Molecular glues targeting GSPT1 in cancers: A potent therapy.

G1 to S phase transition 1 (GSPT1) is a key translation termination factor that significantly overexpressed in various cancer tissues and cells. Molecular glue is a kind of small molecule, which can bind to an E3 ligase such as cereblon (CRBN) and subsequently recruit neosubstrate proteins for ubiquitination-proteasomal degradation. This emerging therapeutic approach shows great potential in treating cancers and other diseases. This review aims to introduce current understanding of antitumor mechanism of molecular glues targeting GSPT1, summarize pharmacology profiles of existing molecular glues, and outline development strategies of novel molecular glues. The insights provided in this review will be valuable for future studies.

Functional Activity of Isoform 2 of Human eRF1.

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome.

Gene Expression Analysis of Yeast Strains with a Nonsense Mutation in the eRF3-Coding Gene Highlights Possible Mechanisms of Adaptation.

In yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence of nonsense mutations in these genes leads to amplification of mutant alleles (sup35-n and sup45-n), which appears to be necessary for the viability of such cells. However, the mechanism of this phenomenon remained unclear. In this study, we used RNA-Seq and proteome analysis to reveal the complete set of gene expression changes that occur during cellular adaptation to the introduction of the sup35-218 nonsense allele. Our analysis demonstrated significant changes in the transcription of genes that control the cell cycle: decreases in the expression of genes of the anaphase promoting complex APC/C (APC9, CDC23) and their activator CDC20, and increases in the expression of the transcription factor FKH1, the main cell cycle kinase CDC28, and cyclins that induce DNA biosynthesis. We propose a model according to which yeast adaptation to nonsense mutations in the translation termination factor genes occurs as a result of a delayed cell cycle progression beyond the G2-M stage, which leads to an extension of the S and G2 phases and an increase in the number of copies of the mutant sup35-n allele.

Recognition of 3' nucleotide context and stop codon readthrough are determined during mRNA translation elongation.

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.

Discovery of a novel role of tumor suppressor PDCD4 in stimulation of translation termination.

Programmed cell death 4 protein (PDCD4) regulates many vital cell processes, although is classified as a tumor suppressor because it inhibits neoplastic transformation and tumor growth. For example, PCDC4 has been implicated in the regulation of transcription and mRNA translation. PDCD4 is known to inhibit translation initiation by binding to eukaryotic initiation factor 4A and elongation of oncogenic c- and A-myb mRNAs. Additionally, PDCD4 has been shown to interact with poly(A)-binding protein (PABP), which affects translation termination, although the significance of this interaction is not fully understood. Considering the interaction between PABP and PDCD4, we hypothesized that PDCD4 may also be involved in translation termination. Using in vitro translation systems, we revealed that PDCD4 directly activates translation termination. PDCD4 stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the PABP, which also stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Using a toe-printing assay, we determined the first stage at which PDCD4 functions-binding of release factors to the A-site of the ribosome. However, preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. Based on these data, we assumed that human PDCD4 controls protein synthesis during translation termination. The described mechanism of the activity of PDCD4 in translation termination provides a new insight into its functioning during suppression of protein biosynthesis.

TaIAA21 represses TaARF25-mediated expression of TaERFs required for grain size and weight development in wheat.

Auxin signaling is essential for the development of grain size and grain weight, two important components for crop yield. However, no auxin/indole acetic acid repressor (Aux/IAA) has been functionally characterized to be involved in the development of wheat (Triticum aestivum L.) grains to date. Here, we identified a wheat Aux/IAA gene, TaIAA21, and studied its regulatory pathway. We found that TaIAA21 mutation significantly increased grain length, grain width, and grain weight. Cross-sections of mutant grains revealed elongated outer pericarp cells compared to those of the wild type, where the expression of TaIAA21 was detected by in situ hybridization. Screening of auxin response factor (ARF) genes highly expressed in early developing grains revealed that TaARF25 interacts with TaIAA21. In contrast, mutation of the tetraploid wheat (Triticum turgidum) ARF25 gene significantly reduced grain size and weight. RNA sequencing analysis revealed upregulation of several ethylene response factor genes (ERFs) in taiaa21 mutants which carried auxin response cis-elements in their promoter. One of them, ERF3, was upregulated in the taiaa21 mutant and downregulated in the ttarf25 mutant. Transactivation assays showed that ARF25 promotes ERF3 transcription, while mutation of TtERF3 resulted in reduced grain size and weight. Analysis of natural variations identified three TaIAA21-A haplotypes with increased allele frequencies in cultivars relative to landraces, a signature of breeding selection. Our work demonstrates that TaIAA21 works as a negative regulator of grain size and weight development via the ARF25-ERFs module and is useful for yield improvement in wheat.

