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Nucleic Acids Research Feb 2017During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been...
During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing.
Topics: Animals; Female; Fertilization; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Male; Paracentrotus; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Sequence Analysis, RNA
PubMed: 28180329
DOI: 10.1093/nar/gkw907 -
Nature Oct 2022Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and...
Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here we use advances in cryo-electron tomography and sub-tomogram analysis to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
Topics: Anti-Bacterial Agents; Cryoelectron Microscopy; Mycoplasma pneumoniae; Peptide Chain Elongation, Translational; Polyribosomes; Protein Biosynthesis; Ribosomal Proteins; Ribosomes
PubMed: 36171285
DOI: 10.1038/s41586-022-05255-2 -
Nature Aug 2021Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies....
Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.
Topics: Cell Line; Collagen; Dengue Virus; Flavivirus; Gene Expression Regulation, Viral; Genomics; Host-Derived Cellular Factors; Host-Pathogen Interactions; Humans; Hydroxylation; Internal Ribosome Entry Sites; Molecular Chaperones; Peptide Chain Initiation, Translational; Poliovirus; Polyribosomes; Procollagen-Proline Dioxygenase; Proline; Protein Aggregates; Protein Biosynthesis; Protein Folding; Protein Interaction Maps; Proteolysis; Proteomics; Zika Virus
PubMed: 34408324
DOI: 10.1038/s41586-021-03851-2 -
ELife May 2022Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the...
Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and activation. Importantly, non-catalytic mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.
Topics: Aspartate Aminotransferases; Oxidative Stress; Polyribosomes; Protein Biosynthesis; RNA-Binding Proteins; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae Proteins
PubMed: 35621265
DOI: 10.7554/eLife.73466 -
Scientific Reports Apr 2022Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the...
Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages anti-rRNA ribosome immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.
Topics: High-Throughput Nucleotide Sequencing; Polyribosomes; Protein Biosynthesis; Ribosomes; Sequence Analysis, RNA
PubMed: 35383235
DOI: 10.1038/s41598-022-09638-3 -
Biochemical and Biophysical Research... Aug 2022Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain....
Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation.
Topics: Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factor-4G; Poly(A)-Binding Proteins; Polyribosomes; Protein Binding; Protein Biosynthesis; RNA, Messenger; Ribonucleoproteins
PubMed: 35716598
DOI: 10.1016/j.bbrc.2022.06.017 -
Journal of Visualized Experiments : JoVE Jun 2021Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being...
Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.
Topics: Cell Fractionation; Centrifugation, Density Gradient; Molecular Biology; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Ribosomes; Saccharomyces cerevisiae
PubMed: 34152326
DOI: 10.3791/62680 -
Molecular Biology of the Cell Oct 2022It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation...
It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 d in the circulatory system. Here, using a highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [S]methionine labeling, and RiboPuromycylation. [S]methionine labeling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative real-time PCR performed on polysome fractions of these cells revealed that (α-globin) and (β-globin) transcripts are translated. Using a luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (α- and β-) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.
Topics: 5' Untranslated Regions; Erythrocytes; Humans; Methionine; Polyribosomes; Protein Biosynthesis; alpha-Globins; beta-Globins
PubMed: 35976696
DOI: 10.1091/mbc.E21-09-0437 -
Journal of Molecular Cell Biology Oct 2019The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Its mutation often leads to...
The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Its mutation often leads to aberrant gene expression in cancer. IDH1 was reported to bind thousands of RNA transcripts in a sequence-dependent manner; yet, the functional significance of this RNA-binding activity remains elusive. Here, we report that IDH1 promotes mRNA translation via direct associations with polysome mRNA and translation machinery. Comprehensive proteomic analysis in embryonic stem cells (ESCs) revealed striking enrichment of ribosomal proteins and translation regulators in IDH1-bound protein interactomes. We performed ribosomal profiling and analyzed mRNA transcripts that are associated with actively translating polysomes. Interestingly, knockout of IDH1 in ESCs led to significant downregulation of polysome-bound mRNA in IDH1 targets and subtle upregulation of ribosome densities at the start codon, indicating inefficient translation initiation upon loss of IDH1. Tethering IDH1 to a luciferase mRNA via the MS2-MBP system promotes luciferase translation, independently of the catalytic activity of IDH1. Intriguingly, IDH1 fails to enhance luciferase translation driven by an internal ribosome entry site. Together, these results reveal an unforeseen role of IDH1 in fine-tuning cap-dependent translation via the initiation step.
Topics: Animals; CRISPR-Cas Systems; Embryonic Stem Cells; Isocitrate Dehydrogenase; Ketoglutaric Acids; Mice; Polyribosomes; RNA, Messenger; Ribosomes
PubMed: 31408165
DOI: 10.1093/jmcb/mjz082 -
Journal of Visualized Experiments : JoVE Apr 2018Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time,...
Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.
Topics: Animals; Gene Expression Profiling; Humans; Leishmania; Male; Mice; Polyribosomes; Testis
PubMed: 29683462
DOI: 10.3791/57600