Ribosomes in the balance: structural equilibrium ensures translational fidelity and proper gene expression

At equilibrium, empty ribosomes freely transit between the rotated and un-rotated states. In the cell, the binding of two translation elongation factors to the same general region of the ribosome stabilizes one state over the other. These stabilized states are resolved by expenditure of energy in the form of GTP hydrolysis. A prior study employing mutants of a late assembling peripheral ribosomal protein suggested that ribosome rotational status determines its affinity for elongation factors, and hence translational fidelity and gene expression. Here, mutants of the early assembling integral ribosomal protein uL2 are used to test the generality of this hypothesis. rRNA structure probing analyses reveal that mutations in the uL2 B7b bridge region shift the equilibrium toward the rotated state, propagating rRNA structural changes to all of the functional centers of ribosome. Structural disequilibrium unbalances ribosome biochemically: rotated ribosomes favor binding of the eEF2 translocase and disfavor that of the elongation ternary complex. This manifests as specific translational fidelity defects, impacting the expression of genes involved in telomere maintenance. A model is presented describing how cyclic intersubunit rotation ensures the unidirectionality of translational elongation, and how perturbation of rotational equilibrium affects specific aspects of translational fidelity and cellular gene expression.     

Identification of a novel alternatively spliced isoform of the ribosomal uL10 protein

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10β. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases – trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10β protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10β is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10β undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10β, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.     

The uL10 protein,a component of the ribosomal P-stalk,is released from the ribosome in nucleolar stress

The ribosomal uL10 protein, formerly known as P0, is an essential element of the ribosomal GTPase-associated center responsible for the interplay with translational factors during various stages of protein synthesis. In eukaryotic cells, uL10 binds two P1/P2 protein heterodimers to form a pentameric P-stalk, described as uL10-(P1-P2)₂, which represents the functional form of these proteins on translating ribosomes. Unlike most ribosomal proteins, which are incorporated into pre-ribosomal particles during early steps of ribosome biogenesis in the nucleus, P-stalk proteins are attached to the 60S subunit in the cytoplasm. Although the primary role of the P-stalk is related to the process of translation, other extraribosomal functions of its constituents have been proposed, especially for the uL10 protein; however, the list of its activities beyond the ribosome is still an open question. Here, by the combination of biochemical and advanced fluorescence microscopy techniques, we demonstrate that upon nucleolar stress induction the uL10 protein accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein. Importantly, using a novel approach, FRAP-AC (FRAP after photoConversion), we have shown that the ribosome-free pool of uL10 represents a population of proteins released from pre-existing ribosomes. Taken together, our data indicate that the presence of uL10 on the ribosomes is affected in stressed cells, thus it might be considered as a regulatory element responding to environmental fluctuations.     

Heat Shock-Induced Accumulation of Translation Elongation and Termination Factors Precedes Assembly of Stress Granules in S. cerevisiae

In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.     

A novel single amino acid change in small subunit ribosomal protein S5 has profound effects on translational fidelity

S5 is a small subunit ribosomal protein (r-protein) linked to the functional center of the 30S ribosomal subunit. In this study we have identified a unique amino acid mutation in Escherichia coli S5 that produces spectinomycin-resistance and cold sensitivity. This mutation significantly alters cell growth, folding of 16S ribosomal RNA, and translational fidelity. While translation initiation is not affected, both +1 and -1 frameshifting and nonsense suppression are greatly enhanced in the mutant strain. Interestingly, this S5 ribosome ambiguity-like mutation is spatially remote from previously identified S5 ribosome ambiguity (ram) mutations. This suggests that the mechanism responsible for ram phenotypes in the novel mutant strain is possibly distinct from those proposed for other known S5 (and S4) ram mutants. This study highlights the importance of S5 in ribosome function and cell physiology, and suggests that translational fidelity can be regulated in multiple ways.     

