Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance.
The classical view on eukaryotic gene expression proposes the scheme of a forward flow for which fluctuations in mRNA levels upon a stimulus contribute to determine variations in mRNA availability for translation. Here we address this issue by simultaneously profiling with microarrays the total mRNAs (the transcriptome) and the polysome-associated mRNAs (the translatome) after EGF treatment of human cells, and extending the analysis to other 19 different transcriptome/translatome comparisons in mammalian cells following different stimuli or undergoing cell programs.
Triggering of the EGF pathway results in an early induction of transcriptome and translatome changes, but 90% of the significant variation is limited to the translatome and the degree of concordant changes is less than 5%. The survey of other 19 different transcriptome/translatome comparisons shows that extensive uncoupling is a general rule, in terms of both RNA movements and inferred cell activities, with a strong tendency of translation-related genes to be controlled purely at the translational level. By different statistical approaches, we finally provide evidence of the lack of dependence between changes at the transcriptome and translatome levels.
We propose a model of diffused independency between variation in transcript abundances and variation in their engagement on polysomes, which implies the existence of specific mechanisms to couple these two ways of regulating gene expression.
Polysomal; Profiling; Transcriptome; Translational; Control; Translatome
Toxicity of the environmental carcinogen chromate is known to involve sulfur starvation and also error-prone mRNA translation. Here we reconcile those facts using the yeast model. We demonstrate that: (i) cysteine and methionine starvation mimic Cr-induced translation errors, (ii) genetic suppression of S starvation suppresses Cr-induced mistranslation, and (iii) mistranslation requires cysteine and methionine biosynthesis. Therefore, Cr-induced S starvation is the cause of mRNA mistranslation. This establishes a single, novel pathway mediating the toxicity of chromate.
Chromate toxicity; sulfate transport; frameshift; Sul1; Saccharomyces cerevisiae
The trichothecene deoxynivalenol (DON), a common contaminant of cereal-based foods, is a ribotoxic mycotoxin known to activate innate immune cells in vivo and in vitro. Although it is recognized that DON induces transcription and mRNA stabilization of inflammation-associated mRNAs in mononuclear phagocytes, it is not known if this toxin affects translation of selected mRNA species in the cellular pool. To address this question, we employed a focused inflammation/autoimmunity PCR array to compare DON-induced changes in profiles of polysome-associated mRNA transcripts (translatome) to total cellular mRNA transcripts (transcriptome) in the RAW 264.7 murine macrophage model. Exposure to DON at 250ng/ml (0.84 µM) for 6h induced robust expression changes in inflammatory response genes including cytokines, cytokine receptors, chemokines, chemokine receptors, and transcription factors, with 73% of the changes being highly comparable within transcriptome and translatome populations. When expression changes of selected representative inflammatory response genes in the polysome and cellular mRNA pools were quantified in a follow-up study by real-time PCR, closely coordinated regulation of the translatome and transcriptome was confirmed; however, modest but significant differences in the relative expression of some genes within the two pools were also detectable. Taken together, DON’s capacity to alter translation expression of inflammation-associated genes appears to be driven predominantly by selective transcription and mRNA stabilization that have been reported previously; however, a small subset of these genes appear to be further regulated at the translational level.
macrophage; inflammation; deoxynivalenol; trichothecene mycotoxin; translatome.
