Relative Translational Efficiencies Research Articles

Search and match across spatial omics samples at single-cell resolution.

Spatial omics technologies characterize tissue molecular properties with spatial information, but integrating and comparing spatial data across different technologies and modalities is challenging. A comparative analysis tool that can search, match and visualize both similarities and differences of molecular features in space across multiple samples is lacking. To address this, we introduce CAST (cross-sample alignment of spatial omics), a deep graph neural network-based method enabling spatial-to-spatial searching and matching at the single-cell level. CAST aligns tissues based on intrinsic similarities of spatial molecular features and reconstructs spatially resolved single-cell multi-omic profiles. CAST further allows spatially resolved differential analysis (∆Analysis) to pinpoint and visualize disease-associated molecular pathways and cell-cell interactions and single-cell relative translational efficiency profiling to reveal variations in translational control across cell types and regions. CAST serves as an integrative framework for seamless single-cell spatial data searching and matching across technologies, modalities and sample conditions.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2&Delta, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are up-regulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

Global and gene-specific translational regulation in Escherichia coli across different conditions

How well mRNA transcript levels represent protein abundances has been a controversial issue. Particularly across different environments, correlations between mRNA and protein exhibit remarkable variability from gene to gene. Translational regulation is likely to be one of the key factors contributing to mismatches between mRNA level and protein abundance in bacteria. Here, we quantified genome-wide transcriptome and relative translation efficiency (RTE) under 12 different conditions in Escherichia coli. By quantifying the mRNA-RTE correlation both across genes and across conditions, we uncovered a diversity of gene-specific translational regulations, cooperating with transcriptional regulations, in response to carbon (C), nitrogen (N), and phosphate (P) limitations. Intriguingly, we found that many genes regulating translation are themselves subject to translational regulation, suggesting possible feedbacks. Furthermore, a random forest model suggests that codon usage partially predicts a gene's cross-condition variability in translation efficiency; such cross-condition variability tends to be an inherent quality of a gene, independent of the specific nutrient limitations. These findings broaden the understanding of translational regulation under different environments and provide novel strategies for the control of translation in synthetic biology. In addition, our data offers a resource for future multi-omics studies.

The High Mutational Sensitivity of ccdA Antitoxin Is Linked to Codon Optimality.

Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, nonactive-site residues are tolerant to mutations. Here, we show that the ccdA antitoxin component of the Escherichia coli ccdAB toxin–antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction (∼80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed ccdA mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and nonsynonymous point mutations than inferred previously.

Down-Regulation of Yeast Helicase Ded1 by Glucose Starvation or Heat-Shock Differentially Impairs Translation of Ded1-Dependent mRNAs.

Ded1 is an essential DEAD-box helicase in yeast that broadly stimulates translation initiation and is critical for mRNAs with structured 5′UTRs. Recent evidence suggests that the condensation of Ded1 in mRNA granules down-regulates Ded1 function during heat-shock and glucose starvation. We examined this hypothesis by determining the overlap between mRNAs whose relative translational efficiencies (TEs), as determined by ribosomal profiling, were diminished in either stressed WT cells or in ded1 mutants examined in non-stress conditions. Only subsets of the Ded1-hyperdependent mRNAs identified in ded1 mutant cells exhibited strong TE reductions in glucose-starved or heat-shocked WT cells, and those down-regulated by glucose starvation also exhibited hyper-dependence on initiation factor eIF4B, and to a lesser extent eIF4A, for efficient translation in non-stressed cells. These findings are consistent with recent proposals that the dissociation of Ded1 from mRNA 5′UTRs and the condensation of Ded1 contribute to reduced Ded1 function during stress, and they further suggest that the down-regulation of eIF4B and eIF4A functions also contributes to the translational impairment of a select group of Ded1 mRNA targets with heightened dependence on all three factors during glucose starvation.

Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803

BackgroundPhotosynthetic cyanobacteria have been studied as potential host organisms for direct solar-driven production of different carbon-based chemicals from CO2 and water, as part of the development of sustainable future biotechnological applications. The engineering approaches, however, are still limited by the lack of comprehensive information on most optimal expression strategies and validated species-specific genetic elements which are essential for increasing the intricacy, predictability and efficiency of the systems. This study focused on the systematic evaluation of the key translational control elements, ribosome binding sites (RBS), in the cyanobacterial host Synechocystis sp. PCC 6803, with the objective of expanding the palette of tools for more rigorous engineering approaches.ResultsAn expression system was established for the comparison of 13 selected RBS sequences in Synechocystis, using several alternative reporter proteins (sYFP2, codon-optimized GFPmut3 and ethylene forming enzyme) as quantitative indicators of the relative translation efficiencies. The set-up was shown to yield highly reproducible expression patterns in independent analytical series with low variation between biological replicates, thus allowing statistical comparison of the activities of the different RBSs in vivo. While the RBSs covered a relatively broad overall expression level range, the downstream gene sequence was demonstrated in a rigorous manner to have a clear impact on the resulting translational profiles. This was expected to reflect interfering sequence-specific mRNA-level interaction between the RBS and the coding region, yet correlation between potential secondary structure formation and observed translation levels could not be resolved with existing in silico prediction tools.ConclusionsThe study expands our current understanding on the potential and limitations associated with the regulation of protein expression at translational level in engineered cyanobacteria. The acquired information can be used for selecting appropriate RBSs for optimizing over-expression constructs or multicistronic pathways in Synechocystis, while underlining the complications in predicting the activity due to gene-specific interactions which may reduce the translational efficiency for a given RBS-gene combination. Ultimately, the findings emphasize the need for additional characterized insulator sequence elements to decouple the interaction between the RBS and the coding region for future engineering approaches.

High-throughput engineering of initial coding regions for maximized production of recombinant proteins

We present a method to rapidly determine the optimal initial sequences for recombinant protein production. This method relies on the competitive translation of two genes in a reaction mixture for cell-free protein synthesis. Genes from a library with randomized +2/+3 codons were individually co-expressed with superfolder green fluorescent protein (sfGFP) in this reaction mixture. As a result of competitive translation, the intensity of sfGFP fluorescence was inversely proportional to the relative translational efficiency of the co-expressed gene. Using this simple method, we identified the +2/+3 codons that markedly enhanced production of recombinant human erythropoietin compared with its wild-type codons. These +2/+3 codons also effectively enhanced protein production in Escherichia coli, indicating that this method is a useful tool to design optimal gene constructs for industrial production of recombinant proteins.

Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff.

Vaccinia virus infection causes a host shutoff that is marked by global inhibition of host protein synthesis. Though the host shutoff may facilitate reallocation of cellular resources for viral replication and evasion of host antiviral immune responses, it poses a challenge for continuous synthesis of cellular proteins that are important for viral replication. It is, however, unclear whether and how certain cellular proteins may be selectively synthesized during the vaccinia virus-induced host shutoff. Using simultaneous RNA sequencing and ribosome profiling, two techniques quantifying genome-wide levels of mRNA and active protein translation, respectively, we analyzed the responses of host cells to vaccinia virus infection at both the transcriptional and translational levels. The analyses showed that cellular mRNA depletion played a dominant role in the shutoff of host protein synthesis. Though the cellular mRNAs were significantly reduced, the relative translation efficiency of a subset of cellular mRNAs increased, particularly those involved in oxidative phosphorylation that are responsible for cellular energy production. Further experiments demonstrated that the protein levels and activities of oxidative phosphorylation increased during vaccinia virus infection, while inhibition of the cellular oxidative phosphorylation function significantly suppressed vaccinia virus replication. Moreover, the short 5' untranslated region of the oxidative phosphorylation mRNAs contributed to the translational upregulation. These results provide evidence of a mechanism that couples translational control and energy metabolism, two processes that all viruses depend on host cells to provide, to support vaccinia virus replication during a host shutoff.IMPORTANCE Many viral infections cause global host protein synthesis shutoff. While host protein synthesis shutoff benefits the virus by relocating cellular resources to viral replication, it also poses a challenge to the maintenance of cellular functions necessary for viral replication if continuous protein synthesis is required. Here we measured the host mRNA translation rate during a vaccinia virus-induced host shutoff by analyzing total and actively translating mRNAs in a genome-wide manner. This study revealed that oxidative phosphorylation mRNAs were translationally upregulated during vaccinia virus-induced host protein synthesis shutoff. Oxidative phosphorylation is the major cellular energy-producing pathway, and we further showed that maintenance of its function is important for vaccinia virus replication. This study highlights the fact that vaccinia virus infection can enhance cellular energy production through translational upregulation in the context of an overall host protein synthesis shutoff to meet energy expenditure.

