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1.  Stress-Dependent Coordination of Transcriptome and Translatome in Yeast 
PLoS Biology  2009;7(5):e1000105.
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.
Author Summary
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.
PMCID: PMC2675909  PMID: 19419242
2.  The Yeast La Related Protein Slf1p Is a Key Activator of Translation during the Oxidative Stress Response 
PLoS Genetics  2015;11(1):e1004903.
The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p ‘closed loop’ complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control.
Author Summary
All organisms must respond to changes in their external environment such as exposure to different stresses. The availability of genome sequences and post-genomic technologies has enabled the analysis of these adaptive responses at the molecular level in terms of altered gene expression profiles. However, relatively few studies have focused on how cells regulate the translation of mRNA into protein in response to stress, despite its fundamental role in gene expression pathways. In this study, we show that a previously identified RNA-binding protein called Slf1p plays a major role in mRNA-specific regulation of translation during oxidative stress conditions and is necessary to promote the translation of stress-responsive mRNAs. This protein is a member of the so-called “La-related” family of proteins that have not been well characterized, although they are conserved throughout evolution. Exposure to oxidants is known to cause a general down-regulation of protein synthesis, although many stress response proteins are able to overcome this inhibition and increase their protein levels following stress by as yet unknown mechanisms. Our experiments offer one possible explanation, as they show that Slf1p plays a critical role in enhancing translation of many of these proteins, including many that are necessary for the cellular stress response.
PMCID: PMC4287443  PMID: 25569619
3.  Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control 
Combining translating ribosome affinity purification with RNA-seq for cell-specific profiling of translating RNAs in developing flowers.Cell type comparisons of cell type-specific hormone responses, promoter motifs, coexpressed cognate binding factor candidates, and splicing isoforms.Widespread post-transcriptional regulation at both the intron splicing and translational stages.A new class of noncoding RNAs associated with polysomes.
What constitutes a differentiated cell type? How much do cell types differ in their transcription of genes? The development and functions of tissues rely on constant interactions among distinct and nonequivalent cell types. Answering these questions will require quantitative information on transcriptomes, proteomes, protein–protein interactions, protein–nucleic acid interactions, and metabolomes at cellular resolution. The systems approaches emerging in biology promise to explain properties of biological systems based on genome-wide measurements of expression, interaction, regulation, and metabolism. To facilitate a systems approach, it is essential first to capture such components in a global manner, ideally at cellular resolution.
Recently, microarray analysis of transcriptomes has been extended to a cellular level of resolution by using laser microdissection or fluorescence-activated sorting (for review, see Nelson et al, 2008). These methods have been limited by stresses associated with cellular separation and isolation procedures, and biases associated with mandatory RNA amplification steps. A newly developed method, translating ribosome affinity purification (TRAP; Zanetti et al, 2005; Heiman et al, 2008; Mustroph et al, 2009), circumvents these problems by epitopetagging a ribosomal protein in specific cellular domains to selectively purify polysomes. We combined TRAP with deep sequencing, which we term TRAP-seq, to provide cell-level spatiotemporal maps for Arabidopsis early floral development at single-base resolution.
Flower development in Arabidopsis has been studied extensively and is one of the best understood aspects of plant development (for review, see Krizek and Fletcher, 2005). Genetic analysis of homeotic mutants established the ABC model, in which three classes of regulatory genes, A, B and C, work in a combinatorial manner to confer organ identities of four whorls (Coen and Meyerowitz, 1991). Each class of regulatory gene is expressed in a specific and evolutionarily conserved domain, and the action of the class A, B and C genes is necessary for specification of organ identity (Figure 1A).
Using TRAP-seq, we purified cell-specific translating mRNA populations, which we and others call the translatome, from the A, B and C domains of early developing flowers, in which floral patterning and the specification of floral organs is established. To achieve temporal specificity, we used a floral induction system to facilitate collection of early stage flowers (Wellmer et al, 2006). The combination of TRAP-seq with domain-specific promoters and this floral induction system enabled fine spatiotemporal isolation of translating mRNA in specific cellular domains, and at specific developmental stages.
Multiple lines of evidence confirmed the specificity of this approach, including detecting the expression in expected domains but not in other domains for well-studied flower marker genes and known physiological functions (Figures 1B–D and 2A–C). Furthermore, we provide numerous examples from flower development in which a spatiotemporal map of rigorously comparable cell-specific translatomes makes possible new views of the properties of cell domains not evident in data obtained from whole organs or tissues, including patterns of transcription and cis-regulation, new physiological differences among cell domains and between flower stages, putative hormone-active centers, and splicing events specific for flower domains (Figure 2A–D). Such findings may provide new targets for reverse genetics studies and may aid in the formulation and validation of interaction and pathway networks.
Beside cellular heterogeneity, the transcriptome is regulated at several steps through the life of mRNA molecules, which are not directly available through traditional transcriptome profiling of total mRNA abundance. By comparing the translatome and transcriptome, we integratively profiled two key posttranscriptional control points, intron splicing and translation state. From our translatome-wide profiling, we (i) confirmed that both posttranscriptional regulation control points were used by a large portion of the transcriptome; (ii) identified a number of cis-acting features within the coding or noncoding sequences that correlate with splicing or translation state; and (iii) revealed correlation between each regulation mechanism and gene function. Our transcriptome-wide surveys have highlighted target genes transcripts of which are probably under extensive posttranscriptional regulation during flower development.
Finally, we reported the finding of a large number of polysome-associated ncRNAs. About one-third of all annotated ncRNA in the Arabidopsis genome were observed co-purified with polysomes. Coding capacity analysis confirmed that most of them are real ncRNA without conserved ORFs. The group of polysome-associated ncRNA reported in this study is a potential new addition to the expanding riboregulator catalog; they could have roles in translational regulation during early flower development.
Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.
PMCID: PMC2990639  PMID: 20924354
Arabidopsis; flower; intron; transcriptome; translation
4.  Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting 
PLoS Genetics  2012;8(8):e1002902.
Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.
Author Summary
Chronic diseases including obesity and associated metabolic abnormalities have become the greatest threat to human health worldwide. How metabolic organs and organelles adapt to nutritional fluctuations, or fail to do so, remains incompletely understood. To explore these issues, we developed a new platform to explore translational responses in the liver, a critical organ for metabolic homeostasis. In this translatomic platform, we integrated polysome profiling and global analysis of polysome-associated mRNAs to systematically quantify protein synthesis on each transcript in obesity and during fasting. Our analysis demonstrated for the first time that protein synthesis is progressively suppressed in the obese liver and that the overall translatome profile of obese liver markedly resembles that of fasting lean mice, particularly in mitochondrial function and bile metabolism. We also examined the physiological impact of some of these alterations and concluded that aberrant bile acid metabolism in the obese liver represents a novel mechanism contributing to hyperglycemia and continuous weight gain. Together, our work reveals abnormal translational regulation as a novel aspect of obesity that could impact future directions in metabolic disease treatment, and we believe translatome profiling represents a new approach to unravel complex mechanisms regulating cellular function and disease pathology.