Cells rapidly alter gene expression in response to environmental stimuli such as nutrients, hormones, and drugs. During the imposed “remodeling” of gene expression, changes in the levels of particular mRNAs do not necessarily correlate with those of the encoded proteins, which could in part rely on the differential recruitment of mRNAs to translating ribosomes. To systematically address this issue, we have established an approach to rapidly access the translational status of each mRNA in the yeast Saccharomyces cerevisiae by affinity purification of endogenously formed ribosomes and the analysis of associated mRNAs with DNA microarrays. Using this method, we compared changes in total mRNA levels (transcriptome) with ribosome associations (translatome) after the application of different conditions of cellular stress. Severe stresses, induced by amino acid depletion or osmotic shock, stimulated highly correlated responses affecting about 15% of both total RNA levels and translatome. Many of the regulated messages code for functionally related proteins, thus reflecting logical responses to the particular stress. In contrast, mild stress provoked by addition of Calcofluor-white and menadione altered the translatome of approximately 1% of messages with only marginal effects on total mRNA, suggesting largely uncorrelated responses of transcriptome and translatome. Among these putative translationally regulated messages were most components of the mitochondrial ATPase. Increased polysome associations of corresponding messages and higher mitochondrial ATPase activities upon treatment confirmed the relevance for regulation of this macromolecular complex. Our results suggest the presence of highly sensitive translational regulatory networks that coordinate functionally related messages. These networks are preferentially activated for rapid adaptation of cells to minor environmental perturbations.
Organisms respond to environmental or physiological changes by altering the amounts and activities of specific proteins that are necessary for their adaptation and survival. Importantly, protein levels can be modulated by changing either the rate of synthesis or the stability of the messenger RNA (mRNA or transcript), or the synthesis or stability of the protein itself. Scientists often measure global mRNA levels upon changing conditions to identify transcripts that are differentially regulated, and often the assumption is made that changes in transcript levels lead to corresponding changes in protein levels. Here, we systematically compared global transcript levels (transcriptome) with global alterations in the levels of ribosome association of transcripts (translatome) when yeast cells are exposed to different stresses to determine how significant the discrepancy between transcript and protein levels can be. We found that changes in the transcriptome correlate well with those in the translatome after application of harsh stresses that arrest cell growth. However, this correlation is generally lost under more mild stresses that do not affect cell growth. In this case, remodeling of gene expression is mainly executed at the translational level by modulating mRNA association with ribosomes. As one example, we show that expression for many components of the mitochondrial ATPase, the major energy production machinery in cells, is translationally but not transcriptionally activated under a specific mild stress condition. Our results therefore show that alteration of protein synthesis can be the dominant mediator of changes of gene expression during adaptation to minor changes in cellular needs.
During cellular adaptation to changing growth conditions, the extent of correlation between changes in transcriptional and translational regulation varies with the severity of the stress.
Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome).
Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region.
Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.
In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome) of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth.
Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy) and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation.
We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein synthesis.
Translational regulation; mRNA Ribosome; Translatome; Statistical modeling; Lactococcus lactis
In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed.
Post-transcriptional control of mRNA transcript processing by RNA binding proteins (RBPs) is an important step in the regulation of gene expression and protein production. The post-transcriptional regulatory network is similar in complexity to the transcriptional regulatory network and is thought to be organized in RNA regulons, coherent sets of functionally related mRNAs combinatorially regulated by common RBPs. We integrated genome-wide transcriptional and translational expression data in yeast with large-scale regulatory networks of transcription factor and RBP binding interactions to analyze the functional organization of post-transcriptional regulation and RNA regulons at a system level. We found that post-transcriptional feedback loops and mixed bifan motifs are overrepresented in the integrated regulatory network and control the coordinated translation of RNA regulons, manifested as clusters of functionally related mRNAs which are strongly coexpressed in the translatome data. These translatome clusters are more functionally coherent than transcriptome clusters and are expressed with higher mRNA and protein levels and less noise. Our results show how the post-transcriptional network is intertwined with the transcriptional network to regulate gene expression in a coordinated way and that the integration of heterogeneous genome-wide datasets allows to relate structure to function in regulatory networks at a system level.
mRNA degradation is coupled with the process of mRNA translation. For example, an mRNA molecule, on which translation is prematurely terminated because of a nonsense codon, may be rapidly degraded. This nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae is mediated by the Upf1 and Upf2 proteins. Yeast mRNAs can also be selectively destabilized by limiting the rate of translation initiation. Two such destabilized mRNAs, from the SSA1 and SSA2 genes, have been identified using temperature-sensitive mutations affecting the Prt1 component of eukaryotic initiation factor 3. For SSA1 and SSA2 mRNAs, and for structurally modified SSA mRNA derivatives, I show here that degradation is triggered when translation initiation is limited but ongoing. This initiation-dependent mRNA degradation is limited to a subset of mRNAs that includes at least those from the SSA1 and SSA2 genes, and occurs through Upf1- and Upf2-mediated processes, although sequence elements characteristic of nonsense-mediated decay are not evident in these mRNAs.