Assessing translational efficiency by a reporter protein co-expressed in a cell-free synthesis system

We demonstrate the use of a cell-free protein synthesis system as a convenient tool for assessing the relative translational efficiencies of genes. When sfGFP was used as a common reporter gene and co-expressed with a series of target genes, the intensities of sfGFP fluorescence from the co-expression reactions were highly correlated with the individual expression levels of the co-expressed genes. The relative translational efficiencies of genes estimated by this method were reproducible when the same genes were expressed in transformed Escherichia coli, suggesting that this method could be used as a universal tool for prognostic assessment of translational efficiency.

TRNA Shifts the G-quadruplex-Hairpin Conformational Equilibrium in RNA towards the Hairpin Conformer.

Non-coding RNAs play important roles in cellular homeostasis and are involved in many human diseases including cancer. Intermolecular RNA-RNA interactions are the basis for the diverse functions of many non-coding RNAs. Herein, we show how the presence of tRNA influences the equilibrium between hairpin and G-quadruplex conformations in the 5' untranslated regions of oncogenes and model sequences. Kinetic and equilibrium analyses of the hairpin to G-quadruplex conformational transition of purified RNA as well as during co-transcriptional folding indicate that tRNA significantly shifts the equilibrium toward the hairpin conformer. The enhancement of relative translation efficiency in a reporter gene assay is shown to be due to the tRNA-mediated shift in hairpin-G-quadruplex equilibrium of oncogenic mRNAs. Our findings suggest that tRNA is a possible therapeutic target in diseases in which RNA conformational equilibria is dysregulated.

TRNA Shifts the G‐quadruplex–Hairpin Conformational Equilibrium in RNA towards the Hairpin Conformer

AbstractNon‐coding RNAs play important roles in cellular homeostasis and are involved in many human diseases including cancer. Intermolecular RNA–RNA interactions are the basis for the diverse functions of many non‐coding RNAs. Herein, we show how the presence of tRNA influences the equilibrium between hairpin and G‐quadruplex conformations in the 5′ untranslated regions of oncogenes and model sequences. Kinetic and equilibrium analyses of the hairpin to G‐quadruplex conformational transition of purified RNA as well as during co‐transcriptional folding indicate that tRNA significantly shifts the equilibrium toward the hairpin conformer. The enhancement of relative translation efficiency in a reporter gene assay is shown to be due to the tRNA‐mediated shift in hairpin‐G‐quadruplex equilibrium of oncogenic mRNAs. Our findings suggest that tRNA is a possible therapeutic target in diseases in which RNA conformational equilibria is dysregulated.

Gene Regulation in Developing Chloroplasts Disentangled.

Gene Regulation in Developing Chloroplasts Disentangled.

Coaxing a Viral RNA Out Of Its Shell: How Does a Viral RNA Genome Initiate Contact With Its Host?

A conceptual challenge in understanding the life cycles of all viruses is how a rigid protein shell – the “capsid” – can both protect the viral genome from nucleases and other insults and, at the appropriate downstream stage, make its genetic information available to the host cell machinery. For double-stranded DNA viruses, like bacteriophages or Herpes Simplex Virus, genomes are packaged into pre-formed empty capsids, and released by pressure-driven ejection, with the capsid remaining intact: work is done to package the genome, but its delivery is spontaneous. In contrast, for single-stranded RNA viruses, like polio or SARS, packaging of their genomes is spontaneous, with capsid protein organizing around the RNA in a co-self-assembly process. These RNA-containing viral capsids are relatively stable, inert particles while on their journey to the next host cell, but must eventually release their RNA to its host to make it available for further viral life cycle processes. This immediately raises the question of whether work is needed to release the RNA from its protective capsid. In the case of positive-strand RNA viruses, whose genomes are directly translated into the viral replication machinery following their entry into the host cell cytoplasm, ribosomes have been implicated in the delivery/release of the viral RNA. We report here on the synthesis of constructs designed to measure the force needed to pull RNA out of its capsid, using virus-like particles reconstituted in vitro from RNA and purified capsid protein from a particularly well-studied virus, cowpea chlorotic mottle virus (CCMV). We also investigate the extent to which the ends of a packaged RNA are accessible for interaction with probes impermeable to the interior of the capsid. The probing techniques offer keen insights regarding the ability for RNA to fluctuate outside of its intact capsid in order to engage the host ribosomal machinery. These results are used to interpret corresponding relative translation efficiencies of naked and packaged forms of RNA genes in cell-free extracts, in an effort to determine how ribosomes and their associated factors are involved in this viral RNA uncoating process. Support or Funding Information Research was supported by the NSF, and for R.W.S. by USPHS of the National Institute of Heath, National Research Service Award 5T32GM008496-20 and the UCLA Graduate Office. An in vitro self-assembled virus-like particle, the cherry bomb, a construct used for generating a handle on an encapsidated viral RNA genome. We are using this construct such that we have access to the viral RNA, can pull it out of its capsid and to measure the force necessary to do so.