PMCID: PMC3426552  PMID: 22927828
5.  Integrative “Omics”-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses 
PLoS Genetics  2013;9(6):e1003576.
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.
Author Summary
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.
PMCID: PMC3688512  PMID: 23818867
6.  Impact of translational error-induced and error-free misfolding on the rate of protein evolution 
Theoretical calculations suggest that, in addition to translational error-induced protein misfolding, a non-negligible fraction of misfolded proteins are error free.We propose that the anticorrelation between the expression level of a protein and its rate of sequence evolution be explained by an overarching protein-misfolding-avoidance hypothesis that includes selection against both error-induced and error-free protein misfolding, and verify this model by a molecular-level evolutionary simulation.We provide strong empirical evidence for the protein-misfolding-avoidance hypothesis, including a positive correlation between protein expression level and stability, enrichment of misfolding-minimizing codons and amino acids in highly expressed genes, and stronger evolutionary conservation of residues in which nonsynonymous changes are more likely to increase protein misfolding.
The rate of protein sequence evolution has long been of central interest to molecular evolutionists. Different proteins of the same species evolve at vastly different rates, which is commonly explained by a variation in functional constraint among different proteins (Kimura and Ohta, 1974). However, it is unclear how to quantify the functional constraint of a protein from the knowledge of its function. In the past decade, various types of genomic data from model organisms have been examined to look for the determinants of the rate of protein sequence evolution. The most unexpected discovery was a very strong anticorrelation between the expression level and evolutionary rate of a protein (E–R anticorrelation) (Pal et al, 2001). The prevailing explanation of the E–R anticorrelation is the translational robustness hypothesis (Drummond et al, 2005). This hypothesis posits that mistranslation induces protein misfolding, which is toxic to cells (Figure 1). Consequently, highly expressed proteins are under stronger pressures to be translationally robust and thus are more constrained in sequence evolution. However, the impact of the other source of misfolded proteins, translational error-free proteins (Figure 1), has not been evaluated. By theoretical calculation, computer simulation, and empirical data analysis, we examined the role of selection against both error-induced and error-free protein misfolding in creating the E–R correlation.
Our theoretical calculations suggested that a non-negligible fraction of misfolded proteins are error free. We estimated that when a protein is not very stable, on average ∼20% of misfolded molecules are error free. However, when a protein is very stable, this fraction reduces to ∼5%, which is probably a result of natural selection against protein misfolding.
We conducted a molecular-level evolutionary simulation (Figure 2A) using three different schemes: error-induced misfolding only, error-free misfolding only, and both types of misfolding. As expected, results from the first simulation are similar to those from a previous study that considers only error-induced misfolding (Drummond and Wilke, 2008). Interestingly, the second and third simulations can also generate the same patterns, including a positive correlation between the protein expression level and the unfolding energy (ΔG) of the error-free protein (Figure 2B), a negative correlation between the expression level and the fraction of protein molecules that misfold after being mistranslated (Figure 2C), a negative correlation between ΔG and the evolutionary rate (Figure 2D), and a negative correlation between the expression level and the evolutionary rate (i.e., the E–R anticorrelation) (Figure 2E). Furthermore, we found that selection against protein misfolding is more effective in reducing error-free misfolding than error-induced misfolding.
Based on these results, we propose that an overarching protein-misfolding-avoidance hypothesis that includes both sources of misfolding is superior to the prevailing translational robustness hypothesis, which considers only error-induced misfolding. We tested three key predictions of the protein-misfolding-avoidance hypotheses using yeast data. First, we showed that, consistent with our prediction, a positive correlation exists between the protein expression level and stability, which is measured by the unfolding energy or melting temperature. In addition, protein expression level is negatively correlated with protein aggregation propensity. Second, we found that codons minimizing protein misfolding are used more frequently in highly expressed proteins than in lowly expressed ones. Third, we showed that, within the same protein, amino acid residues in which random nonsynonymous mutations are more likely to increase protein misfolding are evolutionarily more conserved.
Together, these results provide unambiguous evidence that avoidance of both error-induced and error-free protein misfolding is a major source of the E–R anticorrelation and that protein stability and mistranslation have important roles in protein evolution.
What determines the rate of protein evolution is a fundamental question in biology. Recent genomic studies revealed a surprisingly strong anticorrelation between the expression level of a protein and its rate of sequence evolution. This observation is currently explained by the translational robustness hypothesis in which the toxicity of translational error-induced protein misfolding selects for higher translational robustness of more abundant proteins, which constrains sequence evolution. However, the impact of error-free protein misfolding has not been evaluated. We estimate that a non-negligible fraction of misfolded proteins are error free and demonstrate by a molecular-level evolutionary simulation that selection against protein misfolding results in a greater reduction of error-free misfolding than error-induced misfolding. Thus, an overarching protein-misfolding-avoidance hypothesis that includes both sources of misfolding is superior to the translational robustness hypothesis. We show that misfolding-minimizing amino acids are preferentially used in highly abundant yeast proteins and that these residues are evolutionarily more conserved than other residues of the same proteins. These findings provide unambiguous support to the role of protein-misfolding-avoidance in determining the rate of protein sequence evolution.
PMCID: PMC2990641  PMID: 20959819
evolutionary rate; expression level; mistranslation; protein misfolding
7.  The Yeast PNC1 Longevity Gene Is Up-Regulated by mRNA Mistranslation 
PLoS ONE  2009;4(4):e5212.
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.
PMCID: PMC2667667  PMID: 19381334
8.  GroEL dependency affects codon usage—support for a critical role of misfolding in gene evolution 
Integrating genome-scale sequence, expression, structural and protein interaction data from E. coli we establish an interaction between chaperone (GroEL) dependency and optimal codon usage.Highly expressed sporadic substrates of GroEL employ more optimal codons than expected, show enrichment for optimal codons at structurally sensitive sites and greater conservation of codon optimality under conditions of relaxed purifying selection.We suggest that highly expressed genes cannot routinely utilize GroEL for error control so that codon usage has evolved to provide complementary error limitation, whereas obligate GroEL substrates experience relaxed selection on codon usage.Our results support a critical role of misfolding prevention in gene evolution.
Errors during gene expression are relatively commonplace, which has prompted speculations that many features of gene and genome anatomy and organization have evolved to reduce or mitigate such errors. One type of error that can be particularly costly occurs when the polypeptide chain that emerges from the ribosome fails to fold into its native structure. Some aberrantly folded proteins, exposing hydrophobic residues that would normally be buried, may begin to promiscuously interact with other proteins, become toxic to the cell and thus pose a substantial fitness concern (Gregersen et al, 2006).
