and rps0b∆) comparable in degree to the depletion of 40S subunits observed in our rps26∆∆ mutant. Compared to the two WT strains analyzed in that study, the rps29b∆ and rps0b∆ mutants do not exhibit reduced relative TEs for the 20% of all mRNAs with the shortest CDS lengths (Figure 5—figure supplement 5A, 1st pentile); nor for the group of RPG mRNAs, which have very short CDS lengths and high basal TE values in WT cells (Figure 5—figure supplement 5B, RPG mRNAs). Whereas rps0b∆ confers a small increase in relative TE for the pentile of mRNAs with longest CDS lengths (Figure 5—figure supplement 5A, 5th pentile), this mutation conferred a comparable increase for all mRNAs (Figure 5—figure supplement 5B, all mRNAs). Thus, although these results are incongruent with our findings on the rps26∆∆ mutant, they also differ from the findings of Khajuria et al., 2018 on the effects of depleting ribosomal subunits in human HSPCs.

In summary, our findings suggest that reducing 43S PICs in yeast either by eliminating the 40S ribosome recycling factors, inducing eIF2α phosphorylation, or depleting a 40S subunit protein, all contribute to a similar realignment of TEs wherein inherently strong mRNAs tend to undergo an increase in relative TE at the expense of weak mRNAs. As these trends are predicted by the Lodish model, which should apply generally to all eukaryotic cells, they might be expected to occur in the context of human mutations that reduce the concentration of ribosomal subunits (ribosomopathies) or that reduce the abundance or function of general initiation factors. As pointed out previously (Mills and Green, 2017), it may be possible therefore to produce broad changes in the TEs of many cellular mRNAs without the presence of specific regulatory sequences in the sensitive mRNAs by lowering the abundance of the initiation machinery and intensifying the competition between strong and weak mRNAs. It should be kept in mind, however, that other regulatory mechanisms present in mammalian cells but lacking in yeast could alter the outcome from the expectations of the Lodish model for TE changes in ribosomopathies, such as mTORC-mediated control of RPG mRNA translation via 5’-terminal oligopyrimidine (5’TOP) sequences (Meyuhas and Kahan, 2015). It is also important to realize that an increase in relative TE does not necessarily result in higher translational output, as it may coincide with a reduction in absolute TE in a situation where the median translation rate is dramatically lowered; or if it is buffered by a reduction in mRNA abundance, as observed here for the RPG mRNAs in amino acid starved yeast cells. If the function of a critical gene product is highly dose-sensitive, then a small decrease in absolute translational output could be pathological even if the relative TE of its mRNA is elevated in a disease state that involves attenuated 43S PIC assembly.

Sequencing data from this study have been deposited to the NCBI Gene Expression Omnibus (GEO) under GEO accession number GSE166987.

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Author details

1. Swati Gaikwad

   Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review and editing

   Contributed equally with

   Fardin Ghobakhlou

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1438-9497

2. Fardin Ghobakhlou

   Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Present address

   Department of Microbiology, Infectiology & Immunology, Faculty of Medicine, University of Montreal, Montreal, Canada

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Contributed equally with

   Swati Gaikwad

   Competing interests

   No competing interests declared

3. David J Young

   1. Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States
   2. Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Jyothsna Visweswaraiah

   Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Present address

   Pandion Therapeutics, Watertown, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   Jyosthna Visweswaraiah is affiliated with Pandion Therapeutics; however, the author has no financial interests to declare.

5. Hongen Zhang

   Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Software, Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6871-8463

6. Alan G Hinnebusch

   Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   ahinnebusch@nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1627-8395

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