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Commun Biol
Commun Biol
Communications Biology
2399-3642
Nature Publishing Group UK London

39232119
6761
10.1038/s42003-024-06761-x
Article
Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling
http://orcid.org/0009-0006-2635-6867
Jendruchová Kristína 12
http://orcid.org/0000-0002-1438-9497
Gaikwad Swati 3
Poncová Kristýna 1
Gunišová Stanislava 1
http://orcid.org/0000-0001-8123-8667
Valášek Leoš Shivaya valasekl@biomed.cas.cz

1
http://orcid.org/0000-0002-1627-8395
Hinnebusch Alan G. alanh@mail.nih.gov

3
1 https://ror.org/02p1jz666 grid.418800.5 0000 0004 0555 4846 Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague, Czech Republic
2 https://ror.org/024d6js02 grid.4491.8 0000 0004 1937 116X Faculty of Science, Charles University, Albertov 6, 128 00 Prague, Czech Republic
3 grid.94365.3d 0000 0001 2297 5165 Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 USA
4 9 2024
4 9 2024
2024
7 10836 3 2024
21 8 2024
© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

A study of ribosome recycling in the budding yeast shows that 40S subunit recycling factors influence translation reinitiation independently of their function in bulk recycling, which distinguishes them from their mammalian homologues

Subject terms

Gene regulation
tRNAs
https://doi.org/10.13039/100000009 Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.) issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Synthesis of proteins is carried out by ribosomes, molecular machines that assemble from the small (40S) and large (60S) ribosomal subunits. During translation initiation, the 40S subunit acquires the ternary complex (TC) composed of the methionyl initiator Met-tRNAiMet, GTP and the eukaryotic Initiation Factor (eIF) 2, forming the 43S pre-initiation complex (PIC). Upon mRNA recruitment, the 48S PIC thus formed scans the mRNA 5’ leader region to locate the authentic initiation codon, generally an AUG triplet in a favorable “Kozak” sequence context. Subsequently, subunit joining occurs to produce the 80S initiation complex and elongation commences. Upon stop codon recognition, translation terminates, and the nascent peptide is released. This is followed by ribosomal recycling, i.e., splitting of both ribosomal subunits and dissociation of the 40S and the deacylated tRNA that decoded the last sense codon from mRNA, to start the new translational cycle (reviewed in refs. 1–4). In some cases, the recycling step is incomplete, leaving the post-termination 40S mRNA-bound, which allows it to resume traversing downstream and, upon reacquisition of the TC, reinitiate translation of a downstream ORF (reviewed in ref. 5).

Translation termination and ribosome recycling are intrinsically linked. When the stop codon enters the ribosomal A site, it is recognized by a heterodimer composed of eukaryotic Release Factors 1 and 3 (eRF1/eRF3). Stop codon recognition results in GTP hydrolysis on eRF3 followed by its release. eRF1 then catalyzes hydrolysis of the nascent polypeptide chain, which generates the post-termination 80S ribosome harboring deacylated tRNA base-paired with the penultimate codon in the P site6. The ATP-binding cassette protein Rli1/ABCE1 (yeast/mammals) facilitates the dissociation of the 60S subunit both in vitro in yeast and mammalian reconstituted systems7,8 and in vivo in yeast cells9, aided by the eIF3-associated factor eIF3j10,11. Studies in vitro revealed that after subunit splitting, the deacylated tRNA and mRNA are dissociated from the 40S post-termination complex by the non-canonical initiation factor eIF2D or its functionally and structurally related heterodimer MCTS1/DENR12,13. DENR and MCTS1 bear sequence similarity to the N-terminal and C-terminal regions of eIF2D, respectively. The MCTS1 and the eIF2D N-terminal region harbor DUF1974 and PUA domains, whereas DENR and eIF2D’s C-terminal region contain SWIB/MDM2 and SUI/eIF1 domains. eIF2D additionally contains a central winged-helix domain4.

Ribosome profiling studies demonstrated that the yeast orthologs of eIF2D, MCTS1 and DENR, known as Tma64, Tma20 and Tma22, respectively14, function in 40S post-termination complex recycling in vivo15,16. Thus, deleting the corresponding genes led to accumulation of unrecycled 40S subunits at the majority of stop codons, with the largest defect observed in ∆tma20∆tma64 (tma∆∆) cells. Quantifying the accumulation of unrecycled 40S subunits at all individual stop codons in tma∆∆ vs. wild-type (WT) cells revealed that certain penultimate codons result in significantly higher accumulation of unrecycled 40S subunits in the mutant cells—a phenomenon dubbed Tma-dependence. Furthermore, comparison of the double deletant to single deletant strains showed that Tma64 is largely dispensable, whereas both subunits of the Tma20/Tma22 heterodimer are required for the majority of 40S recycling events in vivo. A notable consequence of defective 40S recycling in mutants lacking an intact Tma20/Tma22 heterodimer is increased REI downstream of stop codons of main ORFs within the 3’ untranslated regions (3’ UTRs) of mRNAs. Such non-canonical REI frequently occurs by conventional scanning of the unrecycled post-termination 40S complexes to 3’ UTR-situated AUG codons, preferentially in optimum Kozak context15,16. Absence of the Tma20/Tma22 heterodimer also conferred increased canonical REI downstream of the stop codons of upstream open reading frames (uORFs) in the 5’ UTRs of reporter mRNAs in yeast cell-free translation extracts15.

Whereas yeast Tma20/Tma22 were shown to inhibit REI, work on their mammalian orthologs indicated that human DENR conversely promotes REI after translation of certain uORFs, including 1aa-long uORFs comprised of only a start and stop codon, referred to here as “start-stops”, with their ATG start codons in optimum Kozak context17,18. In the absence of DENR/MCTS1, mRNAs containing start-stops exhibit reduced translation of the downstream main ORF. DENR-dependence for REI was also established for certain other short uORFs in a manner generally dictated by the presence of particular penultimate codons that, in 40S ribosome profiling data, were found to confer heightened dependence on DENR for bulk 40S recycling at stop codons throughout the translatome13. Indeed, the degree of DENR-dependence often inversely correlates with the propensity of the deacylated tRNA decoding the penultimate codon to dissociate spontaneously from post-termination 40S complexes in reconstituted termination/recycling systems13,19. Accordingly, it was proposed that only those uORFs harboring penultimate codons with low rates of spontaneous dissociation of the cognate tRNAs require DENR/MCTS1 to accelerate release of the deacylated tRNA and allow the post-termination 40S to resume traversing downstream and reacquire the TC at the now-empty P site, enabling REI. The DENR-requirement for REI included the regulatory short uORF that stimulates REI on ATF4 mRNA (uORF1), allowing scanning ribosomes to bypass a more distally positioned uORF (uORF2) that inhibits initiation at the main CDS, both in mammalian cells13 and fruit flies20.

To reconcile the apparent discrepancy between yeast Tma factors and their mammalian counterparts, it was suggested13 that release of the deacylated tRNA by MCTS1/DENR does not lead to dissociation of post-termination 40S subunits at relatively short uORFs in mammalian cells owing to retention of eIF3, eIF4G1, or eIF4E by 80S ribosomes translating the uORFs and continued occupancy of these factors on the post-termination 40S subunits21–23. These retained eIFs presumably impede the 40S dissociation function of MCTS1/DENR, making the only observable consequence of depleting DENR a reduction in REI at the subset of uORFs requiring DENR for efficient release of the deacylated tRNA, whose continued presence in the P site in the absence of DENR then prevents the 40S post-termination complexes from traversing downstream or recruiting TC to the still-occupied P site. Depleting DENR has no effect on REI at the short uORFs where deacylated tRNA dissociates independently of MCTS1/DENR. These alternative outcomes are depicted schematically in Fig. 1A(i)-(ii). At typical uORFs in yeast, by contrast, release of the deacylated tRNA, whether or not it is highly dependent on the Tma proteins, would lead to subsequent dissociation of post-termination 40S subunits from mRNA and low-level REI following most uORFs in WT cells, because eIFs are generally not retained by 80S ribosomes translating yeast uORFs24–27. As such, a further reduction in REI in cells lacking Tma factors is not expected at typical yeast uORFs even when they harbor penultimate codons highly dependent on the Tma factors to stimulate release of deacylated tRNAs from the post-termination 40S subunits. These expected outcomes for typical uORFs in yeast are shown in Fig. 1B(i)-(ii).Fig. 1 Predicted mechanisms governing MCTS1/DENR mediated recycling and REI at mammalian short uORFs and expected functions of yeast Tma20/Tma22 at typical REI-non-permissive yeast uORFs.