In trans, molecular chaperones have long been recognized to play crucial roles in misfolding prevention and remedy. In cis, it has recently been suggested that the use of optimal codons limits mistranslation-induced protein misfolding (Drummond and Wilke, 2008). Evidence for the latter is centred on the argument that synonymous codons differ in their propensity to cause mistranslation. Translationally optimal codons, typically represented by more abundant cognate tRNAs (Duret, 2000), are thought less likely to cause ribosomal stalling and/or incorporation of the wrong amino acid.
Here, we suggest that the role, if any, of error limitation in cis can be revealed by studying its interaction with well-established error management systems in trans (chaperones). If codon usage does indeed play a tangible role in misfolding prevention, we would expect selection on codon identity to vary with the degree to which a protein can rely on other error control mechanisms, namely chaperones. We use the E. coli chaperonin GroEL as a model system to explore whether there is any interaction between optimal codon usage and chaperone dependency.
Kerner et al (2005) had previously determined GroEL substrates on a genome-wide scale. Based on enrichment in GroEL complexes the authors assigned ∼250 proteins to three classes reflecting GroEL dependency: class-I proteins, only a small fraction of which (<1%) associates with GroEL and which spontaneously regain some activity; class-II proteins, which only exhibit spontaneous refolding at more permissive temperatures and class-III proteins, which are obligate substrates of GroEL and largely fail to refold even under more benign conditions. Notably, although on average less abundant than class-I/II proteins (‘sporadic clients'), class-III proteins (‘obligate clients') occupy ∼80% of GroEL's capacity in vivo. Consequently, a higher proportion (∼100% versus ∼20% for class-II and ∼1% for class-I) of these proteins is routinely processed by the GroEL system.
We demonstrate that sporadic but not obligate clients of GroEL exhibit enhanced codon adaptation, carefully controlling for possible confounding factors, notably expression level and protein length (Figure 1). We also point out that genes that recently entered the E. coli genome via horizontal gene transfer will distort equilibrium analyses of codon usage in bacteria and should thus be routinely eliminated from analysis.
Building on earlier work by Zhou et al (2009), we further show that sporadic substrates are conspicuously enriched for optimal codons at structurally sensitive sites, consistent with more severe fitness implications of codon choice for these proteins.
Lastly, we reveal that codon optimality in sporadic clients is more highly conserved in S. dysenteriae. S. dysenteriae is an E. coli clone that has diverged relatively recently from the E. coli K12 strain and has adopted an intracellular lifestyle (Balbi et al, 2009). Concomitant with that lifestyle, Shigella has experienced a lower effective population size and therefore reduced efficiency of purifying selection. This has generated conditions where, overall, codon optimality has started to decay. However, when we followed the fate of ancestrally optimal codons at buried sites in the S. dysenteriae and E. coli K12 genomes, we found that a lower fraction of buried sites has lost codon optimality in sporadic substrates (Figure 4), again consistent with greater structural importance of codon choice in these substrates.
Based on the these findings, we suggest the following explanation: As mentioned above, class-III substrates are defined not only by GroEL being critical for proper folding, but also by occupying most of GroEL's capacity (∼80%). With a high proportion of class-III protein passaged through the GroEL system, mistranslation errors in these proteins weigh less severely as GroEL can remedy at least some misfolding that ensues. In contrast, class-I and II genes are more highly expressed and cannot routinely rely on GroEL to rectify folding errors. Yet class-I/II proteins are clearly liable to misfold as testified by their sporadic association with GroEL. We argue that augmenting GroEL's capacity to address the misfolding propensity of these genes would be prohibitively costly to the organism and that, as an alternative strategy, these genes employ optimal codons to reduce the rate of misfolding error.
Our findings (a) reveal a cis–trans interaction between codon usage and chaperones in providing an integrated error management system, (b) provide independent evidence for a role of misfolding in shaping gene evolution and (c) suggest that the burden of deleterious mutations in long-term bottlenecking populations like that of the insect endosymbiont Buchnera not only comprises unfavourable amino-acid (Moran, 1996) but also synonymous substitutions.
It has recently been suggested that the use of optimal codons limits mistranslation-induced protein misfolding, yet evidence for this remains largely circumstantial. In contrast, molecular chaperones have long been recognized to play crucial roles in misfolding prevention and remedy. We propose that putative error limitation in cis can be elucidated by examining the interaction between codon usage and chaperoning processes. Using Escherichia coli as a model system, we find that codon optimality covaries with dependency on the chaperonin GroEL. Sporadic but not obligate substrates of GroEL exhibit higher average codon adaptation and are conspicuously enriched for optimal codons at structurally sensitive sites. Further, codon optimality of sporadic clients is more conserved in the E. coli clone Shigella dysenteriae. We suggest that highly expressed genes cannot routinely use GroEL for error control so that codon usage has evolved to provide complementary error limitation. These findings provide independent evidence for a role of misfolding in shaping gene evolution and highlight the need to co-characterize adaptations in cis and trans to unravel the workings of integrated molecular systems.
PMCID: PMC2824523  PMID: 20087338
codon bias; GroEL; misfolding
9.  Polysome profiling reveals broad translatome remodeling during endoplasmic reticulum (ER) stress in the pathogenic fungus Aspergillus fumigatus 
BMC Genomics  2014;15:159.
The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain a balance between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence indicates that several pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential loading of ribosomes onto mRNAs. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress.
ER stress was induced by treating the fungus with dithiothreitol, tunicamycin, or a thermal up-shift. The mRNAs were then fractionated on the basis of ribosome occupancy into an under-translated pool (U) and a well-translated pool (W). The mRNAs were used to interrogate microarrays and the ratio of the hybridization signal (W/U) was used as an indicator of the relative translational efficiency of a mRNA under each condition. The largest category of translationally upregulated mRNAs during ER stress encoded proteins involved in translation. Components of the ergosterol and GPI anchor biosynthetic pathways also showed increased polysome association, suggesting an important role for translational regulation in membrane and cell wall homeostasis. ER stress induced limited remodeling of the secretory pathway translatome. However, a select group of transcription factors was translationally upregulated, providing a link to subsequent modification of the transcriptome. Finally, we provide evidence that one component of the ER stress translatome is a novel mRNA isoform from the yvc1 gene that is induced by ER stress in a UPR-dependent manner.
Together, these findings define a core set of mRNAs subject to translational control during the adaptive response to acute ER stress in A. fumigatus and reveal a remarkable breadth of functions that are needed to resolve ER stress in this organism.
PMCID: PMC3943501  PMID: 24568630
Aspergillus fumigatus; UPR; Unfolded protein response; ER stress; Translational regulation; Polysome profiling; Yvc1
10.  The HOG Pathway Dictates the Short-Term Translational Response after Hyperosmotic Shock 
Molecular Biology of the Cell  2010;21(17):3080-3092.
In the global osmoshock translational response in yeast, some gene products were translationally mobilized without transcriptional up-regulation. Conversely, other transcriptionally up-regulated mRNAs were translationally inhibited. Analogous changes occurred on the protein level. These translational responses were strongly dependent on Hog1 and Rck2.
Cellular responses to environmental changes occur on different levels. We investigated the translational response of yeast cells after mild hyperosmotic shock by isolating mRNA associated with multiple ribosomes (polysomes) followed by array analysis. Globally, recruitment of preexisting mRNAs to ribosomes (translational response) is faster than the transcriptional response. Specific functional groups of mRNAs are recruited to ribosomes without any corresponding increase in total mRNA. Among mRNAs under strong translational up-regulation upon shock, transcripts encoding membrane-bound proteins including hexose transporters were enriched. Similarly, numerous mRNAs encoding cytoplasmic ribosomal proteins run counter to the overall trend of down-regulation and are instead translationally mobilized late in the response. Surprisingly, certain transcriptionally induced mRNAs were excluded from ribosomal association after shock. Importantly, we verify, using constructs with intact 5′ and 3′ untranslated regions, that the observed changes in polysomal mRNA are reflected in protein levels, including cases with only translational up-regulation. Interestingly, the translational regulation of the most highly osmostress-regulated mRNAs was more strongly dependent on the stress-activated protein kinases Hog1 and Rck2 than the transcriptional regulation. Our results show the importance of translational control for fine tuning of the adaptive responses.
PMCID: PMC2930000  PMID: 20587780
11.  Translation Reinitiation Relies on the Interaction between eIF3a/TIF32 and Progressively Folded cis-Acting mRNA Elements Preceding Short uORFs 
PLoS Genetics  2011;7(7):e1002137.
Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5′ and 3′ cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5′ cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5′ sequences making up the 5′ enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5′ enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5′ enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5′ enhancer's stimulatory activity is strictly dependent on and thus follows the 3′ enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5′ leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.
Author Summary
Protein synthesis is a fundamental mechanism capturing the rejuvenation of DNA–encoded genetic information by its translation into molecular effectors—proteins. Its regulation can be used to change the protein content and thus to adapt a cell to changing environmental conditions. Translation requires mRNAs delivering genetic information of corresponding genes, tRNAs carrying amino-acids, ribosomes as the molecular translators, and accessory proteins/factors facilitating the entire process. There are numerous regulatory mechanisms that modulate translation at its various stages. Here we describe one such a translational control mechanism called reinitiation. Most eukaryotic mRNAs contain only a single translatable gene (ORF); however, in many of them this gene is preceded by a short coding sequence (uORF) that is in some cases translated first. In order to reinitiate translation on the downstream main ORF, a ribosome has to stay bound to mRNA after it has terminated short uORF translation. This requires a concerted action of specific mRNA elements surrounding the uORF and selected initiation factors. Our results delineate how these key players interact with each other and suggest a sequence of general events that ought to take place on short uORF to enable the ribosome to reach and translate the main ORF downstream.
PMCID: PMC3131280  PMID: 21750682
12.  A dynamic model of proteome changes reveals new roles for transcript alteration in yeast 
By characterizing dynamic changes in yeast protein abundance following osmotic shock, this study shows that the correlation between protein and mRNA differs for transcripts that increase versus decrease in abundance, and reveals physiological reasons for these differences.
The correlation between protein and mRNA change is very high at transcripts that increase in abundance, but negligible at reduced transcripts following NaCl shock.Modeling and experimental data suggest that reducing levels of high-abundance transcripts helps to direct translational machinery to newly made transcripts.The transient burst of transcript increase serves to accelerate changes in protein abundance.Post-transcriptional regulation of protein abundance is pervasive, although most of the variance in protein change is explained by changes in mRNA abundance.
Natural microenvironments change rapidly, and living creatures must respond quickly and efficiently to thrive within this flux. At all cellular levels—signaling, transcription, translation, metabolism, cell growth, and division—the response is dynamic and coordinated. Some aspects of this response, such as dynamic changes of the transcriptome, are well understood. But other aspects, like the response of the proteome, have remained obscured primarily because of previous limitations in technology. Without coordinated time-course data, it has remained impossible to correctly characterize the correlations and dependencies between these two essential levels of cell biology.
This work presents an extended picture of the coordinated response of the transcriptome and proteome as cells respond to an abrupt environmental change. To assay proteomic dynamics, we developed a strategy for large-scale, multiplexed quantitation using isobaric tags and high mass accuracy mass spectrometry. This sensitive yet efficient platform allows for the expedient collection of quantitative time-course proteomic data at six time points, sufficiently reproducible to permit meaningful interpretation of variation across biological replicates. Time-course transcriptome data were generated from paired biological samples, allowing us to examine the relationships between changes in mRNA and protein for each gene in terms of direction and intensity, as well as the characteristics of the temporal profiles for each gene.
It was immediately obvious that a single measure of correlation across the entire data set was a meaningless metric. We therefore analyzed relationships between mRNA and protein for different subsets of data. In response to osmotic shock, hundreds of transcripts are highly induced, and their temporal pattern reveals a transient peak of maximal induction, which resolves into a new elevated level as cells acclimate (Figure 2). For this group of genes, there is extremely high correlation between peak mRNA change and protein change (R2∼0.8). But the dynamics of the molecules differ: while mRNA levels transiently overshoot their final levels, proteins gradually rise in abundance toward their new, elevated state. We observed, however, that a measure of efficiency connects the two profiles. The time it takes for a protein to acclimate to its new state correlates with the magnitude of the excess mRNA induction. Thus, the cell imparts an urgency to protein induction by transiently producing excess transcript.
The most surprising result, however, involves transcripts that decrease in abundance. In response to osmotic shock, the cell transiently reduces over 600 transcripts, many of which are among the most highly expressed in unstressed cells. But protein levels for these genes remain, for the most part, almost completely unchanged. The stark absence of protein repression is independent of basal protein abundance, independent of reported protein half-lives, reproducible across biological replicates, and validated by quantitative western blots. Furthermore, since we do detect a handful of proteins whose abundance is significantly reduced, our technology is capable of identifying protein loss. Thus, we conclude that transcript reduction serves another purpose besides reducing protein levels.
To explore alternate interpretations of the consequence of transcriptional repression, we devised a mass-action kinetic model, which describes protein changes based on mRNA dynamics in the context of transient changes in the rates of cell division. The model successfully recapitulated the observed data, allowing us to alter modeling parameters to test various hypotheses.
In response to osmotic shock, overall rates of translation temporarily decrease and cell growth transiently arrests before resuming at a slower rate. We reasoned that mRNA reduction might lower the rate of new protein synthesis, but that retarded production is balanced by reduced cell division. We explored both aspects of this logic with our model.
As expected, removing cell division from our model led to a calculated decrease of protein levels, indicating that reduced growth is necessary for maintaining protein levels. However, when we computationally held mRNA levels stable and calculated protein levels in the absence of mRNA repression, we did not find the expected increase in protein abundance.
We then considered the possibility that one function of the regulated repression of these highly abundant transcripts was to liberate proteins essential for translation, such as ribosomes or translation initiation factors. To explore this, we examined a mutant lacking the Dot6p/Tod6p transcriptional repressors, which fails to properly repress ∼250 genes in response to osmotic shock. In the wild type, the mRNA for a Dot6p/Tod6p target (ARX1) decreased seven-fold, and the remaining transcript was generally unassociated with poly-ribosomes. In the mutant, however, the mRNA levels were reduced only two-fold, while the remaining transcript continued to bind ribosomes. Therefore, failure to reduce transcript levels led to a persistent association with poly-ribosomes, thereby consuming translational machinery.
Our hypothesis is, therefore, that widespread changes in the transcriptome promote efficient translation of new proteins. Transcript increase serves to increase abundance of the encoded proteins, while reduction of some of the most abundant and highly translated mRNAs supports this project by liberating translational capacity. While it is not clear what factors are the limiting elements, it is clear that a full picture of cellular biology requires exploring the dynamics of the cellular response.
The transcriptome and proteome change dynamically as cells respond to environmental stress; however, prior proteomic studies reported poor correlation between mRNA and protein, rendering their relationships unclear. To address this, we combined high mass accuracy mass spectrometry with isobaric tagging to quantify dynamic changes in ∼2500 Saccharomyces cerevisiae proteins, in biological triplicate and with paired mRNA samples, as cells acclimated to high osmolarity. Surprisingly, while transcript induction correlated extremely well with protein increase, transcript reduction produced little to no change in the corresponding proteins. We constructed a mathematical model of dynamic protein changes and propose that the lack of protein reduction is explained by cell-division arrest, while transcript reduction supports redistribution of translational machinery. Furthermore, the transient ‘burst' of mRNA induction after stress serves to accelerate change in the corresponding protein levels. We identified several classes of post-transcriptional regulation, but show that most of the variance in protein changes is explained by mRNA. Our results present a picture of the coordinated physiological responses at the levels of mRNA, protein, protein-synthetic capacity, and cellular growth.
PMCID: PMC3159980  PMID: 21772262
dynamics; modeling; proteomics; stress; transcriptomics
13.  Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells 
PLoS ONE  2012;7(5):e35915.
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.
PMCID: PMC3344847  PMID: 22574127
14.  Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome 
eLife  2013;2:e00308.
Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97.
eLife digest
Ribosomes are complex molecular machines that translate the sequence of bases in a messenger RNA (mRNA) transcript into a polypeptide that subsequently folds to form a protein. Each ribosome is composed of two major subunits: the small subunit reads the mRNA transcript, and the large subunit joins amino acids together to form the polypeptide. This process stops when the ribosome encounters a stop codon and releases the completed polypeptide.
It is critical that cells perform some form of quality control on the polypeptides as they are translated to prevent a build up of incomplete, incorrect or toxic proteins in cells. Problems can occur if a ribosome stalls while translating the mRNA transcript, or if the mRNA transcript is defective. For example, most mRNA transcripts contain a stop codon, but some do not, and these non-stop mRNA transcripts result in a non-stop polypeptide that remains tethered to the ribosome. It is important that the cell identifies and removes these faulty polypeptides so as to leave the ribosome free to translate other (non-faulty) mRNA transcripts. A regulatory protein called ubiquitin is responsible for marking and sending proteins that are faulty, or are no longer needed by the cell, to a molecular machine called the proteasome, where they are degraded by a process called proteolysis. In 2010 researchers identified Ltn1 as the enzyme that attaches ubiquitin to non-stop proteins in yeast.
Now, building on this work, Verma et al. identify additional proteins involved in this process. In particular, an ATPase enzyme called Cdc48 (known as p97 or VCP in human cells) and two co-factors—Ufd1 and Npl4—promote release of the ubiquitinated non-stop polypeptides from the ribosomes, thus committing the marked polypeptide to destruction by the proteasome. Verma et al. also show that the Cdc48-Ufd1-Npl4 complex is involved in other aspects of quality control of newly synthesized proteins within cells. Collectively these processes are known as ribosome-associated degradation.
Mutations of the gene that codes for human p97 can cause a number of diseases, including Paget's disease of the bone and frontotemporal dementia, so an improved understanding of ribosome-associated degradation could provide new insights into these diseases.
PMCID: PMC3552423  PMID: 23358411
ubiquitin; ribosome; Cdc48; S. cerevisiae
15.  Codon-by-Codon Modulation of Translational Speed and Accuracy Via mRNA Folding 
PLoS Biology  2014;12(7):e1001910.
Secondary structure in mRNAs modulates the speed of protein synthesis codon-by-codon to improve accuracy at important sites while ensuring high speed elsewhere.
Rapid cell growth demands fast protein translational elongation to alleviate ribosome shortage. However, speedy elongation undermines translational accuracy because of a mechanistic tradeoff. Here we provide genomic evidence in budding yeast and mouse embryonic stem cells that the efficiency–accuracy conflict is alleviated by slowing down the elongation at structurally or functionally important residues to ensure their translational accuracies while sacrificing the accuracy for speed at other residues. Our computational analysis in yeast with codon resolution suggests that mRNA secondary structures serve as elongation brakes to control the speed and hence the fidelity of protein translation. The position-specific effect of mRNA folding on translational accuracy is further demonstrated experimentally by swapping synonymous codons in a yeast transgene. Our findings explain why highly expressed genes tend to have strong mRNA folding, slow translational elongation, and conserved protein sequences. The exquisite codon-by-codon translational modulation uncovered here is a testament to the power of natural selection in mitigating efficiency–accuracy conflicts, which are prevalent in biology.
Author Summary
Protein synthesis by ribosomal translation is a vital cellular process, but our understanding of its regulation has been poor. Because the number of ribosomes in the cell is limited, rapid growth relies on fast translational elongation. The accuracy of translation must also be maintained, and in an ideal scenario, both speed and accuracy should be maximized to sustain rapid and productive growth. However, existing data suggest a tradeoff between speed and accuracy, making it impossible to simultaneously maximize both. A potential solution is slowing the elongation at functionally or structurally important sites to ensure their translational accuracies, while sacrificing accuracy for speed at other sites. Here, we show that budding yeast and mouse embryonic stem cells indeed use this strategy. We discover that a codon-by-codon adaptive modulation of translational elongation is accomplished by mRNA secondary structures, which serve as brakes to control the elongation speed and hence translational fidelity. Our findings explain why highly expressed genes tend to have strong mRNA folding, slow translational elongation, and conserved protein sequences. The exquisite translational modulation reflects the power of natural selection in mitigating efficiency–accuracy conflicts, and our study offers a general framework for analyzing similar conflicts, which are widespread in biology.
PMCID: PMC4106722  PMID: 25051069
16.  Nutrient-Regulated Antisense and Intragenic RNAs Modulate a Signal Transduction Pathway in Yeast 
PLoS Biology  2008;6(12):e326.
The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes) is expressed to activate inorganic phosphate (Pi) metabolism for adaptation to Pi starvation. Pi starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus and enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4, thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription) of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system in that Pho85 monitors whether environmental conditions are adequate for cell growth and represses inadequate (untimely) responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4 and Rpo21 binding (Rpo21 being the largest subunit of RNA polymerase II) using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP) species (IP7) that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates the transcription of antisense and intragenic RNAs in the KCS1 locus to down-regulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 mRNA and translation of the intragenic RNA, thereby enabling Vip1 to utilize more IP6 to synthesize IP7 functioning in low-Pi signaling. Because Kcs1 also can phosphorylate these IP7 species to synthesize IP8, reduction in Kcs1 activity can ensure accumulation of the IP7 species, leading to further stimulation of low-Pi signaling (i.e., forming a positive feedback loop). We also report that genes apparently not involved in the PHO system are regulated by Pho4 either dependent upon or independent of the Pi conditions, and many of the latter genes are involved in stress response. In S. cerevisiae, a large-scale cDNA analysis and mapping of RNA polymerase II binding using a high-resolution tiling array have identified a large number of antisense RNA species whose functions are yet to be clarified. Here we have shown that nutrient-regulated antisense and intragenic RNAs as well as direct regulation of structural gene transcription function in the response to nutrient availability. Our findings also imply that Pho4 is present in the nucleus even under high-Pi conditions to activate or repress transcription, which challenges our current understanding of Pho4 regulation.
Author Summary
How does a microorganism adapt to changes in its environment? Phosphate metabolism in the budding yeast Saccharomyces cerevisiae serves as a model for investigating mechanisms involved in physiological adaptation. The nutrient inorganic phosphate (Pi) is essential for building nucleic acids and phospholipids; when yeast cells are deprived of Pi, genes required for scavenging the nutrient are activated. This activation is mediated by the Pho4 transcription factor through its migration into or out of nucleus. The Pi-starvation (low-Pi) signal is transmitted by a class of inositol polyphosphate (IP) species, IP7, which is synthesized by one of two IP6 kinases, Vip1 or Kcs1. However, the IP7 made primarily by Vip1 is key in the signaling pathway. Here we report that under Pi starvation Pho4 binds within the coding sequence of KCS1 to activate transcription of both intragenic and antisense RNAs, resulting in the production of a truncated Kcs1 protein and the likely down-regulation of Kcs1 activity. Consequently Vip1 can produce more IP7 to enhance the low-Pi signaling and thus form a positive feedback loop. We have also demonstrated that Pho4 regulates, both positively and negatively, transcription of genes apparently uninvolved in cellular response to Pi starvation and that it sometimes does so independently of Pi conditions. These findings reveal mechanisms that go beyond the currently held model of Pho4 regulation.
During nutritional adaptation, transcriptional activation in yeast produces noncoding RNAs that allow the formation of a positive-feedback regulatory loop.
PMCID: PMC2605928  PMID: 19108609
17.  Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression 
PLoS Genetics  2012;8(6):e1002782.
RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.
Author Summary
Control of mRNA stability is crucial for bacteria to survive and rapidly adapt to environmental changes and stress conditions. The molecular players and the degradation pathways involved in these adaptive processes are poorly understood in Staphylococcus aureus. The universally conserved double-strand-specific endoribonuclease III (RNase III) in S. aureus is known to repress the synthesis of several virulence factors and was recently implicated in genome-wide mRNA processing mediated by antisense transcripts. We present here the first global map of direct RNase III targets in S. aureus. Deep sequencing was used to identify RNAs associated with epitope-tagged wild-type RNase III and two catalytically impaired but binding-competent mutant proteins in vivo. Experimental validation revealed an unexpected variety of structured RNA transcripts as novel RNase III substrates. In addition to rRNA operon maturation, autoregulation, degradation of structured RNAs, and antisense regulation, we propose novel mechanisms by which RNase III increases mRNA translation. Overall, this study shows that RNase III has a broad function in gene regulation of S. aureus. We can now address more specifically the roles of this universally conserved enzyme in gene regulation in response to stress and during host infection.
PMCID: PMC3386247  PMID: 22761586
18.  The insulin receptor cellular IRES confers resistance to eIF4A inhibition 
eLife  2013;2:e00542.
Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions.
eLife digest
Protein synthesis in eukaryotes occurs in two stages: transcription of DNA into messenger RNA (mRNA) in the nucleus, and then translation of that mRNA into a protein by ribosomes in the cytoplasm. These processes are regulated by a complex network of signaling pathways that enables cells to tailor protein synthesis to match current conditions. This involves regulating the expression of the genes that code for these proteins.
When cells experience stressful events, such as a shortage of oxygen or nutrients, they reduce the synthesis of most proteins. This response is regulated, in part, by a signaling pathway known as the insulin and insulin-like receptor pathway. In particular, stressful events inhibit a protein complex called eIF4F, which normally initiates the translation of mRNA molecules by binding to a structure on one end of the mRNA called the 5′ cap. Despite this general inhibition, the production of certain other proteins—including the insulin receptor itself—is actually increased in response to stress.
Olson et al. have carried out a series of experiments to explore how inhibition of the eIF4F protein complex influences the translation of the mRNA for the insulin receptor. The eIF4F complex is made up of three proteins, including one that binds to the 5′ cap and a helicase that unwinds the RNA. Previous work in the fruit fly Drosophila showed that translation of this mRNA can continue even if formation of the eIF4F complex is inhibited by targeting the cap binding protein. Olsen et al. now show that translation of this mRNA is also independent of the helicase. Instead, translation is maintained under these conditions because the insulin receptor mRNA contains a sequence called an internal ribosome entry site, which allows ribosomes to bind to the mRNA without the influence of the 5′ cap.
Olson et al. reveal the details of this regulatory pathway in Drosophila and show that similar mechanisms are at work in mammalian cells, suggesting this pathway represents a crucial regulatory process that has been conserved during evolution. A key question for future research is whether other genes within the insulin and insulin-receptor like signaling pathway use this same trick to evade translational inhibitors.