[Role of Proteins Interacting with the eRF1 and eRF3 Release Factors in the Regulation of Translation and Prionization].

The review discusses the role that proteins interacting with the translation termination factors eRF1 and eRF3 play in the control of protein synthesis and prionization. These proteins interact not only with each other, but also with many other proteins involved in controlling the efficiency of translation termination, and associate translation termination with other cell processes. The termination of translation is directly related not only to translation re-initiation and ribosome recycling, but also to mRNA stability and protein quality control. This connection is ensured by the interaction of eRF1 and eRF3 with proteins participating in various cell metabolic processes, such as mRNA transport from the nucleus into the cytoplasm (Dbp5/DDX19 and Gle1), ribosome recycling (Rli1/ABCE1), mRNA degradation (Upf proteins), and translation initiation (Pab1/PABP). In addition to genetic control, there is epigenetic control of translation termination. This mechanism is associated with prion polymerization of the Sup35 protein to form the [PSI^(+)] prion. The maintenance of the [PSI^(+)] prion, like other yeast prions, requires the operation of a system of molecular chaperones and protein sorting factors. The review considers in detail the interaction of the translation termination factors with proteins involved in various cellular processes.

Nsp1 of SARS-CoV-2 stimulates host translation termination.

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

Polyadenylate-binding protein-interacting proteins PAIP1 and PAIP2 affect translation termination.

Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination in vitro However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.

Divergent effects of translation termination factor eRF3A and nonsense-mediated mRNA decay factor UPF1 on the expression of uORF carrying mRNAs and ribosome protein genes.

In addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5' leader sequence. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3A or UPF1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3A and UPF1 and to compare the effect of each of these factors on gene expression. We find that eRF3A and UPF1 have very different impacts on the human transcriptome, less than 250 transcripts being targeted by both factors. We show that eRF3A depletion globally derepresses the expression of mRNAs containing translated uORFs while UPF1 knockdown derepresses only the mRNAs harbouring uORFs with an AUG codon in an optimal context for translation initiation. Finally, we also find that eRF3A and UPF1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome.

Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae.

Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination.

Distinct mechanisms of mutant huntingtin toxicity in different yeast strains.

Expansion of polyglutamine stretches in several proteins causes neurodegenerative amyloidoses, including Huntington disease. In yeast, mutant huntingtin (mHtt) with a stretch of 103 glutamine residues (HttQ103) forms toxic aggregates. A range of yeast strains have been used to elucidate the mechanisms of mHtt toxicity, and have revealed perturbations of various unrelated processes. HttQ103 aggregates can induce aggregation of cellular proteins, many of which contain glutamine/asparagine-rich regions, including Sup35 and Def1. In the strain 74-D694 HttQ103, toxicity is related to aggregation-mediated depletion of soluble Sup35 and its interacting partner Sup45. Def1 was also implicated in mHtt toxicity, since its lack detoxified HttQ103 in another yeast strain, BY4741. Here we show that in BY4742, deletion of DEF1 lowers HttQ103 toxicity and decreases the amount of its polymers, but does not affect copolymerization of Sup35. Furthermore, in contrast to 74-D694, increasing the levels of soluble Sup35 and Sup45 does not alleviate toxicity of HttQ103 in BY4742. These data demonstrate a difference in the mechanisms underlying mHtt toxicity in different yeast strains and suggest that in humans with Huntington disease, neurons of different brain compartments and cells in other tissues can also be damaged by different mechanisms.

Studies on human eRF3-PABP interaction reveal the influence of eRF3a N-terminal glycin repeat on eRF3-PABP binding affinity and the lower affinity of eRF3a 12-GGC allele involved in cancer susceptibility.

The eukaryotic release factor 3 (eRF3) has been involved in the control of mRNA degradation through its association with the cytoplasmic Poly(A) Binding Protein, PABP. In mammals, eRF3 N-terminal domain contains two overlapping PAM2 motifs which specifically recognize the MLLE domain of PABP. In humans, eRF3a/GSPT1 gene contains a stable GGC repeat encoding a repeat of glycine residues in eRF3a N-terminus. There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines. Several studies have reported that the presence of eRF3a 12-GGC allele is correlated with an increased risk of cancer development. Using surface plasmon resonance, we have studied the interaction of the various allelic forms of eRF3a with PABP alone or poly(A)-bound PABP. We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP. Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.

