Functional redundancy in tRNA dihydrouridylation.

Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.

Poly(A) tail degradation in human cells: ATF4 mRNA as a model for biphasic deadenylation.

Eukaryotic mRNA deadenylation is generally considered as a two-step process in which the PAN2-PAN3 complex initiates the poly(A) tail degradation while, in the second step, the CCR4-NOT complex completes deadenylation, leading to decapping and degradation of the mRNA body. However, the mechanism of the biphasic poly(A) tail deadenylation remains enigmatic in several points such as the timing of the switch between the two steps, the role of translation termination and the mRNAs population involved. Here, we have studied the deadenylation of endogenous mRNAs in human cells depleted in either PAN3 or translation termination factor eRF3. Among the mRNAs tested, we found that only the endogenous ATF4 mRNA meets the biphasic model for deadenylation and that eRF3 prevents the shortening of its poly(A) tail. For the other mRNAs, the poor effect of PAN3 depletion on their poly(A) tail shortening questions the mode of their deadenylation. It is possible that these mRNAs experience a single step deadenylation process. Alternatively, we propose that a very short initial deadenylation by PAN2-PAN3 is followed by a rapid transition to the second phase involving CCR4-NOT complex. These differences in the timing of the transition from one deadenylation step to the other could explain the difficulties encountered in the generalization of the biphasic deadenylation model.

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.

C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system.

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.

Translation termination-dependent deadenylation of MYC mRNA in human cells.

The earliest step in the mRNA degradation process is deadenylation, a progressive shortening of the mRNA poly(A) tail by deadenylases. The question of when deadenylation takes place remains open. MYC mRNA is one of the rare examples for which it was proposed a shortening of the poly(A) tail during ongoing translation. In this study, we analyzed the poly(A) tail length distribution of various mRNAs, including MYC mRNA. The mRNAs were isolated from the polysomal fractions of polysome profiling experiments and analyzed using ligase-mediated poly(A) test analysis. We show that, for all the mRNAs tested with the only exception of MYC, the poly(A) tail length distribution does not change in accordance with the number of ribosomes carried by the mRNA. Conversely, for MYC mRNA, we observed a poly(A) tail length decrease in the fractions containing the largest polysomes. Because the fractions with the highest number of ribosomes are also those for which translation termination is more frequent, we analyzed the poly(A) tail length distribution in polysomal fractions of cells depleted in translation termination factor eRF3. Our results show that the shortening of MYC mRNA poly(A) tail is alleviated by the silencing of translation termination factor eRF3. These findings suggest that MYC mRNA is co-translationally deadenylated and that the deadenylation process requires translation termination to proceed.

Unveiling structural and functional divergences of bacterial tRNA dihydrouridine synthases: perspectives on the evolution scenario.

Post-transcriptional base modifications are important to the maturation process of transfer RNAs (tRNAs). Certain modifications are abundant and present at several positions in tRNA as for example the dihydrouridine, a modified base found in the three domains of life. Even though the function of dihydrourine is not well understood, its high content in tRNAs from psychrophilic bacteria or cancer cells obviously emphasizes a central role in cell adaptation. The reduction of uridine to dihydrouridine is catalyzed by a large family of flavoenzymes named dihydrouridine synthases (Dus). Prokaryotes have three Dus (A, B and C) wherein DusB is considered as an ancestral protein from which the two others derived via gene duplications. Here, we unequivocally established the complete substrate specificities of the three Escherichia coli Dus and solved the crystal structure of DusB, enabling for the first time an exhaustive structural comparison between these bacterial flavoenzymes. Based on our results, we propose an evolutionary scenario explaining how substrate specificities has been diversified from a single structural fold.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly.

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2ß phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2ß mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2ß and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

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.

Translation termination efficiency modulates ATF4 response by regulating ATF4 mRNA translation at 5' short ORFs.

The activating transcription factor 4 (ATF4) promotes transcriptional upregulation of specific target genes in response to cellular stress. ATF4 expression is regulated at the translational level by two short open reading frames (uORFs) in its 5'-untranslated region (5'-UTR). Here, we describe a mechanism regulating ATF4 expression in translation termination-deficient human cells. Using microarray analysis of total RNA and polysome-associated mRNAs, we show that depletion of the eucaryotic release factor 3a (eRF3a) induces upregulation of ATF4 and of ATF4 target genes. We show that eRF3a depletion modifies ATF4 translational control at regulatory uORFs increasing ATF4 ORF translation. Finally, we show that the increase of REDD1 expression, one of the upregulated targets of ATF4, is responsible for the mTOR pathway inhibition in eRF3a-depleted cells. Our results shed light on the molecular mechanisms connecting eRF3a depletion to mammalian target of rapamycin (mTOR) pathway inhibition and give an example of ATF4 activation that bypasses the signal transduction cascade leading to the phosphorylation of eIF2α. We propose that in mammals, in which the 5'-UTR regulatory elements of ATF4 mRNA are strictly conserved, variations in translation termination efficiency allow the modulation of the ATF4 response.