MCTS1/DENR or Tma20/Tma22 are depicted as linked blue ovals whose distinct functions in releasing the penultimate deacylated tRNA (red “L”) and subsequently dissociating the empty 40S from the mRNA are labeled with boxes 1 and 2, respectively, and solid or dashed arrows. A DENR-mediated recycling at short mammalian uORFs depending on the presence of (i) DENR-hyperdependent penultimate codons requiring the recycling factors for efficient tRNA release, or (ii) DENR-hypodependent codons where tRNA can be released at high levels spontaneously. (i) DENR-hyperdependent uORFs: in WT cells, “(+) DENR”, MCTS1/DENR efficiently releases the penultimate-codon tRNA (function 1) but its second function in 40S dissociation is impeded by eIF3 (green oval) retained on post-termination 40S subunits to enable high-level REI at the main CDS. In cells depleted of DENR, “(−) DENR”, diminished release of the penultimate-codon tRNA lowers REI. (ii) DENR-hypodependent uORFs: efficient release of the tRNA and high-level REI occur both in the presence (upper) or absence (lower) of DENR, yielding no decrease in REI on DENR depletion. B Tma-mediated recycling at typical yeast short uORFs depending on the presence of (i) Tma-hyperdependent or (ii) Tma-hypodependent penultimate codons. (i) Tma-hyperdependent uORFs: in WT cells (upper), Tma factors efficiently release the penultimate-codon tRNA (function 1) but also dissociate the empty 40S subunit from mRNA (function 2), conferring low-level REI. In tma∆∆ cells (lower), the absence of Tma-mediated tRNA release helps to ensure low-level REI, yielding no change in REI in tma∆∆ vs. WT cells. (ii) Tma-hypodependent uORFs: Tma-independent tRNA release occurs efficiently in both WT and tma∆∆ cells, but REI is low in both cases because, lacking eIF3, the empty 40S subunits dissociate from the mRNA independently of Tma factors, for no change in REI in tma∆∆ vs. WT cells.

A different outcome would be predicted for a handful of atypical uORFs in yeast, where 40S dissociation is counteracted by initiation factors that remain associated with post-termination 40S complexes. The 5’-proximal AUG-initiated uORF in GCN4 mRNA, uORF1, is the best characterized such uORF in yeast. It is optimized for eIF3 and eIF4G binding and retention of post-termination 40S subunits by the presence of cis-acting REI promoting elements (RPEs) rendering it highly permissive for REI downstream (Fig. 2)5,27,28. Accordingly, the 40S post-termination complexes at the uORF1 stop codon should not spontaneously dissociate from mRNA on release of the deacylated tRNA, as described above for short uORFs in mammalian cells (Fig. 1A). This in turn should enable us to determine whether eliminating the Tma proteins confers reduced REI at uORF1 variants equipped with penultimate triplets with heightened dependence on Tma factors for release of deacylated tRNA in bulk 40S recycling. At the same time, we should observe little or no effect at uORF1 variants containing penultimate codons that are less dependent on the Tma proteins in bulk 40S recycling, in the manner depicted in Fig. 1A(i)-(ii) for DENR-depletion in mammalian cells.Fig. 2 Summary of all cis-determinants that either promote or inhibit REI on GCN4 mRNA after translation of its four short uORFs.

Schematics of the 5′ enhancers of uORF1 and 2 containing their respective RPEs, some of which functionally interact with eIF3 to promote resumption of scanning. Green color-coding generally indicates stimulatory effects of the corresponding cis-factors on efficiency of REI, whereas red color-coding indicates inhibitory effects (with the exception of RPE ii. of uORF1, which is also stimulatory); the number of asterisks below the inhibitory elements of the uORF2 and uORF3 3′ sequences depicts the degree of their inhibition as determined experimentally. Mutations converting uORFs 1, 3, or 4 to Start-stop elements uSt-st1, uSt-st3, or uSt-st4, described later in RESULTS, are given below the respective WT uORF schematics. Reprinted and modified with permission from ref. 33.

Importantly, GCN4 uORF1 is functionally equivalent to uORF1 in ATF4 mRNA, and GCN4 and ATF4 translation are governed by very similar REI mechanisms5. However, whereas ATF4 uORF1 is DENR-dependent for efficient REI13,20, we determined previously that translational control of WT GCN4 mRNA occurs normally in the tma20∆tma64∆ mutant29. This finding implies that the Tma20/Tma22 heterodimer is not required to dissociate deacylated tRNACys from the native penultimate UGC codon of WT GCN4 uORF1. Consistently, UGC was judged to be hypodependent on DENR in mammalian cells13 and one of the least Tma-dependent codons for bulk 40S recycling in yeast16. There is also evidence that the cognate tRNACys is weakly associated with mammalian 40S post-termination complexes19. Here, using uORF1 as a genetic tool, we examined whether substituting the native UGC codon in GCN4 uORF1 with different codons judged to be Tma-hyperdependent for bulk 40S recycling will reduce REI in tma∆∆ cells in the manner observed for DENR-dependent short uORFs in DENR-depleted mammalian cells (Fig. 1A(i)-(ii)).

In contrast to REI-permissive uORF1, the fourth AUG-initiated uORF in GCN4 mRNA (uORF4) does not allow retention of eIFs on post-termination 40S subunits, owing to multiple, surrounding cis-acting sequences, thus making it non-permissive for REI and repressive to GCN4 translation in WT cells26,27,30–33. We reasoned that uORF4 is a typical example of short uORFs in yeast that do not allow REI owing to efficient dissociation of 40S post-termination complexes regardless of the requirement for Tma factors in releasing tRNA (Fig. 1B(i)-ii)). uORF4 contains a penultimate CCG proline codon, whose dependence on DENR for recycling and REI in human cells is ambiguous13, and was neither unusually dependent nor independent of the Tma proteins for bulk 40S recycling in yeast16. Since elimination of the Tma20/Tma22 heterodimer did not impact translational control of WT GCN4 mRNA29, as mentioned above, it seems unlikely that CCG contributes to low-level REI at WT uORF4 by imposing a heightened requirement for Tma factors for tRNA release. Here, using uORF4 as another genetic tool, we tested the expectation that replacing the native CCG codon with other codons known to be hyperdependent on Tma proteins for bulk recycling will not reduce REI in tma∆∆ cells because, lacking eIF3, the 40S subunits will dissociate and fail to reinitiate regardless of the tRNA release kinetics (Fig. 1B(i), (ii)). We also examined whether converting the native 3-codon uORF1 and uORF4 to start-stop elements by eliminating their second and third coding triplets confers a dependence on the Tma proteins for REI in the manner observed for DENR with these specialized uORFs in mammalian cells17,18.

Our results depart from the expected outcomes for GCN4 uORF4 shown in Fig. 1B and suggest instead a role for Tma factors at this uORF in dissociating 40S post-termination complexes from mRNA following release of deacylated tRNA, regardless of whether the penultimate triplets are Tma-hyperdependent in bulk recycling. Our findings for GCN4 uORF1 also differ from those obtained for short, REI-permissive uORFs in mammalian cells13 (Fig. 1A(i), (ii)) in revealing that uORF1 generally allows efficient REI independently of Tma factors even when equipped with Tma-hyperdependent penultimate codons. Overall, the effects of Tma factors on REI at the functionally distinct GCN4 regulatory uORFs equipped with different penultimate codons are not principally dictated by the Tma-dependence of these codons in bulk recycling. We also found that, unlike in mammalian cells, REI following start-stop uORFs is Tma-independent; and we further show that Tma64 plays a supportive role to that of Tma20/Tma22 in impeding REI at uORF4, consistent with its auxiliary role in bulk 40S recycling at most stop codons in yeast.

Results

Tma64 partially substitutes for the Tma20/Tma22 heterodimer in suppressing REI after translation of the GCN4 uORF4 variant containing the Tma-hyperdependent TTGLeu codon

To examine the effects of replacing the penultimate codons of uORF1 or uORF4 with triplets that differ in their dependence on Tma factors in bulk 40S recycling, we employed the well-established GCN4-lacZ reporter system, which faithfully recapitulates the functions of the cis and trans regulatory elements/factors involved in GCN4 translational control24,25,30–37. This reporter contains the entire GCN4 transcription unit, including the ~600 nt leader harboring the four uORFs (Fig. 2), with the lacZ coding sequences inserted in-frame with the GCN4 main ORF. To study uORF1, we employed the “uORF1-only” reporter lacking the AUGs of uORFs 2–4. As it was shown previously that leaky-scanning of the WT uORF1 AUG codon is extremely infrequent36, expression of β-galactosidase from this reporter provides a read-out of REI following termination at uORF1. Similarly, uORF4 variants were examined in the “uORF4-only” reporter lacking the AUGs of uORFs 1–3, which produces low levels of β-galactosidase owing to a lack of leaky-scanning of the uORF4 AUG codon in the absence of the other uORFs and highly inefficient REI downstream following uORF4 translation34. Both uORF1 and uORF4 are present at their normal positions in the leader of these reporters.