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

Coaxing a Viral RNA Out of its Shell: How does a Viral RNA Genome Initiate Contact with its Host?

A conceptual challenge in understanding the life cycles of all viruses is how a rigid protein shell - the “capsid” - can both protect the viral genome from nucleases and other insults and, at the appropriate downstream stage, make its genetic information available to the host cell machinery. For double-stranded DNA viruses, like bacteriophages or Herpes Simplex Virus, genomes are packaged into preformed empty capsids, and released by pressure-driven ejection, with the capsid remaining intact: work is done to package the genome, but its delivery is spontaneous. In contrast, for single-stranded RNA viruses, like polio or SARS, packaging of their genomes is spontaneous, with capsid protein organizing around the RNA in a self-assembly process. This immediately raises the question of whether work is needed to release the RNA from its protective capsid. In the case of positive-strand RNA viruses, whose genomes are directly translated following their entry into the host cell cytoplasm, ribosomes have been implicated in the delivery/release of the viral RNA. We report here on the synthesis of constructs designed to measure the force needed to pull RNA out of its capsid, using virus-like particles reconstituted in vitro from RNA and purified capsid protein from a particularly well-studied virus, cowpea chlorotic mottle virus (CCMV). We also investigate the extent to which the ends of a packaged RNA are accessible for interaction with probes impermeable to the interior of the capsids. These results are used to interpret relative translation efficiencies of naked and packaged forms of RNA genes in cell-free extracts, in an effort to determine how ribosomes are involved in this RNA uncoating process.

Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

Robust, quantitative analysis of proteins using peptide immunoreagents, in vitro translation, and an ultrasensitive acoustic resonant sensor.

A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-xL. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications.

Polysome profiling reveals broad translatome remodeling during endoplasmic reticulum (ER) stress in the pathogenic fungus Aspergillus fumigatus

BackgroundThe 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.ResultsER 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.ConclusionsTogether, 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.

Length variants of the 5′ untranslated region of p53 mRNA and their impact on the efficiency of translation initiation of p53 and its N-truncated isoform ΔNp53

Recently, we have determined the secondary structure of the 5′-terminal region of p53 mRNA that starts from the P1 transcription initiation site and includes two major translation initiation codons responsible for the synthesis of p53 and ΔNp53 isoform. Here, we showed that when this region was extended into 5′ direction to the P0 transcription start site, the two characteristic hairpin motifs found in this region were preserved. Moreover, the presence of alternatively spliced intron 2 did not interfere with the formation of the larger hairpin in which the initiation codon for p53 was embedded. The impact of the different variants of p53 5′-terminal region, which start at P0 or P1 site and end with the initiation codon for p53 or ΔNp53, on the translation of luciferase reporter protein was compared. Strikingly, the efficiency of translation performed in rabbit reticulocyte lysate differed by two orders of magnitude. The toe-printing analysis was also applied to investigate the formation of the ribosomal complex on the model mRNA constructs. The relative translation efficiencies in HeLa and MCF-7 cells were similar to those observed in the cell lysate, although some differences were noted in comparison with cell-free conditions. The results were discussed in terms of the role of secondary structure folding of the 5′-terminal region of p53 mRNA in translation and possible modes of p53 and ΔNp53 translation initiation.