PMCID: PMC3713452  PMID: 23878722
Foxo; IRES; Insulin receptor; PDCD4; eIF4A; IGFR; D. melanogaster; Mouse
19.  Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis 
PLoS Biology  2013;11(11):e1001703.
This study describes, for the first time, the rhythmic translational program within circadian clock cells. The results indicate that most clock cell mRNAs are translated at low-energy times of either mid-day or mid-night, and also that related cellular functions are coordinately regulated by the synchronized translation of relevant mRNAs at the same time of day.
Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity.
Author Summary
The circadian clock controls daily rhythms in physiology and behavior via mechanisms that regulate gene expression. While numerous studies have examined the clock regulation of gene transcription and documented rhythms in mRNA abundance, less is known about how circadian changes in protein synthesis contribute to the orchestration of physiological and behavioral programs. Here we have monitored mRNA ribosomal association (as a proxy for translation) to globally examine the circadian timing of protein synthesis specifically within clock cells of Drosophila. The results reveal, for the first time in any organism, the complete circadian program of protein synthesis (the “circadian translatome”) within these cells. A novel finding is that most mRNAs within clock cells are translated at one of two predominant circadian phases—midday or mid-night—times of low energy expenditure. Our work also finds that many clock cell processes, including metabolism, redox state, signaling, neurotransmission, and even protein synthesis itself, are coordinately regulated such that mRNAs required for similar cellular functions are translated in synchrony at the same time of day.
PMCID: PMC3864454  PMID: 24348200
20.  Posttranscriptional Expression Regulation: What Determines Translation Rates? 
PLoS Computational Biology  2007;3(3):e57.
Recent analyses indicate that differences in protein concentrations are only 20%–40% attributable to variable mRNA levels, underlining the importance of posttranscriptional regulation. Generally, protein concentrations depend on the translation rate (which is proportional to the translational activity, TA) and the degradation rate. By integrating 12 publicly available large-scale datasets and additional database information of the yeast Saccharomyces cerevisiae, we systematically analyzed five factors contributing to TA: mRNA concentration, ribosome density, ribosome occupancy, the codon adaptation index, and a newly developed “tRNA adaptation index.” Our analysis of the functional relationship between the TA and measured protein concentrations suggests that the TA follows Michaelis–Menten kinetics. The calculated TA, together with measured protein concentrations, allowed us to estimate degradation rates for 4,125 proteins under standard conditions. A significant correlation to recently published degradation rates supports our approach. Moreover, based on a newly developed scoring system, we identified and analyzed genes subjected to the posttranscriptional regulation mechanism, translation on demand. Next we applied these findings to publicly available data of protein and mRNA concentrations under four stress conditions. The integration of these measurements allowed us to compare the condition-specific responses at the posttranscriptional level. Our analysis of all 62 proteins that have been measured under all four conditions revealed proteins with very specific posttranscriptional stress response, in contrast to more generic responders, which were nonspecifically regulated under several conditions. The concept of specific and generic responders is known for transcriptional regulation. Here we show that it also holds true at the posttranscriptional level.
Author Summary
Large-scale mRNA concentration measurements are a hallmark of our post-genomic era. Usually they are taken as a surrogate for the corresponding protein concentrations. For most genes, proteins are the actual cellular players, but up to now it has been much more difficult to measure protein concentrations than mRNA concentrations. However, due to numerous posttranscriptional regulation mechanisms, mRNA levels only partly correlate with protein concentrations. Based on thoroughly composed reference datasets for protein and mRNA concentrations in yeast under standard growth conditions, we report the best corresponding correlation so far. We took into account additional factors, beyond mRNA concentrations, that influence protein levels in order to improve protein level predictions. Extending our previous approach, where ribosome occupancy and ribosome density were considered, we now also consider ORF-specific translation elongation rates. Different measures for elongation velocity were examined, and the codon adaptation index was found to be most appropriate. Moreover, saturation kinetics were introduced to better describe the translation process. The general findings were also applied to four stress conditions. Three new concepts, translation on demand, just-in-time translation, and general and specific posttranscriptional stress responders, are discussed.
PMCID: PMC1829480  PMID: 17381238
21.  Metabolomic and transcriptomic stress response of Escherichia coli 
GC-MS-based analysis of the metabolic response of Escherichia coli exposed to four different stress conditions reveals reduction of energy expensive pathways.Time-resolved response of E. coli to changing environmental conditions is more specific on the metabolite as compared with the transcript level.Cease of growth during stress response as compared with stationary phase response invokes similar transcript but dissimilar metabolite responses.Condition-dependent associations between metabolites and transcripts are revealed applying co-clustering and canonical correlation analysis.
The response of biological systems to environmental perturbations is characterized by a fast and appropriate adjusting of physiology on every level of the cellular and molecular network.
Stress response is usually represented by a combination of both specific responses, aimed at minimizing deleterious effects or repairing damage (e.g. protein chaperones under temperature stress) and general responses which, in part, comprise the downregulation of genes related to translation and ribosome biogenesis. This in turn is reflected by growth cessation or reduction observed under essentially all stress conditions and is an important strategy to adjust cellular physiology to the new condition.
E. coli has been intensively investigated in relation to stress responses. Thus far, however, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. To better understand system response to perturbation, we designed a time-resolved experiment to compare and integrate metabolic and transcript changes of E. coli using four stress conditions including non-lethal temperature shifts, oxidative stress, and carbon starvation relative to cultures grown under optimal conditions covering both states before and directly after stress application, resumption of growth after stress-induced lag phase, and finally the stationary phase.
Metabolic changes occurring after stress application were characterized by a reduction in metabolites of central metabolism (TCA cycle and glycolysis) as well as an increase in free amino acids. Whereas the latter is probably due to protein degradation and stalling of translation, the former supports and extends conclusions based on transcriptome data demonstrating a major decrease in energy-consuming processes as a general stress response. Further comparative analysis of the response on the metabolome and transcriptome, however, revealed in addition to these similarities major differences. Thus, the response on the metabolome displayed a significantly higher specificity towards the specific stress as compared with the transcriptome. Further, when comparing the metabolome of cells ceasing growth due to stress application with cells ceasing growth due to reaching stationary phase the metabolome response differed to a significant extent between both growth arrest phases, whereas the transcriptome response showed significant overlap again, suggesting that the response of E. coli on the metabolome level displays a higher level of significance as compared with the transcriptome level.