We first examined the relative contribution of the Tma20/Tma22 heterodimer and Tma64 to the efficiency of REI following uORF4 translation, which we regard as an exemplar of typical REI-non-permissive uORFs in budding yeast3,13 (Fig. 1B(i), (ii)). To this end, uORF4-only reporters were generated (Fig. 3A) with the native penultimate CCGPro codon replaced by either the TTGLeu codon or TGGTrp codon, judged to be the most or least dependent on Tma factors, respectively, for bulk 40S recycling in yeast16. (Tma-dependence for each triplet was assigned previously as the ratio of 40S occupancies in tma20∆tma64∆ vs. WT cells averaged across the stop codons of all genes. Here, we designate penultimate codons as being “hyperdependent” or “hypodependent” on Tma factors for recycling if they showed, respectively, greater or less than average tma∆∆/WT 40S ratios at a 99% confidence level; with all other triplets regarded as displaying “average” Tma-dependency16.) Assaying the reporters in the three single deletion strains lacking TMA20, TMA22, or TMA64, the tma20∆tma22∆tma64∆ triple deletion mutant, and the isogenic WT revealed that the tma20Δ and tma22Δ single deletion strains, and the triple deletion mutant (tma∆∆∆) produced a very modest, albeit significant change in expression of the reporter with the Tma-hypodependent TGGTrp codon (Fig. 3B). This result matches the expected outcome depicted in Fig. 1B(ii). At odds with the expectations in Fig. 1B(i), however, the single deletions of TMA20 or TMA22 conferred significant ≥2-fold derepression of the reporter containing the Tma-hyperdependent TTGLeu codon. Although the single deletion of TMA64 had only a small effect of lower significance, the triple mutant showed even higher expression of the TTGLeu reporter compared to the tma20∆ and tma22∆ single mutants, at >3-fold above the level in WT (Fig. 3B). One way to explain these unexpected findings on the TTGLeu reporter is to propose that eliminating Tma20/Tma22’s second function in dissociating post-termination 40S subunits from mRNA allows the retained subunits to traverse the leader and reinitiate downstream at the GCN4 AUG codon, even though the first function of the heterodimer in releasing the deacylated leucyl tRNA is absent in tma∆ mutant cells. As elaborated below, this in turn implies the occurrence of Tma-independent release of the deacylated tRNA at the TTGLeu codon, allowing increased REI to occur when dissociation of the post-termination 40S subunit is impaired by tma20∆ or tma22∆ mutations. This proposal of both Tma-dependent and -independent mechanisms of tRNA release at uORF4 variants harboring Tma-hyperdependent penultimate codons is supported by additional evidence presented below.Fig. 3 Differential effects of various TMA deletions on expression of uORF4-only GCN4-lacZ reporters equipped with Tma-hypodependent or Tma-hyperdependent penultimate codons.

A Schematic of the uORF4-only GCN4-lacZ reporters harboring either WT or mutant penultimate codons. B Yeast strains YSG181 (tma20∆), YSG184 (tma22∆), YSG178 (tma64∆), or YKJ3 (tma20∆tma22∆tma64∆), deleted for one or all three TMA genes and the corresponding WT strain YSG142 (WT BY4741) were transformed with uORF4-only reporters containing WT uORF4 (p226) or uORF4 variants with the penultimate Tma-average codon CCG exchanged for Tma-hyperdependent TTGLeu (pKJ34) or Tma-hypodependent TGGTrp triplet (pKJ36). Reporter activity was assayed in whole cell extracts for at least five independent transformants (with the specific n denoted in each graph) and activities in the tma∆ transformants were normalized as described in “Methods”. Data are represented as ratios of normalized mean values ± SD of β-galactosidase activities in tma∆ strains/WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant; for details of statistical analysis, see “Materials and methods”. The exact p values can be found in Supplementary Data 1. C Same analysis as in (B) except comparing transformants of the double mutants YKJ6 (tma20∆tma22∆) and YSG196 (tma20∆tma64∆) to the triple mutant YKJ3 (tma20∆tma22∆tma64∆). D Serial dilutions of strains of the indicated genotypes from (C) were spotted on minimal SD medium and grown for 48 h at 30 °C.

The results on the TTGLeu reporter in Fig. 3B further suggest that although Tma64 cannot fully substitute for the Tma20/Tma22 heterodimer it still contributes to inhibiting REI in cells lacking the heterodimer, as eliminating all three Tma proteins in the triple mutant conferred significantly greater derepression of the TTGLeu reporter compared to the tma20∆ or tma22∆ single mutants (Fig. 3B). This inference was confirmed by our finding that the triple mutant and tma20∆tma64∆ double mutant, in which both the heterodimer and Tma64 are missing, exhibit significantly greater derepression of the TTGLeu reporter compared to the tma20∆tma22∆ double mutant still containing Tma64 (Fig. 3C). Consistent with this, no discernible difference in growth rate was observed between the tma20∆tma64∆ double mutant and the triple mutant, both of which grow slightly more slowly than the WT (Fig. 3D). Accordingly, in subsequent experiments, we considered the tma20∆tma64∆ and tma20∆tma22∆tma64∆ strains to be equivalent in completely lacking the functions of both the heterodimer and Tma64 in regulating REI.

Tma proteins suppress REI at uORF4 variants containing Tma-hypodependent as well as Tma-hyperdependent penultimate codons

To extend our analysis of the impact of different penultimate codons on Tma-dependent 40S recycling and REI, we examined uORF4-only reporters carrying the three classes of penultimate codons listed in Table 1 that are Tma-hypodependent (Fig. 4B), Tma-average (Fig. 4C), or Tma-hyperdependent (Fig. 4D) for 40S recycling in the translatome16. To this end, we measured their expression in both the tma20∆tma22∆ double mutant and the triple mutant to provide insights into the codon dependence of Tma64 vs. the Tma20/Tma22 heterodimer in suppressing REI. Considering first results for the tma∆∆∆ triple mutant, we observed that all of the reporters with Tma-hypodependent codons (Fig. 4B, black bars) or Tma-average codons (Fig. 4C, black bars) showed statistically significant derepression in the triple mutant, ranging from 1.2-fold to 3.1-fold for different codons (Table 1, Tma-hypodependent & Tma-average data in col. 3). Derepression was also observed for the four reporters with Tma-hyperdependent codons (Fig. 4D; black bars), ranging from 2.4-fold to 4.6-fold (Table 1, Tma-hyperdependent data in col. 3). Thus, strong derepression in the triple mutant was observed for multiple reporters representing Tma-hypodependent codons (CAAGln, GCTAla) and Tma-average codons (ATGMet, CCAPro) in addition to the four Tma-hyperdependent codons we examined.Table 1 Fold-changes in uORF4-only and uORF1-only reporter expression in tma mutant vs. WT strainsa

Penultimate codon	tma20∆ tma22∆/WT ratio (uORF4) ± SD	tma∆∆∆/WT ratio (uORF4) ± SD	tma20∆ tma64∆/WT ratio (uORF1) ± SEM	
Tma-hypodependent	
TACTyr	2.33 ± 0.64	1.47 ± 0.19	0.73 ± 0.24	
TGGTrp	1.30 ± 0.43	1.23 ± 0.28	1.15 ± 0.58	
CAAGln	1.42 ± 0.07	2.48 ± 0.45	1.06 ± 0.21	
GCTAla	2.10 ± 0.23	2.83 ± 0.57	1.04 ± 0.12	
GACAsp	N/A	N/A	1.03 ± 0.13	
GAGGlu	N/A	N/A	0.92 ± 0.02	
CACHis	N/A	N/A	0.84 ± 0.17	
TTTPhe	N/A	N/A	0.94 ± 0.32	
TGTCys	N/A	N/A	0.85 ± 0.10	
Tma-average	
CCGPro (uORF4 WT)	1.50 ± 0.53	1.67 ± 0.53	0.92 ± 0.11	
GCGAla	2.92 ± 0.07	1.55 ± 0.19	0.79 ± 0.03	
CTGLeu	4.13 ± 1.42	1.59 ± 0.34	0.76 ± 0.09	
ATGMet	2.55 ± 0.33	2.73 ± 0.39	0.40 ± 0.03	
CCAPro	2.46 ± 0.11	3.13 ± 0.55	0.44 ± 0.16	
TGCCys (uORF1 WT)	N/A	N/A	0.78 ± 0.17	
GAAGlu	N/A	N/A	0.87 ± 0.07	
GTCVal	N/A	N/A	0.69 ± 0.04	
GGGGly	N/A	N/A	1.01 ± 0.13	
CGGArg	N/A	N/A	0.65 ± 0.19	
CCCPro	N/A	N/A	0.91 ± 0.18	
TCGSer	N/A	N/A	0.84 ± 0.05	
AGGArg	N/A	N/A	1.01 ± 0.12	
CGCArg	N/A	N/A	0.67 ± 0.20	
Tma-hyperdependent	
TTGLeu	1.91 ± 0.58	3.43 ± 0.47	0.59 ± 0.06	
ATTIle	3.36 ± 1.26	2.40 ± 0.55	0.85 ± 0.33	
TATTyr	1.55 ± 0.33	2.92 ± 0.36	0.96 ± 0.05	
AATAsn	2.26 ± 0.70	4.56 ± 0.78	0.83 ± 0.13	
AAALys	N/A	N/A	0.76 ± 0.11	
AAGLys	N/A	N/A	0.86 ± 0.07	
N/A not available.