[Identification of new genes that affect [PSI

Translation termination is an important step in gene expression. Its correct processing is governed by eRF1 (Sup45) and eRF3 (Sup35) proteins. In Saccharomyces cerevisiae, mutations in the corresponding genes, as well as Sup35 aggregation in [PSI^(+)] cells that propagate the prion form of Sup35 lead to inaccurate stop codon recognition and, consequently, nonsense suppression. The presence of stronger prion variants results in the more efficient suppression of nonsense mutations. Previously, we proposed a synthetic lethality test that enables the identification of genes that may influence either translation termination factors or [PSI^(+)] manifestation. This is based on the fact that the combination of sup45 mutations with the strong [PSI^(+)] prion variant in diploids is lethal. In this work, a set of genes that were previously shown to enhance nonsense suppression was analyzed. It was found that ABF1, FKH2, and REB1 overexpression decreased the growth of strains in a prion-dependent manner and, thus, might influence [PSI^(+)] prion toxicity. It was also shown that the synthetic lethality of [PSI^(+)] and sup45 mutations increased with the overexpression of GLN3 and MOT3 that encode Q/N-rich transcription factors. An analysis of the effects of their expression on the transcription of the release factors genes revealed an increase in SUP35 transcription in both cases. Since SUP35 overexpression is known to be toxic in [PSI^(+)] strains, these genes apparently enhance [PSI^(+)] toxicity via the regulation of SUP35 transcription.

The translation termination factor eRF1 (Sup45p) of Saccharomyces cerevisiae is required for pseudohyphal growth and invasion.

Mutations in the essential genes SUP45 and SUP35, encoding yeast translation termination factors eRF1 and eRF3, respectively, lead to a wide range of phenotypes and affect various cell processes. In this work, we show that nonsense and missense mutations in the SUP45, but not the SUP35, gene abolish diploid pseudohyphal and haploid invasive growth. Missense mutations that change phosphorylation sites of Sup45 protein do not affect the ability of yeast strains to form pseudohyphae. Deletion of the C-terminal part of eRF1 did not lead to impairment of filamentation. We show a correlation between the filamentation defect and the budding pattern in sup45 strains. Inhibition of translation with specific antibiotics causes a significant reduction in pseudohyphal growth in the wild-type strain, suggesting a strong correlation between translation and the ability for filamentous growth. Partial restoration of pseudohyphal growth by addition of exogenous cAMP assumes that sup45 mutants are defective in the cAMP-dependent pathway that control filament formation.

The Hbs1-Dom34 protein complex functions in non-stop mRNA decay in mammalian cells.

In yeast, aberrant mRNAs lacking in-frame termination codons are recognized and degraded by the non-stop decay (NSD) pathway. The recognition of non-stop mRNAs involves a member of the eRF3 family of GTP-binding proteins, Ski7. Ski7 is thought to bind the ribosome stalled at the 3'-end of the mRNA poly(A) tail and recruit the exosome to degrade the aberrant message. However, Ski7 is not found in mammalian cells, and even the presence of the NSD mechanism itself has remained enigmatic. Here, we show that unstable non-stop mRNA is degraded in a translation-dependent manner in mammalian cells. The decay requires another eRF3 family member (Hbs1), its binding partner Dom34, and components of the exosome-Ski complex (Ski2/Mtr4 and Dis3). Hbs1-Dom34 binds to form a complex with the exosome-Ski complex. Also, the elimination of aberrant proteins produced from non-stop transcripts requires the RING finger protein listerin. These findings demonstrate that the NSD mechanism exists in mammalian cells and involves Hbs1, Dom34, and the exosome-Ski complex.

The processed isoform of the translation termination factor eRF3 localizes to the nucleus to interact with the ARF tumor suppressor.

The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61-71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.

Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP).

Affinity purification using the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide synthase (iNOS) mRNA identified the cytosolic poly(A)-binding protein (PABP) as a protein interacting with the human iNOS 3'-UTR. Downregulation of PABP expression by RNA interference resulted in a marked reduction of cytokine-induced iNOS mRNA expression without changes in the expression of mRNAs coding for the major subunit of the RNA polymerase II (Pol 2A) or ß2-microglobuline (ß2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1α, NF-κB p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16kb iNOS promoter in siPABP cells. In contrast, the siPABP-mediated decline of iNOS expression correlated with a reduction in the stability of the iNOS mRNA. As the stability of the Pol 2A and ß2M mRNA was not changed, siPABP-treatment seems to have a specific effect on iNOS mRNA decay. UV-crosslinking experiments revealed that PABP interacts with one binding site in the 5'-UTR and two different binding sites in the 3'-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5'-UTR but not in the 3'-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5'-UTR or iNOS-3'-UTR luciferase reporter constructs. In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5'-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression.