-
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 -
Cell Cycle (Georgetown, Tex.) 2017
Topics: Polyribosomes; RNA Stability; RNA, Nuclear
PubMed: 28933589
DOI: 10.1080/15384101.2017.1377501 -
Biochemistry. Biokhimiia Jan 2018Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has... (Review)
Review
Polyribosomes in cells usually have a certain structural organization whose significance has not yet been elucidated. The development of cryo electron tomography has provided a new approach to study polyribosome structure. New data confirm or correct observations made earlier by classical techniques of electron microscopy. The existence of circular and linear (zigzag) topology of polyribosomes was confirmed, and their relationship with the frequently observed two-row forms was clarified. Contacts between ribosomes have been identified in densely packed three-dimensional helical polyribosomes. At the same time, modern cell-free translation systems have opened the possibility of investigating polyribosomes on mRNA of a given structure to elucidate the mechanism of polyribosome structure formation, especially of circular polyribosomes. There is an increasing amount of data supporting the idea of interdependence between polyribosome structure and their translational activity. Moreover, participation of polyribosomes in mRNA transport and localization of protein synthesis in the cell has been shown. Improvement of the resolution and the development of the cryo electron tomography technique for the analysis of polyribosomes in situ will enable further progress in understanding the process of protein synthesis in cells.
Topics: Cryoelectron Microscopy; Polyribosomes
PubMed: 29544430
DOI: 10.1134/S0006297918140055 -
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 -
Trends in Biochemical Sciences Dec 2018Great progress has been made toward solving the atomic structure of the ribosome, which is the main biosynthetic machine in cells, but we still do not have a full... (Review)
Review
Great progress has been made toward solving the atomic structure of the ribosome, which is the main biosynthetic machine in cells, but we still do not have a full picture of exactly how cellular ribosomes function. Based on the analysis of crystallographic and electron microscopy data, we propose a basic model of the structural organization of ribosomes into a compartment. This compartment is regularly formed by arrays of ribosomal tetramers made up of two dimers that are actually facing in opposite directions. The compartment functions as the main 'factory' for the production of cellular proteins. The model is consistent with the existing biochemical and genetic data. We also consider the functional connections of such a compartment with cellular transcription and ribosomal biogenesis.
Topics: DNA-Directed RNA Polymerases; Microscopy, Electron; Polyribosomes; Ribosomal Proteins; Ribosomes
PubMed: 30337135
DOI: 10.1016/j.tibs.2018.09.017 -
Methods (San Diego, Calif.) Mar 2017Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs... (Review)
Review
Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs translated in this way are referred to 'translatome.' Translation determines a cell's overall gene expression profile. Studying translatome leads to a better understanding of the translational machinery and of its complex regulatory pathways. Given its fundamental role in cell homeostasis and division, bacterial translation is an important target for antibiotics. However, there are no detailed protocols for polysome purification from Staphylococcus aureus, the human pathogen responsible for the majority of multi-drug resistance issues. We therefore developed methods for the isolation of active polysomes, ribosomes, and ribosomal subunits, examining the purity and quality of each fraction and monitoring polysomal activity during protein synthesis. These steps are mandatory for the use of purified S. aureus polysomes and ribosomes for structural studies or for genome-scale analysis of most translated mRNAs.
Topics: Cell Fractionation; Electrophoresis, Agar Gel; Microscopy, Electron; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Ribosome Subunits, Large, Bacterial; Ribosome Subunits, Small, Bacterial; Staphylococcus aureus
PubMed: 27729294
DOI: 10.1016/j.ymeth.2016.10.003 -
BMC Genomics Mar 2019Cardiac cell fate specification occurs through progressive steps, and its gene expression regulation features are still being defined. There has been an increasing...
BACKGROUND
Cardiac cell fate specification occurs through progressive steps, and its gene expression regulation features are still being defined. There has been an increasing interest in understanding the coordination between transcription and post-transcriptional regulation during the differentiation processes. Here, we took advantage of the polysome profiling technique to isolate and high-throughput sequence ribosome-free and polysome-bound RNAs during cardiomyogenesis.
RESULTS
We showed that polysome-bound RNAs exhibit the cardiomyogenic commitment gene expression and that mesoderm-to-cardiac progenitor stages are strongly regulated. Additionally, we compared ribosome-free and polysome-bound RNAs and found that the post-transcriptional regulation vastly contributes to cardiac phenotype determination, including RNA recruitment to and dissociation from ribosomes. Moreover, we found that protein synthesis is decreased in cardiomyocytes compared to human embryonic stem-cells (hESCs), possibly due to the down-regulation of translation-related genes.
CONCLUSIONS
Our data provided a powerful tool to investigate genes potentially controlled by post-transcriptional mechanisms during the cardiac differentiation of hESC. This work could prospect fundamental tools to develop new therapy and research approaches.
Topics: Biomarkers; Cell Differentiation; Cells, Cultured; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Human Embryonic Stem Cells; Humans; Myocytes, Cardiac; Organogenesis; Polyribosomes; RNA, Messenger
PubMed: 30876407
DOI: 10.1186/s12864-019-5550-3 -
Biochemistry. Biokhimiia Sep 2021"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a... (Review)
Review
"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.
Topics: Cryoelectron Microscopy; Eukaryota; Nucleic Acid Conformation; Poly A; Polyribosomes; RNA Caps; RNA, Messenger; Ribosome Subunits
PubMed: 34565311
DOI: 10.1134/S0006297921090030 -
American Journal of Physiology.... Dec 2009Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is...
Liver regeneration after 70% partial hepatectomy (PH) in rats induces >95% of hepatocytes to undergo two rounds of semisynchronous cell replication. Gene expression is controlled primarily by posttranscriptional processing, including changes in mRNA stability. However, the translational activity of a specific mRNA can also be modulated after PH, resulting in significant uncoupling of protein and transcript levels relative to quiescent liver for many genes including c-myc and p53. Although the precise mechanism by which this uncoupling occurs is unknown, the polysomal association of mRNA and microRNA (miRNA) can significantly modulate rate of decay as well as translational activity. Thus we characterized the association of c-myc and p53 mRNAs and miRNAs in free and cytoskeleton- and membrane-bound polysome populations 3, 6, and 24 h after PH. The transcripts for c-myc and p53 were differentially distributed in the three discrete polysome populations, and this was dramatically modulated during liver regeneration. Nascent polysome-associated p53 and c-myc proteins were also differentially expressed in the free and cytoskeleton- and membrane-bound polysomes and significantly uncoupled from transcript levels relative to nonresected liver. At least 85 miRNAs were associated with the three polysome populations, and their abundance and distribution changed significantly during liver regeneration. These data suggest that posttranscriptional control of c-myc and p53 protein expression is associated with the translocation of transcripts between the different polyribosomes. The alteration of expression for the same transcript in different polysome populations may, in part, be due to the action of miRNAs.
Topics: Animals; Biological Transport; Cell Proliferation; Gene Expression Profiling; Hepatectomy; Liver; Liver Regeneration; Male; MicroRNAs; Models, Animal; Oligonucleotide Array Sequence Analysis; Polyribosomes; Proto-Oncogene Proteins c-myc; RNA Processing, Post-Transcriptional; RNA, Messenger; Rats; Rats, Sprague-Dawley; Time Factors; Tumor Suppressor Protein p53
PubMed: 19779017
DOI: 10.1152/ajpgi.90636.2008 -
Methods in Molecular Biology (Clifton,... 2022Protein synthesis is a highly regulated essential process. As such, it is subjected to substantial regulation in response to stress. One hallmark of the Integrated...
Protein synthesis is a highly regulated essential process. As such, it is subjected to substantial regulation in response to stress. One hallmark of the Integrated Stress Response (ISR) is the immediate shutdown of most translation through phosphorylation of the alpha subunit of translation initiation factor eIF2 and activation of eIF4E binding proteins. While these posttranslational modifications largely inhibit cap-dependent translation, many mRNA resist this inhibition by alternative translation mechanisms involving cis-regulatory sequences and structures in 5' transcript leaders, including upstream Open Reading Frames (uORFs), Internal Ribosome Entry Sites (IRESes), and Cap-Independent Translation Elements (CITEs). Studies of uORF and IRES activity are often performed on a gene-by-gene basis; however, high-throughput methods have recently emerged. Here, we describe a protocol for Polysome Library Sequencing (PoLib-Seq; Fig. 1), a multiplexed assay of reporter gene translation that can be used during the ISR. A designer library of reporter RNAs are transfected into tissue-culture cells, and their translation is assayed via sucrose gradient fractionation followed by high-throughput sequencing. As an example, we include PoLib-seq results simultaneously assaying translation of wildtype and uORF mutant human ATF4 reporter RNAs, recapitulating the known function of uORF1 in resisting translational inhibition during the ISR.
Topics: Humans; Open Reading Frames; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Ribosomes
PubMed: 35171472
DOI: 10.1007/978-1-0716-1975-9_3