aList of the used constructs classified according to the Tma-dependency of their penultimate codons according to Young et al.16. This table contains average fold-change ± SD in reporter expression from biological replicates in either tma20∆ tma22∆ tma64∆ strain (YKJ3) and tma20∆ tma22∆ strain (YKJ6) (uORF4) or tma20∆ tma64∆ (H4520) (uORF1) vs. corresponding WT.

Fig. 4 Differential effects of various TMA double and triple deletions on expression of uORF4-only GCN4-lacZ reporters equipped with Tma-hypodependent, Tma-average or Tma-hyperdependent penultimate codons.

A Schematic of the uORF4-only GCN4-lacZ reporters harboring either WT or mutant penultimate codons. B–D Yeast strains YKJ6 (tma20∆tma22∆) and YKJ3 (tma20∆tma22∆tma64∆) and the corresponding WT strain were transformed with the WT uORF4-only GCN4-lacZ reporter construct (p226) and variants with the WT uORF4 penultimate codon (CCG) exchanged for the indicated Tma-hypodependent, Tma-average or Tma-hyperdependent codons. Reporter activity was assayed in whole cell extracts for at least four independent transformants (with the specific n denoted in each graph) and activities in the tma∆ transformants were normalized as described in “Methods”. Data are represented as GCN4-LacZ reporter enzyme activities in WT strain and tma∆ strains. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant; for details of statistical analysis, see “Materials and methods”. The exact p values can be found in Supplementary Data 1.

Despite this apparent discrepancy with the previously published data on the Tma-dependency of different penultimate codons16, plotting the expression changes for Tma-hyperdependent, Tma-hypodependent or Tma-average penultimate codons measured in the triple mutant vs. WT (Fig. 5A) revealed a significant tendency for the four Tma-hyperdependent codons to exhibit greater derepression in the triple mutant compared to the four Tma-hypodependent codons tested (p = 0.0404). Nonetheless, since the Tma-average reporters containing ATGMet or CCAPro and the Tma-hypodependent GCTAla and CAAGln reporters did not exhibit statistically smaller derepression ratios in the triple mutant compared to the Tma-hyperdependent reporters for codons TTGLeu, ATTIle, and TATTyr (Table 1, col. 3), we conclude that the Tma-dependence of penultimate codons determined previously16 is not a strong indicator of Tma-mediated suppression of REI following uORF4 translation.Fig. 5 Changes in reporter expression for groups of Tma-hyperdependent, Tma-hypodependent or Tma-average penultimate codons in tma mutant vs. WT cells.

A Violin plot of the average fold-changes in expression of uORF4-only GCN4-lacZ reporters harboring the four Tma-hyperdependent (Hyperdep), four Tma-hypodependent (Hypodep) or five Tma-average (Average) penultimate codons in the triple deletant yeast strain YKJ3 (tma20∆tma22∆tma64∆) vs. isogenic WT strain. The average fold-change of expression determined from biological replicates only was calculated for each construct from results plotted in Fig. 4. Statistical analysis was performed using two-tailed unpaired t-test *p < 0.05. B Violin plot of fold changes in expression of uORF1-only GCN4-lacZ reporters harboring the six Tma-hyperdependent, nine Tma-hypodependent and 14 Tma-average penultimate codons in tma20∆tma64∆ strain H4520 vs. WT strain BY4741. The average fold-change of expression determined from biological replicates was calculated for each construct from the results plotted in Fig. 6. Statistical analysis was performed using the two-tailed unpaired t-test. *p < 0.05. The exact p values can be found in Supplementary Data 1.

Considering next results obtained in the tma20∆tma22∆ double mutant, where Tma64 can potentially substitute for the heterodimer, we found that five of the uORF4-only reporters that showed marked derepression in the triple mutant displayed substantially less derepression in the tma20∆tma22∆ double mutant, including reporters with CAAGln and GCTAla (Fig. 4B), CCAPro (Fig. 4C), TTGLeu, TATTyr, and AATAsn (Fig. 4D) penultimate codons (summarized in Table 1, cf. cols. 2–3). For these six reporters, containing Tma-hypodependent, -average, or -hyperdependent codons, it appears that Tma64 can partially substitute for heterodimer function in suppressing REI. The Tma-hypodependent CAAGln (Fig. 4B) and Tma-hyperdependent TATTyr (Fig. 4D) codons illustrate the most complete functional substitution by Tma64, with only slight derepression in the tma20∆tma22∆ double mutant despite marked derepression in the triple mutant. Reporters with TACTyr (Tma-hypodependent; Fig. 4B), ATGMet, CTGLeu and GCGAla (Tma-average; Fig. 4C), and ATTIle (Tma-hyperdependent; Fig. 4D) codons appear to represent the other extreme, wherein Tma64 cannot functionally compensate at all and eliminating the heterodimer alone in the double mutant confers derepression equal to or even greater than that found in the triple mutant also lacking Tma64. Finally, the comparatively low-level derepression observed in both mutants for the native uORF4 CCGPro (Fig. 4C) and TGGTrp reporters (Fig. 4B, black bars) makes it difficult to determine whether Tma64 can partially substitute for the heterodimer at these uORFs. Nevertheless, these findings overall raise the interesting possibility that the ability of Tma64 to partially substitute for the heterodimer in suppressing REI depends on the penultimate codon at uORF4, and this codon variability applies to both Tma-hyperdependent and Tma-average codons. If so, then the ability of Tma64 to suppress REI in cells lacking the heterodimer would not appear to be governed by the codon-dependence of Tma factors for bulk 40S recycling16.

Very few penultimate codons confer Tma-dependence at REI-permissive GCN4 uORF1

The mammalian DENR/MCTS1 heterodimer was shown to promote REI after certain short uORFs in mammalian cells, including ATF4 uORF113; whereas the yeast Tma20/Tma22 counterpart functioned oppositely to inhibit REI following uORF translation in yeast extracts16. As mentioned above, it was suggested that the discrepancy between mammals and yeast arises from the inability of post-termination 40S complexes at typical yeast uORFs to remain associated with the mRNA and migrate to downstream start codons following release of the tRNA decoding the penultimate codon13 (Fig. 1B(i)-(ii) vs. 1A(i)-(ii)). To test this hypothesis, we analyzed the effects of the tma mutations on REI at GCN4 uORF1. As mentioned, this atypical uORF is surrounded by cis-acting RPEs, some of which interact with eIF3 subunits and promote retention of post-termination 40S complexes on the mRNA, enabling high-level REI (Fig. 2). Indeed, REI after uORF1 translation is ~20–25-fold higher than after uORF4 translation25,26,32,33. The retention of post-termination 40S subunits at uORF1 makes the uORF1-only GCN4-lacZ reporter (Fig. 6A) ideally suited to examine whether Tma factors are required to dissociate deacylated tRNA and enable REI at the GCN4 start codon in a manner limited to penultimate codons with heightened dependence on the Tma factors for bulk 40S recycling in yeast16. If so, then uORF1-only reporters equipped with Tma-hyperdependent codons should exhibit reduced REI and GCN4-lacZ expression in tma∆∆ vs. WT cells in the manner observed for short uORFs in mammalian cells depleted of DENR (Fig. 1A(i)).Fig. 6 Differential effects of deleting both TMA20 and TMA64 on expression of uORF1-only GCN4-lacZ reporters equipped with Tma-average, Tma-hypodependent or Tma-hyperdependent penultimate codons.

A Schematic of the uORF1-only GCN4-lacZ reporters harboring either WT or mutant penultimate codons. B Yeast strains H4520 (tma20∆tma64∆) and WT strain BY4741 were transformed with uORF1-only GCN4-lacZ reporter constructs with the WT uORF1 penultimate codon (TGC) exchanged for the indicated Tma-average, Tma-hypodependent or Tma-hyperdependent codons. β-galactosidase activities determined for each of three independent transformants (n = 3) of the tma20∆tma64∆ strain were divided by a factor of 1.316 prior to calculating the mean activities and S.E.M. values plotted here to normalize for differences in reporter expression between the two strains that are independent of the uORF1 variants and observed for an uORF-less reporter (as described in “Methods”). The mean values between mutant and WT were compared for each reporter in a two-tailed, unpaired Student’s t test to assign p values (*p < 0.05). The exact p values can be found in Supplementary Data 1.

We exchanged the native uORF1 penultimate codon TGCCys in the uORF1-only reporter with other penultimate codons of all three classes defined above, including six Tma-hyperdependent, nine Tma-hypodependent, and 13 Tma-average codons. The native TGCCys codon is Tma-average, and the native TGTCys codon of the REI-permissive uORF2 of GCN432 was chosen as one of the Tma-hypodependent codons. Expression of the resulting reporters was assayed in the tma20∆tma64∆ double mutant and WT strains (Fig. 6 and Table 1, col. 4). At odds with the prediction that REI would be reduced in the mutant for reporters with Tma-hyperdependent codons, only one of the six reporters of this class, that containing the TTGLeu codon, showed significantly reduced expression in the mutant (Fig. 6B, hyperdependent constructs). While TTGLeu was judged to be highly Tma-hyperdependent for bulk 40S recycling, ATTIle and TATTyr were equally so16, yet the reporters for the latter two codons showed unchanged expression in the mutant strain. Moreover, the constructs containing the Tma-average ATGMet or CCAPro codons were the only reporters besides the TTGLeu construct showing significantly reduced expression in tma20∆tma64∆ cells (Fig. 6B, Tma-average constructs). Fold changes in expression between WT and tmaΔΔ strains can be found summarized in Table 1, col. 4. Plotting the expression changes for the entire set of 29 codons examined at uORF1 (Fig. 5B) revealed a general tendency for reduced expression in the tmaΔΔ mutant vs. WT cells, but no significant difference in median expression ratios between Tma-hyperdependent reporters and either Tma-hypodependent or Tma-average reporters.

Figure 5B does reveal a small but significant difference between the median expression ratios between the Tma-hypodependent and Tma-average groups (p = 0.0394), which reflects the slightly increased REI observed in tma∆∆ cells for the Tma-hypodependent group. None of the uORF1-only reporters, however, showed the ≥2-fold increased REI conferred by the tmaΔΔΔ mutation for most of the uORF4-only reporters (Fig. 5A), even for the ten cases where uORF1-only and uORF4-only reporters share the same penultimate codons (ATT, TTG, AAT, TAT, ATG, GCG, CTG, CAA, GCT, CCA). This is consistent with the prediction that the specialized RPEs at uORF1 allow 40S post-termination complexes to resist ribosome dissociation by the Tma factors that suppress REI at uORF4 lacking RPEs (Fig. 2). In summary, our findings indicate that uORF1 differs from mammalian short uORFs, including the REI-permissive uORF1 of ATF4, in lacking a requirement for Tma factors in dissociating deacylated tRNA from post-termination complexes at penultimate codons that impose a heightened Tma-requirement in bulk 40S recycling. A lack of Tma-dependence on REI also applies to eight of the nine DENR-dependent codons13 represented among our uORF1-only reporters (ATT, GCG, CTG, CCG, CGG, CCC, AGG, and GCT). In fact, ATG is the sole DENR-dependent codon we tested that confers a dependence on Tma factors for REI at uORF1, but as noted above, ATG is Tma-average for 40S recycling in yeast.

In summary, neither Tma-dependence nor DENR-dependence of the penultimate codon is an accurate predictor of Tma-dependence for REI at GCN4 uORF1 variants, despite the functional similarity between GCN4 uORF1 and ATF4 uORF1.

Function of yeast “start-stop” elements is not Tma-dependent

As mentioned earlier, mammalian DENR was shown to be required for REI after start-stop uORFs consisting of ATG followed by a stop codon13,18. To test the Tma-dependence of start-stops for downstream REI in yeast, we deleted the 2nd and 3rd codons from native uORF1-, uORF3- and uORF4-only constructs, without altering any other sequences, producing uStart-stop-only constructs uSt-st1, uSt-st3 and uSt-st4, respectively. (See bottom of Fig. 2 for sequences of the three uSt-st uORFs.)

First, we noticed that all three start-stop elements are more inhibitory to REI than their native uORF counterparts, significantly reducing GCN4-lacZ expression in WT cells by ~50-fold, ~3-fold, and ~0.6-fold compared to the levels observed for native uORF1-only, uORF3-only, and uORF4-only reporters, respectively (Fig. 7A). Based on findings in mammalian cells, we reasoned that converting native uORF1 to uSt-st1 would confer a dependence on REI by the Tma proteins not observed for native uORF1 with its penultimate codon TGCCys (Fig. 6, TGC(WT)), reducing expression in tma∆∆ cells. This was not observed, however (Fig. 7B, cols. 1–4), even though we found above that ATGMet as the penultimate codon for 3-codon uORF1 conferred Tma-dependence for efficient REI (Fig. 6, ATG). Interestingly, converting uORF4 to uSt-st4 eliminated the significant derepression observed in tma∆∆ cells for the native uORF4-only reporter (Fig. 7B, cols. 9–12). This suggests that conversion to a start-stop element eliminates the requirement for Tma factors for highly efficient 40S recycling at uORF4 and repression of REI downstream. The Tma proteins appear to make no contribution to recycling and REI at native uORF3, as the uORF3-only reporter expression is unaffected by the tma20∆tma64∆ mutations; whereas its conversion to uSt-st3 confers a modestly significant Tma-dependence for recycling and suppression of REI (Fig. 7B, cols. 5–8). Overall, our findings suggest that, unlike their mammalian counterparts, the Tma proteins play little role in controlling REI after start-stop elements in yeast.Fig. 7 Effects of start-stop elements on downstream REI in the GCN4-lacZ reporter system and differential effects of TMA20 and TMA64 deletion on re-initiation downstream of start-stop elements.

A WT strain BY4741 was transformed with GCN4-lacZ reporter plasmids containing the following single WT uORFs: uORF1-only (p209), uORF3-only (pSG61_(2)), or uORF4-only (p226); or containing single Start-stop elements uSt-st1 (pKP78), uSt-st3 (pKP76), or uSt-st4 (pKP77). β-galactosidase activities were assayed in at least four biological replicates (with the specific n denoted in each graph) and data are presented as GCN4-LacZ reporter enzyme activities. B Strains YSG196 (tma20∆ tma64∆) and the corresponding WT were transformed with the same set of GCN4-lacZ reporter plasmids described in (A) and reporter expression was measured as described there. Reporter activity values in the tma∆∆ strain were normalized as described in “Methods”. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant; for details of statistical analysis, see “Materials and methods”. The exact p values can be found in Supplementary Data 1.

Discussion

As yeast Tma20/Tma22 and, to a lesser degree, Tma64 have been implicated in 40S ribosome recycling in vivo15,16, we used an established reporter system based on GCN4 translational control to examine the influence of yeast Tma20/MCTS1, Tma22/DENR and Tma64/eIF2D on REI downstream of short uORFs optimized for either REI (uORF1) or for recycling and suppression of REI (uORF4)5. In addition, we dissected the functional interplay between the Tma factors in regulating these alternative post-termination outcomes. To do so, we employed variants of uORF1 or uORF4 containing various penultimate codons that conferred either unusually strong dependence, average, or lower than average dependence on Tma proteins for bulk 40S recycling at most stop codons in yeast as genetic tools.

Using the uORF4-only GCN4-lacZ reporter, we first demonstrated that inserting Tma-hyperdependent codon TTGLeu substantially increased REI in single mutants lacking TMA20 or TMA22 but only slightly in the mutant lacking TMA64 (Fig. 3A). Deleting TMA64 in the tma20∆tma22∆ mutant significantly enhanced this defect (Fig. 3C), suggesting that Tma64 can partially compensate for the absence of Tma20/Tma22 in suppressing REI. This conclusion agrees with previous 40S profiling data16, wherein a single deletion of TMA20 or TMA22, but not TMA64, led to accumulation of unrecycled 40S subunits at stop codons translatome-wide, and this phenotype was more pronounced in the tma20∆tma64∆ deletion strain lacking both the heterodimer and Tma64. Consistent with this, we observed significantly increased REI for the TTGLeu uORF4 variant in the tma20∆tma64∆ double mutant and tma20∆tma22∆tma64∆ triple mutant compared to the tma20∆tma22∆ double mutant (Fig. 3C). We concluded that intact Tma20/Tma22 heterodimer is indispensable for WT 40S recycling and suppression of REI but receives functional support from Tma64 at the stop codon of this uORF4 variant.

Examining a total of 12 different uORF4 variants with different penultimate codons, plus native uORF4, revealed that the absence of all three Tma proteins in the tma∆∆∆ mutant increased REI by less than 2-fold for native uORF4 containing CCGPro, and for the TACTyr, CTGLeu, GCGAla and TGGTrp uORF4 variants, but by more than 2-fold for the CAAGln, GCTAla, CCAPro and ATGMet variants (Fig. 4B, C and Table 1). Since all these codons showed average or hypodependency on Tma factors in bulk recycling16, these mixed outcomes indicate that the Tma-dependency in bulk 40S recycling determined previously16 is generally a weak predictor of Tma-dependence in promoting 40S recycling and suppressing REI for different penultimate codons at uORF4. On the other hand, our finding that the group of Tma-hyperdependent reporters shows a significantly greater increase in median expression compared to the Tma-hypodependent and Tma-average groups (Figs. 4D, 5A and Table 1) suggests that Tma-hyperdependence in bulk recycling at least partly contributes to the impact of Tma proteins in blocking REI observed for uORF4 variants encompassing a range of Tma-dependencies.

Our finding that REI is elevated in tma∆∆∆ cells at uORF4 variants containing Tma-average or Tma-hypodependent codons is difficult to explain if dissociation of the empty 40S subunit is believed to occur spontaneously at these penultimate codons, which should maintain low-level REI in the presence or absence of Tma factors (Fig. 8A(ii)-(a)). Hence, in the model proposed in Fig. 8A(ii)-(b), we assume that Tma factors must figure prominently in dissociating the empty 40S subunits at Tma-hypodependent codons (recycling step 2 in WT), such that retention of a fraction of 40S subunits in their absence in tma∆∆ cells allows increased REI downstream when release of the deacylated tRNA is achieved by a Tma-independent pathway. Our observation that the Tma proteins also inhibit REI at uORF4 variants containing Tma-hyperdependent codons is likewise difficult to explain. Specifically, the failure to release the deacylated tRNA in the tma∆∆ mutant should block REI even though Tma-catalyzed dissociation of the 40S subunit from mRNA is absent, thus keeping REI low in tma∆∆ cells (Fig. 8A(i)-(a)). Therefore, it seems necessary to propose that the deacylated tRNA eventually dissociates spontaneously or by another pathway in the absence of Tma factors, so that loss of Tma-catalyzed dissociation of the empty 40S subunit can enable REI and confer the increased REI we observed in tma∆∆ cells (Fig. 8A(i)-(b)).Fig. 8 Expected vs. proposed functions for yeast Tma20/Tma22 at REI-permissive GCN4 uORF1 and REI-non-permissive GCN4 uORF4.

Tma20/Tma22 (blue ovals), penultimate deacylated tRNA (red “L”), and the two functions of Tma factors (boxes 1 and 2) in releasing tRNA and dissociating empty 40S subunits, respectively (solid or dashed arrows), are depicted as in Fig. 1. A Tma-mediated recycling at GCN4 uORF4 depending on the presence of (i) Tma-hyperdependent or (ii) Tma-hypodependent penultimate codons. (i) Tma-hyperdependent uORFs. (a) expectation: as depicted exactly as in Fig. 1B(i) (presented again for comparison), in WT cells (upper), Tma factors efficiently release the penultimate-codon tRNA and also dissociate the empty 40S subunits, conferring low-level REI. In tma∆∆ cells (lower), the absence of Tma-mediated tRNA release helps to ensure low-level REI, yielding no change in REI in tma∆∆ vs. WT cells. (b) model: in WT (upper), deacylated tRNA can be released by Tma-hyperdependent or Tma-independent mechanisms, but Tma-mediated 40S dissociation maintains low-level REI. In tma∆∆ cells (lower), Tma-independent tRNA release coupled with absence of Tma-mediated 40S dissociation confers the increased REI we observed in tma∆∆ vs. WT cells. (ii) Tma-hypodependent uORFs. (a) expectation: as depicted exactly in Fig. 1B(ii), Tma-independent tRNA release occurs in both WT and tma∆∆ cells, but REI is low in both cases because the empty 40S subunits dissociate from the mRNA independently of Tma factors, for no change in REI vs. WT. (b) model: in WT (upper), 40S dissociation is catalyzed by Tma factors even at these Tma-hypodependent uORFs, and absence of this function in tma∆∆ cells, coupled with Tma-independent tRNA release, confers the observed increased REI in tma∆∆ vs. WT cells. B Tma-mediated recycling at GCN4 uORF1 depending on the presence of Tma-hyperdependent (i) or Tma-hypodependent (ii) codons. (i) Tma-hyperdependent uORFs. (a) expectation: as depicted for MCTS1/DENR in Fig. 1A(i), In WT cells (upper), Tma factors efficiently release the penultimate-codon tRNA but its second function in 40S dissociation is impeded by eIF3 (green oval) to enable high-level REI. In tma∆∆ cells (lower), diminished release of the penultimate-codon tRNA lowers REI. (b) model: deacylated tRNA can be released from uORF1 variants by both Tma-hyperdependent and Tma-independent mechanisms in WT yeast (upper) and Tma-independent tRNA release maintains high-level REI in tma∆∆ cells (lower), as 40S dissociation is impeded by eIF3 in both WT and tma∆∆ cells, thus accounting for the unchanged REI we observed in tma∆∆ vs. WT cells. (ii) Tma-hypodependent uORFs. (a) expectation: as depicted for MCTS1/DENR in Fig. 1A(ii), Tma-independent release of the tRNA and subsequent high-level REI occurs in the presence (top) or absence (bottom) of Tma factors, yielding no decrease in REI in tma∆∆ vs. WT cells, as we observed.

The models we propose in (b) panels of Fig. 8A(i)-(ii) assume that Tma-independent recycling can operate at all stop codons and that recycling at Tma-hyperdependent penultimate codons simply relies more heavily on Tma factors than at Tma-hypodependent codons. This possibility is consistent with the fact that eliminating all three Tma proteins confers only a small effect on cell growth, implying the frequent occurrence of Tma-independent recycling throughout the translatome.

We also investigated whether the Tma factors function similarly to MCTS1/DENR in promoting, rather than impeding, REI at short uORFs that are permissive for REI owing to retention of eIFs during uORF translation. It was proposed that the persistence of eIFs on 40S post-termination complexes prevents dissociation of the 40S subunits following MCTS1/DENR-catalyzed release of deacylated tRNA, allowing resumption of 40S migration and rebinding of TC for REI downstream. This explained why REI was impaired on depletion of MCTS1/DENR only for uORFs containing DENR-dependent penultimate codons13 (Fig. 1A(i)-(ii)). To investigate this mechanism in yeast, we examined the GCN4 uORF1-only reporter in which RPEs surrounding uORF1 increase eIF3 and eIF4F occupancy of the post-termination 40S subunits and confer high-frequency REI at GCN4 (Fig. 2)25,26,32,33. Consistent with results for MCTS1/DENR, REI was not reduced in the tma∆∆ mutant for native uORF1 nor for 21 other uORF1 variants equipped with Tma-average or Tma-hypodependent penultimate codons but REI was reduced when uORF1 contained the Tma-hyperdependent TTGLeu codon. However, at odds with results for MCTS1/DENR, REI was not reduced in tma∆∆ cells at five other uORF1 variants with Tma-hyperdependent codons. This included the TAT codon present in uORF1 of ATF4 mRNA—functionally equivalent to GCN4 uORF138—that confers DENR-dependent REI on ATF4 mRNA13. Moreover, REI was markedly reduced in tma∆∆ cells when uORF1 contained the Tma-average penultimate codons ATG and CCA. Thus, dependence on the Tma factors for REI was conferred on uORF1 by only three of 29 penultimate codons examined, only one of which (TTG) is Tma-hyperdependent for bulk 40S recycling16. Moreover, there was no significant difference in median tma∆∆ derepression ratios between uORF1-only reporters containing Tma-hyperdependent vs. Tma-average or Tma-hypodependent codons (Fig. 5B). While there is a general tendency for REI to be diminished at uORF1 variants in tma∆∆ vs. WT cells (Fig. 5B), the Tma factors do not stimulate REI preferentially at uORF1 variants whose penultimate codons are Tma-hyperdependent in bulk recycling in the manner described for MCTS1/DENR at REI-permissive uORFs in mammalian cells.