The mistranslation-induced protein misfolding hypothesis predicts that selection should prefer high-fidelity codons at sites at which translation errors are structurally disruptive and lead to protein misfolding and aggregation. To test this hypothesis, we analyzed the relationship between codon usage bias and protein structure in the genomes of four model organisms, Escherichia coli, yeast, fly, and mouse. Using both the Mantel–Haenszel procedure, which applies to categorical data, and a newly developed association test for continuous variables, we find that translationally optimal codons associate with buried residues and also with residues at sites where mutations lead to large changes in free energy (ΔΔG). In each species, only a subset of all amino acids show this signal, but most amino acids show the signal in at least one species. By repeating the analysis on a reduced data set that excludes interdomain linkers, we show that our results are not caused by an association of rare codons with solvent-accessible linker regions. Finally, we find that our results depend weakly on expression level; the association between optimal codons and buried sites exists at all expression levels, but increases in strength as expression level increases.
codon usage bias; optimal codon; protein structure; protein evolution; translational accuracy selection
Light, a dynamic environmental parameter, is an essential regulator of plant growth and development. Light-regulated transcriptional networks are well documented, whereas light-regulated post-transcriptional regulation has received limited attention. In this study, dynamics in translation of cytosolic mRNAs were evaluated at the genome-level in Arabidopsis thaliana seedlings grown under a typical light/dark diurnal regime, shifted to darkness at midday, and then re-illuminated. One-hour of unanticipated darkness reduced levels of polysomes by 17% in a manner consistent with inhibition of initiation of translation. This down-regulation of translation was reversed within 10 min of re-illumination. Quantitative comparison of the total cellular population of transcripts (the transcriptome) to those associated with one or more 80S ribosome (the translatome) identified over 1600 mRNAs that were differentially translated in response to light availability. Unanticipated darkness limited both transcription and translation of mRNAs encoding components of the photosynthetic machinery. Many mRNAs encoding proteins associated with the energy demanding process of protein synthesis were stable but sequestered in the dark, in a rapidly reversible manner. A meta-analysis determined these same transcripts were similarly and coordinately regulated in response to changes in oxygen availability. The dark and hypoxia translationally repressed mRNAs lack highly supported candidate RNA-regulatory elements but are characterized by G + C-rich 5′-untranslated regions. We propose that modulation of translation of a subset of cellular mRNAs functions as an energy conservation mechanism.
light-regulated gene expression; dark-regulated gene expression; re-illumination; post-transcriptional control; mRNA sequence elements; ribosomal proteins; energy status
There is now considerable evidence supporting the view that codon usage is
frequently under selection for translational accuracy. There are, however,
multiple forms of inaccuracy (missense, premature termination, and frameshifting
errors) and pinpointing a particular error process behind apparently adaptive
mRNA anatomy is rarely straightforward. Understanding differences in the fitness
costs associated with different types of translational error can help us devise
critical tests that can implicate one error process to the exclusion of others.
To this end, we present a model that captures distinct features of frameshifting
cost and apply this to 641 prokaryotic genomes. We demonstrate that, although it
is commonly assumed that the ribosome encounters an off-frame stop codon soon
after the frameshift and costs of mis-elongation are therefore limited, genomes
with high GC content typically incur much larger per-error costs. We go on to
derive the prediction, unique to frameshifting errors, that differences in
translational robustness between the 5′ and 3′ ends of genes
should be less pronounced in genomes with higher GC content. This prediction we
show to be correct. Surprisingly, this does not mean that GC-rich organisms
necessarily carry a greater fitness burden as a consequence of accidental
frameshifting. Indeed, increased per-error costs are often more than
counterbalanced by lower predicted error rates owing to more diverse anticodon
repertoires in GC-rich genomes. We therefore propose that selection on tRNA
repertoires may operate to reduce frameshifting errors.