Subsequently, both data sets were jointly analyzed using co-clustering and canonical correlation approaches to identify coordinated changes on the transcriptome and the metabolite level indicative metabolite–transcript associations. A first outcome of this study was that no association was preserved during all conditions analyzed but rather condition-specific associations were observed. One set of associations found was between metabolites from the oxidative pentose phosphate pathway such as glc-6-P, 6-P-gluconic acid, ribose-5-P, and E-4-P and metabolites from the glycolytic pathway (3PGA and PEP in addition to glc-6-P with two genes encoding pathway enzymes, that is rpe encoding ribulose phosphate 3-epimerase and pps encoding PEP synthase.
A second example comprises metabolites of the TCA cycle such as pyruvic acid, 2-ketoglutaric acid, fumaric acid, malic acid, and succinic acid and the mqo gene encoding malate-quinone oxidoreductase (MQO). MQO catalyses the irreversible oxidation of malate to oxaloacetate that in turn regulates the activity of citrate synthase, which is a major rate determining enzyme of the TCA cycle. The strong association between mqo gene expression and multiple members of the TCA cycle as well as pyruvate suggest mqo expression to have a major function for the regulation of the TCA cycle, which need to be experimentally validated.
Multiple further associations identified show on the one hand the power of integrative systems oriented approaches for developing new hypothesis, on the other hand their condition-dependent behavior shows the extreme flexibility of the biological systems studied thus requesting a much more intense effort toward parallel analysis of biological systems under several environmental conditions.
Environmental fluctuations lead to a rapid adjustment of the physiology of Escherichia coli, necessitating changes on every level of the underlying cellular and molecular network. Thus far, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. Here, we incorporate the metabolite composition together with gene expression data to provide a more comprehensive insight on system level stress adjustments by describing detailed time-resolved E. coli response to five different perturbations (cold, heat, oxidative stress, lactose diauxie, and stationary phase). The metabolite response is more specific as compared with the general response observed on the transcript level and is reflected by much higher specificity during the early stress adaptation phase and when comparing the stationary phase response to other perturbations. Despite these differences, the response on both levels still follows the same dynamics and general strategy of energy conservation as reflected by rapid decrease of central carbon metabolism intermediates coinciding with downregulation of genes related to cell growth. Application of co-clustering and canonical correlation analysis on combined metabolite and transcript data identified a number of significant condition-dependent associations between metabolites and transcripts. The results confirm and extend existing models about co-regulation between gene expression and metabolites demonstrating the power of integrated systems oriented analysis.
PMCID: PMC2890322  PMID: 20461071
Escherichia coli; metabolomic; response to stress; time course; transcriptomic
22.  Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression 
BMC Genomics  2012;13:528.
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.
PMCID: PMC3543184  PMID: 23036066
Translational regulation; mRNA Ribosome; Translatome; Statistical modeling; Lactococcus lactis
23.  A Comprehensive, Quantitative, and Genome-Wide Model of Translation 
PLoS Computational Biology  2010;6(7):e1000865.
Translation is still poorly characterised at the level of individual proteins and its role in regulation of gene expression has been constantly underestimated. To better understand the process of protein synthesis we developed a comprehensive and quantitative model of translation, characterising protein synthesis separately for individual genes. The main advantage of the model is that basing it on only a few datasets and general assumptions allows the calculation of many important translational parameters, which are extremely difficult to measure experimentally. In the model, each gene is attributed with a set of translational parameters, namely the absolute number of transcripts, ribosome density, mean codon translation time, total transcript translation time, total time required for translation initiation and elongation, translation initiation rate, mean mRNA lifetime, and absolute number of proteins produced by gene transcripts. Most parameters were calculated based on only one experimental dataset of genome-wide ribosome profiling. The model was implemented in Saccharomyces cerevisiae, and its results were compared with available data, yielding reasonably good correlations. The calculated coefficients were used to perform a global analysis of translation in yeast, revealing some interesting aspects of the process. We have shown that two commonly used measures of translation efficiency – ribosome density and number of protein molecules produced – are affected by two distinct factors. High values of both measures are caused, i.a., by very short times of translation initiation, however, the origins of initiation time reduction are completely different in both cases. The model is universal and can be applied to any organism, if the necessary input data are available. The model allows us to better integrate transcriptomic and proteomic data. A few other possibilities of the model utilisation are discussed concerning the example of the yeast system.
Author Summary
Translation is the production of proteins by decoding mRNA produced in transcription, and is a part of the overall process of gene expression. Although the general theoretical background of translation is known, the process is still poorly characterised at the level of individual proteins. In particular, the quantitative parameters of translation, such as time required to complete it or the number of protein molecules produced from a transcript during its lifetime, are extremely difficult to measure experimentally. To overcome this problem, we developed a computational model that, on the basis of only few datasets and general assumptions, measures quantitatively the translational activity at the level of individual genes. We discussed it concerning the example of the yeast system; however, it can be applied to any organism of known genome. We used the obtained results to study the general characteristics of the yeast translational system, revealing the diversity of strategies of gene expression regulation. We exemplified and discussed other possible ways of model utilisation, as it may help in examining protein-protein interactions, metabolic pathways, gene annotation, ribosome queueing, protein folding, and translation initiation. It also may be crucial for better integration of cell-wide, high-throughput experiments.
PMCID: PMC2912337  PMID: 20686685
24.  Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5′ terminal oligopyrimidine tract 
Oncogene  2010;30(13):1531-1541.
The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and ‘ribosomopathies’. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5′ terminal oligopyrimidine (5′ TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5′ TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5′ TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5′ TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.
PMCID: PMC3227680  PMID: 21102526
RPS6; diffuse large B-cell lymphoma; 5′ terminal oligopyrimidine; p53
25.  Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity 
PLoS Pathogens  2012;8(3):e1002612.
Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection.
Author Summary
Retroviruses are intracellular parasites that utilize the host translation machinery to catalyze viral protein synthesis. The activity of the translation machinery fluctuates during cell cycle progression and is reduced in the G2/M phase. HIV-1 infection causes the cells to arrest in the G2/M phase, which has the potential to alter the activity of the translation machinery. Herein several lines of evidence demonstrated that lymphocyte mRNA translation is suppressed by the action of HIV-1 accessory protein Vpr. The molecular basis of translation suppression is reduced activity of the rate-limiting translation intitation factor, eIF4E. However, synthesis of the viral structural proteins is sustained and is due to the difference in composition of the viral and cellular mRNA-ribonucleoprotein complexes. Both cellular and completely spliced viral mRNAs are predominantly associated with the cytoplasmic cap binding protein, eIF4E. In contrast, unspliced HIV-1 mRNAs are predominantly associated with the components of the nuclear cap binding complex (CBC). The retention of CBC on the viral mRNAs provides a mechanism to sustain viral protein synthesis. This newly characterized interface of the virus-host-protein synthesis machinery is likely a cellular adaptation used to enable synthesis of proteins that reengage the cell cycle and facilitate recovery from stress.
PMCID: PMC3310836  PMID: 22457629

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