One possibility is that the specialized interaction of GCN4 uORF1 RPEs with eIF3 generally overrides the requirement for Tma factors in dissociating the deacylated tRNA at Tma-hyperdependent penultimate codons. By preventing 40S dissociation from the mRNA, eIF3 might provide sufficient time for dissociation of the deacylated tRNA, either spontaneously or by a Tma-independent pathway, to prevent reduced REI in tma∆∆ cells. This scenario is depicted in the model proposed in Fig. 8B(i)-(b) to explain our unexpected findings on uORF1 variants with Tma-hyperdependent codons. In support, numerous tRNAs were found to readily dissociate from the 40S subunit without the help of any dedicated factor19. Furthermore, structural analysis of MCTS1/DENR bound to the 40S subunit indicates that MCTS1 might occupy the same binding site on the ribosome as the eIF3a, b, and c subunits of mammalian eIF339. In addition, the ribosome binding interface in MCTS1 seems to be well conserved from yeast to higher eukaryotes (Fig. 9A), as we also observed when comparing the MCTS1 structure with the Tma20 AlphaFold structure prediction (Fig. 9B). This might indicate competitive ribosome binding between eIF3 and Tma20/Tma22, such that the uORF1 RPEs would promote binding of eIF3a vs. the Tma heterodimer and thereby prevent the latter from influencing REI appreciably at uORF1.Fig. 9 Comparison of S. cerevisiae (yeast), D. melanogaster (fly), H. sapiens (human) and C. elegans (worm) Tma20/MCTS1 proteins.

A Multiple sequence alignment was performed using Clustal Omega. This alignment illustrates the conservation of amino acid residues of the ribosome binding interface described previously39 and marked here by red dots. Similarity score and identity percentage were determined using UniProt BLAST tool. B Comparison of predicted Tma20 structure (upper panel) and structure of MCTS1 in complex with DENR (bottom panel; MCTS1 colored in light gray, DENR colored in dark grey). Conserved residues are depicted in red and regions separating them are colored in blue. Tma20 structure was predicted using the AlphaFold348 algorithm. MCTS1/DENR complex structure was published previously39 (PDB accession 6MS4). PyMOL 3.0 was used for data visualization.

Presumably, interaction of deacylated leucyl tRNA with the TTGLeu codon is too stable to overcome the requirement for Tma-mediated release, leading to the observed reduced REI for the TTGLeu uORF1 variant in tma∆∆ cells, in the manner predicted for DENR-hyperdependent codons (Fig. 1A(i)). The same might be true for interaction of elongator methionyl tRNA and prolyl tRNA with their cognate ATGMet and CCAPro codons, which also displayed Tma-dependence for REI at uORF1, even though ATG and CCA were not identified as being Tma-hyperdependent for bulk 40S recycling16. Supporting this idea, ATG was identified as a DENR-hyperdependent penultimate codon in mammalian cells both in bulk 40S recycling and reporter REI assays13.

Another consequence of introducing ATG as the penultimate codon of GCN4 uORF1 not yet mentioned is a significant reduction in REI in WT cells (p = 0.04), which is further exacerbated in the tma∆∆ mutant (Fig. 6). A reduction in WT cells was also found for GGGGly (p = 0.03), CCGPro, CCCPro, AGGArg and GAGGlu codon replacements (p = 0.09) (Fig. 6B). CCGPro is the evolutionarily conserved penultimate codon at uORFs 3 and 4 and was shown previously to inhibit REI when introduced at uORF133, which might result from delaying decoding or termination at the adjacent stop codon33.

In addition to ATF4 uORF1 and other REI-permissive uORFs, the MCTS1/DENR heterodimer was found to promote REI after translation of the ATF4 start-stop element preceding uORF113 and at various other start-stop elements in optimum Kozak context13,17,18,20. In contrast, we found no evidence for Tma-dependent recycling and REI at the start-stop elements we derived from GCN4 uORFs 1, 3, or 4, all of which contain the optimum A-rich sequence context for initiation in yeast. Converting uORF3 and uORF4 to start-stop elements decreased REI in WT cells and diminished the increased REI observed for native uORF4 in tma∆∆ cells. It appears that uSt-st4 exhibits an elevated level of spontaneous 40S recycling that bypasses the contribution of Tma factors to recycling seen at native uORF4. Converting uORF1 to start-stop element uSt-st1 also dramatically decreased REI in WT cells, apparently disabling the RPEs that enable high-level REI at native uORF1. Although uSt-st1 behaves like a typical inhibitory uORF, 40S recycling appears to occur without any contribution from the Tma factors at this element. The fact that uSt-st1, uSt-st4, and uSt-st3 do not show decreased REI in tma∆∆ cells in the manner we observed for the GCN4 uORF1 variant with ATGMet as penultimate codon (Fig. 6B) is not surprising considering that, similar to uORF4, the St-st elements appear to lack the capacity for eIF3-mediated retention of 40S post-termination complexes (Fig. 2).

In summary, we found that most replacements of the uORF4 penultimate codon confer increased REI in mutants lacking both Tma64 and the Tma20/Tma22 heterodimer compared to the native CCG codon, suggesting a codon-dependent role for the Tma factors in dissociating 40S post-termination complexes at this uORF, even for those penultimate triplets that were shown previously to be Tma-hypodependent for recycling at other stop codons throughout the translatome. These findings imply that the deacylated tRNA can dissociate in the absence of Tma factors at both Tma-hyperdependent and Tma-hypodependent penultimate codons, so that loss of Tma-catalyzed recycling of the empty 40S subunit permits elevated REI at uORF4 in the mutant cells (models in (b) panels of Fig. 8A(i)-(ii)). In contrast to findings on short REI-permissive uORFs in mammalian cells, and ATF4 uORF1 in particular13, we found that only one of six Tma-hyperdependent penultimate codons introduced at GCN4 uORF1 conferred reduced REI in tma20∆tma64∆ cells. These last findings indicate that dissociation of the deacylated tRNA and subsequent REI can generally proceed without Tma factors at uORF1, even at penultimate codons where these proteins markedly enhance bulk 40S recycling, which we again attribute to Tma-independent tRNA release (model in Fig. 8B(i)-(b)). Together, our results on uORF1 and uORF4 account for our previous finding that GCN4 translational control was essentially unperturbed in mutants lacking both Tma20/Tma22 and Tma6429—in stark contrast to the requirement for MCTS1/DENR for induction of ATF4 mRNA translation. Also at odds with findings on mammalian MCTS1/DENR, converting uORF1 to a start-stop element did not confer Tma-dependent REI, and converting uORF4 to a start-stop element decreased REI and diminished the contribution of Tma factors in suppressing REI. Overall, the influence of Tma factors on REI at short regulatory uORFs in yeast cells differs considerably from that observed for mammalian MCTS1/DENR.

Materials and methods

Yeast strains and plasmids

The genotypes of all yeast strains employed in this study are listed in Supplementary Table 1. Yeast mutants lacking one or more TMA genes were constructed using DNA cassettes bearing kanMX4, hphNT1 or natNT2 resistance markers contained in plasmids pFA6a-KanMX440 (acquired from J.H. Hegeman), pZC3 and pZC441, respectively. The cassettes were amplified by PCR using gene-specific primers SG325 and SG326 (TMA64/YDR117C) and KJ1 and KJ2 (TMA22/YJR014W) (see Supplementary Table 2 for complete list of primers). Subsequently, the amplified DNA was used to transform yeast to confer resistance to antibiotics Geneticin G418 (BioConcept), Nourseothricin (Jena Bioscience), or Hygromycin B (Carl Roth GmbH), respectively, on YPD agar medium. YSG178 (tma64∆) was derived from WT BY4741 (YSG142) by transformation with a TMA64-specific kanMX4 deletion cassette. YSG196 (tma20∆tma64∆) was generated from YSG181 (tma20∆) by transformation with a TMA64-specific natNT2 deletion cassette. YKJ3 (tma20∆tma22∆tma64∆) was generated from YSG196 (tma20∆tma64∆) by transformation with a TMA22-specific hphNT1 cassette. YKJ6 (tma20∆tma22∆) was generated from YSG181 (tma20∆) by transformation with a TMA22-specific natNT2 cassette. Deletion of WT alleles and replacement with the appropriate deletion cassettes was verified by colony PCR42 using gene-specific primers SG295 (for TMA20/YER007C-A), SG294 (for TMA64/YDR117C deletions) and SG296 (for TMA22/YJR014W deletions) in combination with universal primer PB238 complementary to all deletion cassettes. Strains YSG181 (tma20∆) and YSG184 (tma22∆) were acquired from the Euroscarf yeast deletion collection.