; translational error; GC content; translational accuracy; codon usage bias; tRNA repertoire
Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level (“expressome”). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.
Bacteria are frequently exposed to disadvantageous conditions, like elevated temperatures or nutrient depletion. The ability to maintain viable populations is based on cellular stress responses, which are regulated in a complex manner with different outputs on different regulatory levels. For example, mRNA levels do not ultimately determine protein amounts since translation of mRNAs can be influenced irrespective of mRNA levels. To appreciate nature and frequency of these regulatory events, multi-layered experimental approaches are required on a global scale. The photo-oxidative stress response of the purple bacterium Rhodobacter sphaeroides was chosen as a model. Changes of total mRNAs (transcriptome) and ribosomal-bound mRNAs (translatome) were monitored by microarrays. The proteome was assessed by mass spectrometry, applying a “bacterial SILAC standard” for indirect quantification, an approach which additionally identified new open reading frames. Integration of the three expression levels provided a comprehensive insight into regulatory events and identified new stress-responsive genes, including genes for transcriptional regulators and for quorum sensing. We found that translational control exceeds simple regulation on the transcriptional level. Furthermore, polar expression patterns within inducible operons point at the possibility of expression fine-tuning by gene positioning.
Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.
Protein synthesis is a template polymerization process composed by three main steps: initiation, elongation, and termination. During translation, ribosomes are engaged into polysomes whose size is used for the quantitative characterization of translatome. However, simultaneous transcription and translation in the bacterial cytosol complicates the analysis of translatome data. We established a procedure for robust estimation of the ribosomal density in hundreds of genes from Lactococcus lactis polysome size measurements. We used a mechanistic model of translation to integrate the information about the ribosomal density and for the first time we estimated the protein synthesis rate for each gene and identified the rate limiting steps. Contrary to conventional considerations, we find significant number of genes to be elongation limited. This number increases during stress conditions compared to optimal growth and proteins synthesized at maximum rate are predominantly elongation limited. Consistent with bacterial physiology, we found proteins with similar rate and control characteristics belonging to the same functional categories. Under stress conditions, we found that synthesis rate of regulatory proteins is becoming comparable to proteins favored under optimal growth. These findings suggest that the coupling of metabolic states and protein synthesis is more important than previously thought.
Post-transcriptional regulation is important for the understanding of gene expression control. Our work is a genome-scale analysis of the translation steps of protein synthesis from transcripts. We have developed a mathematical model to integrate and analyze experimental ribosome density of hundreds of transcripts of Lactococcus lactis, providing robust estimation of polysome sizes. Using a mechanistic approach we have modeled for the first time in bacteria the protein synthesis rate for each gene and determined by control analysis the limiting rate between initiation, elongation and termination. Highly expressed proteins belonged to the group of the proteins with high synthesis rate and were controlled by elongation. Unexpectedly, a significant number of genes under elongation limitation were found although initiation was generally believed to be limiting. In addition, we showed that translation rate and control were in agreement with cellular requirements in cells growing in optimal environment but also in cells under nutritional limitation. This work provided a better understanding of translational regulation in bacteria and demonstrated how protein synthesis control was closely related to cellular metabolic states.