The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.

Selenocysteine insertion into selenoproteins involves the translational recoding of UGA stop codons. In mammals, selenoprotein expression further depends on selenium availability, which has been particularly described for glutathione peroxidase 1 and 4 (Gpx1 and Gpx4). The SECIS element located in the 3'UTR of the selenoprotein mRNAs is a modulator of UGA recoding efficiency in adequate selenium conditions. One of the current models for the UGA recoding mechanism proposes that the SECIS binds SECIS-binding protein 2 (SBP2), which then recruits a selenocysteine-specific elongation factor (EFsec) and tRNA (Sec) to the ribosome, where L30 acts as an anchor. The involvement of the SECIS in modulation of UGA recoding activity was investigated, together with SBP2 and EFsec, in Hek293 cells cultured with various selenium levels. Luciferase reporter constructs, in transiently or stably expressing cell lines, were used to analyze the differential expression of Gpx1 and Gpx4. We showed that, upon selenium fluctuation, the modulation of UGA recoding efficiency depends on the nature of the SECIS, with Gpx1 being more sensitive than Gpx4. Attenuation of SBP2 and EFsec levels by shRNAs confirmed that both factors are essential for efficient selenocysteine insertion. Strikingly, in a context of either EFsec or SBP2 attenuation, the decrease in UGA recoding efficiency is dependent on the nature of the SECIS, GPx1 being more sensitive. Finally, the profusion of selenium of the culture medium exacerbates the lack of factors involved in selenocysteine insertion.

Looking for the functions of RNA granules in ALK-transformed cells.

Numerous cytoplasmic foci containing mRNA s and their associated proteins have been described in mammalian somatic and germ cells. The best studied examples are given by the processing bodies (PBs) that are present in all cell types, and the stress granules (SGs) that are transiently formed following stress stimuli. Those foci are non-membranous dynamic structures that, through the continuous exchange of their content with the cytoplasm, are believed to control mRNA storage, translation and degradation. However, due in part to the fact that their composition has not been fully characterized, their relevance to mRNA regulation and cell survival remains a matter of debate. In a recent study, we described new cytoplasmic foci that form specifically in transformed cells expressing the constitutively active ALK tyrosine kinase. Those granules, further called AGs for ALK granules, contain polyadenylated mRNAs but are distinct from PBs and SGs. Using a method based on sucrose density gradient fractionation, we further purified AGs and identified their mRNA content. We discuss our findings in relation to other granules containing untranslated mRNAs and speculate on the possible contribution of AGs to the oncogenic properties of ALK-expressing cells.

Novel mRNA-containing cytoplasmic granules in ALK-transformed cells.

In mammalian cells, nontranslating messenger RNAs (mRNAs) are concentrated in different cytoplasmic foci, such as processing bodies (PBs) and stress granules (SGs), where they are either degraded or stored. In the present study, we have thoroughly characterized cytoplasmic foci, hereafter called AGs for ALK granules that form in transformed cells expressing the constitutively active anaplastic lymphoma kinase (ALK). AGs contain polyadenylated mRNAs and a unique combination of several RNA binding proteins that so far has not been described in mammalian foci, including AUF1, HuR, and the poly (A(+)) binding protein PABP. AGs shelter neither components of the mRNA degradation machinery present in PBs nor known markers of SGs, such as translation initiation factors or TIA/TIAR, showing that they are distinct from PBs or SGs. AGs and PBs, however, both move on microtubules with similar dynamics and frequently establish close contacts. In addition, in conditions in which mRNA metabolism is perturbed, AGs concentrate PB components with the noticeable exception of the 5' to 3' exonuclease XRN1. Altogether, we show that AGs constitute novel mRNA-containing cytoplasmic foci and we propose that they could protect translatable mRNAs from degradation, contributing thus to ALK-mediated oncogenicity.

Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3'-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5'-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.

eRF3a/GSPT1 12-GGC allele increases the susceptibility for breast cancer development.

It is now widely recognized that translation factors are involved in cancer development and that components of the translation machinery that are deregulated in cancer cells may become targets for cancer therapy. The eukaryotic Release Factor 3 (eRF3) is a GTPase that associates with eRF1 in a complex that mediates translation termination. eRF3a/GSPT1 first exon contains a (GGC)n expansion coding for proteins with different N-terminal extremities. Herein we show that the longer allele (12-GGC) is present in 5.1% (7/137) of the breast cancer patients analysed and is absent in the control population (0/135), corresponding to an increased risk for cancer development, as revealed by Odds Ratio analysis. mRNA quantification suggests that patients with the 12-GGC allele overexpress eRF3a/GSPT1 in tumor tissues relative to the normal adjacent tissues. However, using an in vivo assay for translation termination in HEK293 cells, we do not detect any difference in the activity of the eRF3a proteins encoded by the various eRF3a/GSPT1 alleles. Although the connection between the presence of eRF3a/GSPT1 12-GGC allele and tumorigenesis is still unknown, our data suggest that the presence of the 12-GGC allele provides a potential novel risk marker for various types of cancer.