All plasmids employed in this study are listed in Supplementary Data 2. All uORF4-only reporters were created by replacing the SalI-BstEII fragment of uORF4-only GCN4-lacZ construct p22643 with the appropriate SalI-BstEII fragments generated by either PCR primer mutagenesis or DNA synthesis. Fragments generated by PCR primer mutagenesis were amplified using common forward primer KJ27 (mapping near the SalI cloning site) and the following mutagenic reverse primers (mapping near the BstEII site) for constructing the indicated plasmids: primer KJ25 for plasmid pKJ34, primer KJ26 for pKJ35, primer KJ24 for pKJ36, primer KJ78 for pKJ57, primer KJ80 for pKJ58, primer KJ76 for pKJ59, primer KJ74 for pKJ61, primer KJ77 for PKJ62. DNA fragments for constructing pKJ17, pKJ23, pKJ20 and pKJ21 were synthesized by GeneArt Gene Synthesis (Thermo Fisher). All fragment inserts in the final constructs were verified by Sanger sequencing, and the DNA sequences are listed in Supplementary Data 3.

The start-stop constructs pKP76, pKP77 and pKP78 were created by replacing the SalI-BstEII fragment of the GCN4-lacZ construct pSG19433; a uORF1-only construct created by replacing the coding sequence of uORF1 by that of uORF2) by the appropriate SalI-BstEII fragments generated by GeneArt Gene Synthesis (Thermo Fisher). These fragments contain point mutations in start codons of uORFs (point mutations in uORFs 1, 2 and 4 in pKP76, point mutations in uORFs 1, 2 and 3 in pKP77 and point mutations in uORFs 2, 3 and 4 in pKP78) and deletions of sense codons between start and stop codons in uORF3 (pKP76), uORF4 (pKP77) and uORF1 (pKP78), respectively, which creates start-stop elements uSt-st3, uSt-st4 and uSt-st1. The mutation scheme of these constructs is also shown in Fig. 2. All fragment inserts in the final constructs were verified by Sanger sequencing, and the DNA sequences are listed in Supplementary Data 3.

All uORF1-only reporters were created by replacing the SalI-BstEII fragment of WT GCN4-lacZ construct p18044 with the appropriate SalI-BstEII fragments, generated by DNA synthesis by LifeSct LLC, containing point mutations in the start codons of uORFs 2-4 (uORF2 ATG to CTG, uORF3 ATG to AGG, and uORF4 ATG to AGG) and either WT uORF1 (plasmid pSG61) or uORF1 variants with altered third codons (pSG62-pSG89). The inserts in the resulting plasmids were sequenced in their entirety to confirm the desired sequences. The sequence of the SalI-BstEII fragment of pSG61 (with WT uORF1) is listed in Supplementary Data 3; sequences of all other constructs differ only by the replacements of the third uORF1 codon with the triplet indicated in Table S2 for the corresponding plasmids.

GCN4-lacZ reporter assays

β-galactosidase specific activities (in units of nmol of ONPG cleaved per min per mg of protein) were assayed in whole cell extracts as described previously33 in yeast transformants of uORF4-only and uSt-st GCN4-lacZ reporter plasmids, and as previously described45 for transformants of uORF1-only reporter plasmids and the “uORF-less” GCN4-lacZ reporter p22730. Briefly, cells were transformed with plasmids bearing GCN4-lacZ fusion reporters and grown in synthetic SD media to mid-log phase. Cells were centrifuged, washed with deionized water and broken using glass beads in Breaking Buffer (100 mM Tris 8.0, 4% Glycerol, 1 mM β-mercaptoethanol), then clarified by centrifugation (18,400 × g, 30 min, 4 °C). β-galactosidase activity (measured as nanomoles of o-nitrophenyl-3-Dgalactosidase cleaved per minute per milligram of protein) was normalized to protein concentration of the whole cell extract, which was measured by Bradford assay with BSA as standard.

For the uORF4-only and uSt-st-only reporter assays, the normalized β-galactosidase activity for each reporter construct in transformants of tma∆ strains was determined by dividing the unnormalized β-galactosidase activities by a normalization factor. This factor was calculated separately for each individual experiment by assaying the activity of the “uORF-less” reporter (p227) in WT and tma∆ strains in four transformants under the same conditions as uORF4-only/uSt-st-only reporters. Then, the mean of tma∆ “uORF-less” activities was divided by the mean of WT “uORF-less” activities, determining the normalization factor.

For the uORF1-only reporter assays, β-galactosidase activities determined for each replicate transformant of the tma20∆tma64∆ strain were divided by a factor of 1.316 prior to calculating the mean activities to be compared to the mean unnormalized activities for the replicate transformants of the WT strain in a two-tailed, unpaired Student’s t test to assign p values to differences between the means in mutant vs. WT cells. The normalization factor was determined by assaying six transformants each of the same tma20∆tma64∆ and WT strains harboring the “uORF-less” reporter (p227) under the same conditions employed for uORF1-only reporters. The tma20∆tma64∆/WT ratio of mean expression values was 1.316, which differed significantly from unity in a two-tailed t-test with a p-value of 0.003.

Yeast spotting assay

Yeast strains were spotted onto minimal synthetic defined (SD) medium plates in five serial 10-fold dilutions (starting with OD600 0.5) and grown for 48 h at 30 °C.

Multiple sequence alignment and protein structure visualization

Multiple sequence alignment of S. cerevisiae Tma20 (UniProt accession P89886), D. melanogaster MCTS1 (UniProt accession Q9W445), C. elegans C11D2.7 (UniProt accession Q8MXH7) and H. sapiens MCTS1 (UniProt accession Q9ULC4) protein sequences was performed using Clustal Omega46 with default settings through European Bioinformatics Institute Tools services47. Similarity scores and identity percentages for the sequences were determined using the UniProt BLAST tool.

Tma20 protein structure was predicted using the AlphaFold3 algorithm48 through AlphaFold Server (Google DeepMind) services. MCTS1/DENR complex structure39 was obtained through PDB accession 6MS4. PyMOL 3.0 was used for visualization of the structures.

Statistics and reproducibility

Statistical significance was determined as follows. For all uORF4-only and Start-stop reporter assays, all data were tested for normality by the Shapiro–Wilk test. Based on the normality test, data were compared either by the parametric, unpaired, two-tailed Welch’s t test, or by the non-parametric Mann–Whitney test; **** indicates p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns non-significant. All individual data points are shown as part of bar plots and the number of replicates is shown above each bar. The SD was calculated from all biological replicates for each construct in each strain. For uORF1-only reporter assays, an unpaired, two-tailed Student’s t test was employed. Statistical analysis and visualization were performed in GraphPad Prism, version 9.4.1 (GraphPad Software). All replicates shown in the reporter assays graphs were independent transformants—biological replicates.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Description of Additional Supplementary Materials

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Reporting Summary

Transparent Peer Review file

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-024-06761-x.

Acknowledgements

We are thankful to all members of our laboratories for fruitful discussions. This work was supported in part by a Grant of Excellence in Basic Research (EXPRO 2019) provided by the Czech Science Foundation (19-25821X), the Praemium Academiae grant provided by the Czech Academy of Sciences, CZ.02.01.01/00/22_008/0004575 RNA for therapy by ERDF and MEYS (all to L.S.V.), by GA UK project no. 339022 by Charles University Grant Agency (to K.J.), and by the Intramural Program of the National Institutes of Health (S.G. and A.G.H.).

Author contributions

St.Gu., L.S.V. and A.G.H. conceived and designed the project. K.J. and Sw.Ga. carried out majority of experiments and performed the data analysis; they were assisted by K.P. at the onset of this study. K.J., Sw.Ga., L.S.V. and A.G.H. interpreted the results. K.J., L.S.V. and A.G.H. wrote the paper with input from Sw.Ga.

Peer review

Peer review information

Communications Biology thanks Ayala Shiber and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Elah Pick and David Favero. A peer review file is available.

Funding

Open access funding provided by the National Institutes of Health.

Data availability

All data from this study are available within this paper and its Supplementary Information. All plasmids and strains used in this study are available upon request from the authors. Source data for graphs presented in the main figures can be found at Supplementary Data 1.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Kristína Jendruchová, Swati Gaikwad.
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