Cytoplasmic complex of p53 and eEF2

We have shown previously that cytoplasmic p53 is covalently linked to 5.8S rRNA. The covalent complex is associated with a small subset of polyribosomes, which includes polyribosomes translating p53 mRNA. Because 5.8S rRNA resides in or near the ribosomal P site, our findings suggested involvement of p53 in translational regulation. Ninety-seven kiloDaltons eEF2 was found to coimmunoprecipitate in a salt-stable complex with p53. The 97 kDa species was identified as eEF2, because it was (1) recognized by a polyclonal antiserum specific for eEF2, (2) ADP-ribosylated by diphtheria toxin (DT), and (3) radiolabeled by gamma-32P-azido-GTP and UV-irradiation. p53 and eEF2 sedimented in sucrose gradients in both polyribosomal and subribosomal fractions. Subribosomal p53 can bind eEF2 without the mediation of ribosomes, because (1) it binds subribososomal eEF2, (2) it binds phosphorylated eEF2, and (3) subribosomal p53-bound eEF2 can be ADP-ribosylated by DT. No effect of p53 activation was found on eEF2 expression or phosphorylation. However, the binding of eEF2 to p53 decreased when cytoplasmic p53 migrated to the nucleus. Renaturation of temperature sensitive A135V mutant p53 (ts-p53) was found to alter the sensitivity of p53 mRNA translation, but not bulk mRNA translation, to the translocation-specific elongation inhibitor, cycloheximide (Cx). The association of p53 with two translational components involved in ribosomal translocation, eEF2 and 5.8S rRNA, and the effect of p53 on sensitivity to the translocation inhibitor, Cx, as well as the known molecular interactions of these components in the ribosome suggest involvement of p53 in elongation.     

Impairing the production of ribosomal RNA activates mammalian target of rapamycin complex 1 signalling and downstream translation factors

Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all these processes. Here, we show that, in human cells, impaired rRNA processing, caused by expressing an interfering mutant of BOP1 or by knocking down components of the PeBoW complex elicits activation of mTORC1 signalling. This leads to enhanced phosphorylation of its substrates S6K1 and 4E-BP1, and stimulation of proteins involved in translation initiation and elongation. In particular, we observe both inactivation and downregulation of the eukaryotic elongation factor 2 kinase, which normally inhibits translation elongation. The latter effect involves decreased expression of the eEF2K mRNA. The mRNAs for ribosomal proteins, whose translation is positively regulated by mTORC1 signalling, also remain associated with ribosomes. Therefore, our data demonstrate that disrupting rRNA production activates mTORC1 signalling to enhance the efficiency of the translational machinery, likely to help compensate for impaired ribosome production.     

Eukaryotic initiation factor 6 mediates a continuum between 60S ribosome biogenesis and translation

Eukaryotic ribosome biogenesis and translation are linked processes that limit the rate of cell growth. Although ribosome biogenesis and translation are mainly controlled by distinct factors, eukaryotic initiation factor 6 (eIF6) has been found to regulate both processes. eIF6 is a necessary protein with a unique anti‐association activity, which prevents the interaction of 40S ribosomal subunits with 60S subunits through its binding to 60S ribosomes. In the nucleolus, eIF6 is a component of the pre‐ribosomal particles and is required for the biogenesis of 60S subunits, whereas in the cytoplasm it mediates translation downstream from growth factors. The translational activity of eIF6 could be due to its anti‐association properties, which are regulated by post‐translational modifications; whether this anti‐association activity is required for the biogenesis and nuclear export of ribosomes is unknown. eIF6 is necessary for tissue‐specific growth and oncogene‐driven transformation, and could be a new rate‐limiting step for the initiation of translation.     

A new plant protein interacts with eIF3 and 60S to enhance virus‐activated translation re‐initiation

The plant viral re‐initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re‐initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ;Here, we report a new plant factor—re‐initiation supporting protein (RISP)—that enhances TAV function in re‐initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C‐terminus of ribosomal protein L24. RISP pre‐bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co‐localize in epidermal cells of infected plants, and eIF3–TAV–RISP–L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3‐bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery.     