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation.

In eukaryotes, eRF1 and eRF3 are associated in a complex that mediates translation termination. The regulation of the formation of this complex in vivo is far from being understood. In mammalian cells, depletion of eRF3a causes a reduction of eRF1 level by decreasing its stability. Here, we investigate the status of eRF3a when not associated with eRF1. We show that eRF3a forms altered in their eRF1-binding site have a decreased stability, which increases upon cell treatment with the proteasome inhibitor MG132. We also show that eRF3a forms altered in eRF1 binding as well as wild-type eRF3a are polyubiquitinated. These results indicate that eRF3a is degraded by the proteasome when not associated with eRF1 and suggest that proteasomal degradation of eRF3a controls translation termination complex formation by adjusting the eRF3a level to that of eRF1.

Girolline interferes with cell-cycle progression, but not with translation.

Girolline is a 2-aminoimidazole derivative with cytotoxic activity. It affects the survival of exponentially growing leukaemic cultured cells and has a significant antitumour activity on grafted murine tumours in vivo. In vitro studies showed that girolline affected protein synthesis by interfering with the translation termination process. Here, we investigate the effect of girolline on translation termination in human cultured cells. We show that girolline neither induces an increase in translational readthrough of stop codons nor affects the polysome profile in treated cells. This suggests that girolline does not act on translation in vivo. Then, we examine the effect of girolline on cell-cycle progression and we show that girolline induces an arrest of the cell cycle at the G2 stage.

Human eukaryotic release factor 3a depletion causes cell cycle arrest at G1 phase through inhibition of the mTOR pathway.

Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Studies have related eRF3 with cell cycle regulation, cytoskeleton organization, and tumorigenesis. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. eRF3a is the major factor acting in translation termination, and its expression level controls termination complex formation. Here, we investigate the role of eRF3a in cell cycle progression using short interfering RNAs and flow cytometry. We show that eRF3a depletion induces a G1 arrest and that eRF3a GTP-binding activity, but not the eRF3a N-terminal domain, is required to restore G1-to-S-phase progression. We also show that eRF3a depletion decreases the global translation rate and reduces the polysome charge of mRNA. Finally, we show that two substrates of the mammalian TOR (mTOR) kinase, 4E-BP1 and protein kinase S6K1, are hypophosphorylated in eRF3a-depleted cells. These results strongly suggest that the G1 arrest and the decrease in translation induced by eRF3a depletion are due to the inhibition of mTOR activity and hence that eRF3a belongs to the regulatory pathway of mTOR activity.

Aminoglycosides and other factors promoting stop codon readthrough in human cells.

Enhanced stop codon readthrough is a potential treatment strategy for diseases caused by nonsense mutations. Here, we compare readthrough levels induced by three types of factors: aminoglycoside antibiotics, suppressor tRNAs, and factors decreasing translation termination efficiency. We show that the highest levels of readthrough were obtained by prolonged treatment with aminoglycosides and suppressor tRNAs, whereas prolonged depletion of release factors induced only a moderate increase in readthrough. We discuss the benefits and inconvenients of the three types of factors for their use in the therapy of diseases caused by premature stop codons.

Involvement of human release factors eRF3a and eRF3b in translation termination and regulation of the termination complex formation.

eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.

Stop codon selection in eukaryotic translation termination: comparison of the discriminating potential between human and ciliate eRF1s.

During eukaryotic translation termination, eRF1 responds to three stop codons. However, in ciliates with variant genetic codes, only one or two codons function as a stop signal. To localize the region of ciliate eRF1 implicated in stop codon discrimination, we have constructed ciliate-human hybrid eRF1s by swapping regions of human eRF1 for the equivalent region of ciliate Euplotes eRF1. We have examined the formation of a cross-link between recombinant eRF1s and mRNA analogs containing the photoactivable 4-thiouridine (s(4)U) at the first position of stop and control sense codons. With human eRF1, this cross-link can be detected only when either stop or UGG codons are located in the ribosomal A site. Here we show that the cross-link of the Euplotes-human hybrid eRF1 is restricted to mRNAs containing UAG and UAA codons, and that the entire N-terminal domain of Euplotes eRF1 is involved in discriminating against UGA and UGG. On the basis of these results, we discuss the steps of the selection process that determine the accuracy of stop codon recognition in eukaryotes.