mRNAs that contain premature stop codons are selectively degraded in all eukaryotes tested, a phenomenon termed “nonsense-mediated mRNA decay” (NMD) or “mRNA surveillance.” NMD may function to eliminate aberrant mRNAs so that they are not translated, because such mRNAs might encode deleterious polypeptide fragments. In both yeasts and nematodes, NMD is a nonessential system. Mutations affecting three yeast UPF genes or seven nematode smg genes eliminate NMD. We report here the molecular analysis of smg-2 of Caenorhabditis elegans. smg-2 is homologous to UPF1 of yeast and to RENT1 (also called HUPF1), a human gene likely involved in NMD. The striking conservation of SMG-2, Upf1p, and RENT1/HUPF1 in both sequence and function suggests that NMD is an ancient system, predating the divergence of most eukaryotes. Despite similarities in the sequences of SMG-2 and Upf1p, expression of Upf1p in C. elegans does not rescue smg-2 mutants. We have prepared anti-SMG-2 polyclonal antibodies and identified SMG-2 on Western blots. SMG-2 is phosphorylated, and mutations of the six other smg genes influence the state of SMG-2 phosphorylation. In smg-1, smg-3, and smg-4 mutants, phosphorylation of SMG-2 was not detected. In smg-5, smg-6, and smg-7 mutants, a phosphorylated isoform of SMG-2 accumulated to abnormally high levels. In smg-2(r866) and smg-2(r895) mutants, which harbor single amino acid substitutions of the SMG-2 nucleotide binding site, phosphorylated SMG-2 accumulated to abnormally high levels, similar to those observed in smg-5, smg-6, and smg-7 mutants. We discuss these results with regard to the in vivo functions of SMG-2 and NMD.
Upf1p, Nmd2p, and Upf3p regulate the degradation of yeast mRNAs that contain premature translation termination codons. These proteins also appear to regulate the fidelity of termination, allowing translational suppression in their absence. Here, we have devised a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100), and used it to determine the regulatory roles of the UPF/NMD gene products. Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy-number can1-100 expression in wild-type cells caused an increase in mRNA abundance similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. Our findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay. Consistent with these relationships, Upf1p, Nmd2p, and Upf3p were shown to be present at 1,600, 160, and 80 molecules per cell, levels that underscored the importance of Upf1p but minimized the likelihood that these proteins were associated with all ribosomes or that they functioned as a stoichiometric complex.
Codon usage and codon-pair context are important gene primary structure features that influence mRNA decoding fidelity. In order to identify general rules that shape codon-pair context and minimize mRNA decoding error, we have carried out a large scale comparative codon-pair context analysis of 119 fully sequenced genomes.
We have developed mathematical and software tools for large scale comparative codon-pair context analysis. These methodologies unveiled general and species specific codon-pair context rules that govern evolution of mRNAs in the 3 domains of life. We show that evolution of bacterial and archeal mRNA primary structure is mainly dependent on constraints imposed by the translational machinery, while in eukaryotes DNA methylation and tri-nucleotide repeats impose strong biases on codon-pair context.
The data highlight fundamental differences between prokaryotic and eukaryotic mRNA decoding rules, which are partially independent of codon usage.
Nonsense-mediated mRNA decay (NMD), the loss of mRNAs carrying premature stop codons, is a process by which cells recognize and degrade nonsense mRNAs to prevent possibly toxic effects of truncated peptides. Most mammalian nonsense mRNAs are degraded while associated with the nucleus, but a few are degraded in the cytoplasm; at either site, there is a requirement for translation and for an intron downstream of the early stop codon. We have examined the NMD of a mutant HEXA message in lymphoblasts derived from a Tay-Sachs disease patient homozygous for the common frameshift mutation 1278ins4. The mutant mRNA was nearly undetectable in these cells and increased to approximately 40% of normal in the presence of the translation inhibitor cycloheximide. The stabilized transcript was found in the cytoplasm in association with polysomes. Within 5 h of cycloheximide removal, the polysome-associated nonsense message was completely degraded, while the normal message was stable. The increased lability of the polysome-associated mutant HEXA mRNA shows that NMD of this endogenous mRNA occurred in the cytoplasm. Transfection of Chinese hamster ovary cells showed that expression of an intronless HEXA minigene harboring the frameshift mutation or a closely located nonsense codon resulted in half the normal mRNA level. Inclusion of multiple downstream introns decreased the abundance further, to about 20% of normal. Thus, in contrast to other systems, introns are not absolutely required for NMD of HEXA mRNA, although they enhance the low-HEXA-mRNA phenotype.