Eukaryotic elongation factor 2 can bind to the synthetic oligoribonucleotide that mimics sarcin/ricin domain of rat 28S ribosomal RNA

Eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to P site by binding to the ribosome. In this work, the complex formation of rat liver eEF2 with a synthetic oligoribonucleotide (SRD RNA) that mimics sarcin/ricin domain of rat 28S ribosomal RNA is invested in vitro. Purified eEF2 can specifically bind SRD RNA to form a stable complex. tRNA competes with SRD RNA in binding to eEF2 in a less extent. Pretreatment of eEF2 with GDP or ADP-ribosylation of eEF2 by diphtheria toxin can obviously reduce the ability of eEF2 to form the complex with the synthetic oligoribonucleotide. These results indicate that eEF2 is likely to bind directly to the sarcin/ricin domain of 28S ribosomal RNA in the process of protein synthesis.     

All translation elongation factors and the e, f, and h subunits of translation initiation factor 3 are encoded by 5'-terminal oligopyrimidine (TOP) mRNAs

Terminal oligopyrimidine (TOP) mRNAs (encoded by the TOP genes) are identified by a sequence of 6–12 pyrimidines at the 5′ end and by a growth-associated translational regulation. All vertebrate genes for the 80 ribosomal proteins and some other genes involved, directly or indirectly, in translation, are TOP genes. Among the numerous translation factors, only eEF1A and eEF2 are known to be encoded by TOP genes, most of the others having not been analyzed. Here, we report a systematic analysis of the human genes for translation factors. Our results show that: (1) all five elongation factors are encoded by TOP genes; and (2) among the initiation and termination factors analyzed, only eIF3e, eIF3f, and eIF3h exhibit the characteristics of TOP genes. Interestingly, these three polypeptides have been recently shown to constitute a specific subgroup among eIF3 subunits. In fact, eIF3e, eIF3f, and eIF3h are the part of the functional core of eIF3 that is not conserved in Saccharomyces cerevisiae. It has been hypothesized that they are regulatory subunits, and the fact that they are encoded by TOP genes may be relevant for their function.     

Ribosomal Heterogeneity of Maize Tissues: Insights of Biological Relevance

In recent years, the selective role of ribosomes in the translational process of eukaryotes has been suggested. Evidence indicates that ribosomal heterogeneity at the level of protein stoichiometry and phosphorylation status differs among organisms, suggesting ribosomal specialization according to the state of development and the surrounding environment. During germination, protein synthesis is an active process that begins with the translation of the mRNAs stored in quiescent seeds and continues with the newly synthesized mRNAs. In this study, we identified differences in the abundance of ribosomal proteins (RPs) in maize embryos at different developmental stages. The relative quantification of RPs during germination revealed changes in six small subunit proteins, S3 (uS3), S5 (uS7), S7 (eS7), two isoforms of S17 (eS17), and S18 (uS13), and nine large subunit proteins, L1 (uL1), L5 (uL18), two isoforms of P0 (uL10), L11 (uL5), L14 (eL14), L15 (eL15), L19 (eL19), and L27 (eL27). Further analysis of ribosomal protein phosphorylation during germination revealed that the phosphorylation of PRP0 (uL10) and P1 increased and that of PRS3 (uS3) decreased in germinated versus quiescent embryos. The addition of insulin during germination increased the phosphorylation of the P1 protein, suggesting that its phosphorylation is controlled by the TOR pathway. Our results indicate that a heterogeneous ribosomal population provides to maize ribosomes during germination a different ability to translate mRNAs, suggesting another level of regulation by the ribosomes.     

Increased -1 ribosomal frameshifting efficiency by yeast prion-like phenotype [PSI+]

Programmed -1 ribosomal frameshifting (-1RF) is usually regulated by a slippery sequence and an RNA secondary structure but can be affected by the slippery sequence combined with translational perturbation. Dramatic increases of -1RF efficiencies arise from the slippery sequences in [PSI+], an epigenetic modifier of translation termination fidelity. Curing of [PSI+] abolished such increases of -1RF efficiency. Enhanced -1RF frequency at the slippery sequences could be another physiological effect induced directly or indirectly by the perturbation of the translation process in [PSI+] cells.     

Translation inhibition from a distance: The small RNA SgrS silences a ribosomal protein S1-dependent enhancer

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA–mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5ʹ untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.