Proteins are precisely assembled with amino acids by matching the anticodons of charged transfer RNAs to nucleotide triplets in mRNA sequences. Accurate translation depends on the specific coupling of cognate amino acids and tRNAs – a step carried out by aminoacyl-tRNA synthetases (aaRSs) and that generates the genetic code. Due to their intrinsic similarity, aaRSs developed highly differentiated structures to discriminate between amino acids at the active site for aminoacylation. Because this discrimination is not sufficient to prevent toxic mistranslation, aaRSs developed separate structures to further refine recognition by proofreading. From comprehensive structural studies on aaRSs, many of the molecular details have been elucidated for the recognition of cognate amino acids and for the misactivation and editing of noncognate amino acids, Here we review recent advances in the structural description of the binding, activation and editing of amino acids, which collectively reveal many aspects of the fine-tuned systems that resulted in a robust and universal genetic code.
Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Δ mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Δ cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Δ cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.
A C→U mutation (rdn5) in the conserved sarcin/ricin domain of yeast 25S rRNA has been shown to cause translational suppression and paromomycin resistance. It also separates the killing from the misreading effect of this antibiotic. We confirm these findings and provide in vitro evidence that rdn5 causes a 3-fold increase in translational errors and resistance to paromomycin. The role of this 25S rRNA domain in ribosome's decoding function was further demonstrated when 60S subunits from rdn5 cells were combined with 40S subunits from cells carrying an error-prone mutation in the eukaryotic accuracy center ribosomal protein S23, an homologue of Escherichia coli S12. These hybrids exhibited an error frequency similar to that of rdn5 alone, despite the error-prone mutation in S23. This was accompanied by extreme resistance to paromomycin, unlike the effects of the individual mutations. Furthermore, rdn5 lowers peptidyltransferase activity measured as a second-order rate constant (kcat/Ks) corresponding to the rate of peptide bond formation. This mutation was also found to affect translocation. Elongation factor 2 (EF2)-dependent translocation of Ac-Phe-tRNA from the A- to P-site was achieved at an EF2 concentration 3.5 times lower than in wild type. In conclusion, the sarcin/ricin domain of 25S rRNA influences decoding, peptide bond formation and translocation.
mRNAs are monitored for errors in gene expression by RNA surveillance, in which mRNAs that cannot be fully translated are degraded by the nonsense-mediated mRNA decay pathway (NMD). RNA surveillance ensures that potentially deleterious truncated proteins are seldom made. NMD pathways that promote surveillance have been found in a wide range of eukaryotes. In Saccharomyces cerevisiae, the proteins encoded by the UPF1, UPF2, and UPF3 genes catalyze steps in NMD and are required for RNA surveillance. In this report, we show that the Upf proteins are also required to control the total accumulation of a large number of mRNAs in addition to their role in RNA surveillance. High-density oligonucleotide arrays were used to monitor global changes in the yeast transcriptome caused by loss of UPF gene function. Null mutations in the UPF genes caused altered accumulation of hundreds of mRNAs. The majority were increased in abundance, but some were decreased. The same mRNAs were affected regardless of which of the three UPF gene was inactivated. The proteins encoded by UPF-dependent mRNAs were broadly distributed by function but were underrepresented in two MIPS (Munich Information Center for Protein Sequences) categories: protein synthesis and protein destination. In a UPF+ strain, the average level of expression of UPF-dependent mRNAs was threefold lower than the average level of expression of all mRNAs in the transcriptome, suggesting that highly abundant mRNAs were underrepresented. We suggest a model for how the abundance of hundreds of mRNAs might be controlled by the Upf proteins.
A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.