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European Journal of Biochemistry Feb 1978Techniques allowing the recovery of large free and membrane-bound polysomes in high yield are reported. Subcellular fractions were prepared from rat liver homogenates as...
Techniques allowing the recovery of large free and membrane-bound polysomes in high yield are reported. Subcellular fractions were prepared from rat liver homogenates as described in the preceding paper. Purified microsomal membranes (obtained from the post-lysosomal supernatant) were adjusted to 50 mM Mg(CH3COO)2 and treated with 2% Triton X-100 and 0.3% sodium deoxycholate in the presence of yeast RNA and cell sap, and polysomes were purified by overnight centrifugation through low-ionic-strength discontinuous sucrose gradients containing 2 mg/ml of cell sap proteins. Polysomes were isolated from the mitochondria/endoplasmic reticulum complex (fraction C) by treatment with 2% Triton X-100 and 0.5% sodium deoxycholate in the presence of 50 mM Tris-HCl, pH 7.6, 0.1 M KCl, 0.15 M NH4Cl and 50 mM Mg(CH3COO)2 and purified through sucrose layers of decreasing ionic strength containing 2 mg/ml of cell sap proteins. Analyses of polysomes in isokinetic sucrose gradients showed that the free polysome fraction and both membrane-bound polysome fractions had 14-15 ribosomes per mRNA at the maximum of absorbance. Experiments from which these methods were derived are described.
Topics: Animals; Cell Fractionation; Centrifugation, Density Gradient; Liver; Male; Membranes; Microsomes, Liver; Molecular Weight; Polyribosomes; RNA; Rats
PubMed: 627209
DOI: 10.1111/j.1432-1033.1978.tb12063.x -
Journal of Visualized Experiments : JoVE Mar 2016The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes,...
The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.
Topics: Animals; Brain; Cryoelectron Microscopy; Imaging, Three-Dimensional; Mice; Microscopy, Atomic Force; Polyribosomes
PubMed: 27023752
DOI: 10.3791/53851 -
Methods (San Diego, Calif.) Feb 2019The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be...
The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with traditional biochemical methods of studying translation to extend our understanding of these relationships. Additionally, there is growing importance given to how these processes may be regulated across varied cell types as a means to achieve tissue-specific expression of proteins. Here, we provide an in-depth methodology for polysome profiling to dissect the composition of mRNAs and proteins that make up the translatome from both whole tissues and a specific cell type isolated from mammalian tissue. Also, we provide a detailed computational workflow for the analysis of the next-generation sequencing data generated from these experiments.
Topics: Animals; Brain; Cell Fractionation; Centrifugation, Density Gradient; Computational Biology; Gene Ontology; Gene Regulatory Networks; Hepatocytes; High-Throughput Nucleotide Sequencing; Liver; Mice; Molecular Sequence Annotation; Myocardium; Myocytes, Cardiac; Neurons; Organ Specificity; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Sequence Analysis, RNA
PubMed: 30500367
DOI: 10.1016/j.ymeth.2018.11.015 -
PloS One 2019Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive...
Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome fractionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.
Topics: 3' Untranslated Regions; Escherichia coli; Escherichia coli Proteins; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 30779773
DOI: 10.1371/journal.pone.0212297 -
Biochemistry. Biokhimiia Jan 2018Here I introduce collection of review articles written by members of the Institute of Protein Research of the Russian Academy of Sciences. This collection commemorates...
Here I introduce collection of review articles written by members of the Institute of Protein Research of the Russian Academy of Sciences. This collection commemorates the 50th anniversary of the Institute. The review articles cover a broad range of problems concerning the spatial structure of protein molecules, including the state of the molten globule, protein-RNA interactions, polysome and ribosome structure, the molecular colony method, and the original methods for studying the structure of proteins. Several of the reviews consider the practical use of knowledge about the structure of proteins and protein polymers. They reflect both the long experience of the authors and contemporary scientific data.
Topics: Academies and Institutes; Polyribosomes; Proteins; RNA; Research; Ribosomes; Russia
PubMed: 29544426
DOI: 10.1134/S0006297918140018 -
The Journal of Cell Biology Aug 1981Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal...
Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations.
Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal cytoplasmic ribonucleoprotein. Poly(A)+ RNA fractions isolated from free and bound polysomal RNA (poly(A)+ RNAfree and poly(A)+ RNAbound) by oligo(dT) cellulose chromatography exhibited number-average lengths of 1,600 and 1,200 nucleotides, respectively, on formamide sucrose gradients. Poly(A)+ RNAfree and poly(A)+ RNAbound contain 9.1 +/- 0.55 and 10.7 +/- 0.50% poly(A) as measured by hybridization to [3H]poly(U) and comprise 2.37 and 1.22% of their respective polysomal RNA populations. Homologous poly(A)+ RNA-cDNA hybridizations revealed that greater than 95% of the mass of poly(A)+ RNAfree and poly(A)+ RNAbound contain nucleotide complexities of about 3.4 x 10(7) and 6.0 x 10(6), respectively. This represents about 20,000 and 5,000 poly(A)+ RNA species of average sizes. Heterologous hybridizations suggested that considerable overlap exists between poly(A)+ RNAfree and poly(A)+ RNAbound sequences that cannot be attributed to cross-contamination. This was confirmed by conducting heterologous reactions using kinetically enriched cDNA populations. Heterologous hybridizations involving poly(A)+ RNA derived from tightly bound polysomes and cDNAfree indicated tha most of the overlapping sequences are not contributed by loosely bound (high-salt releasable) polysomes. The ramifications of these findings are discussed.
Topics: Animals; Base Sequence; Cell Fractionation; Centrifugation, Density Gradient; DNA; Liver; Nucleic Acid Hybridization; Poly A; Polyribosomes; RNA, Messenger; Rats
PubMed: 6116718
DOI: 10.1083/jcb.90.2.495 -
PLoS Pathogens Jun 2019Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV...
Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5' cap-structure and 3'-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. Consistent with an overwhelming abundance of viral mRNA in the polysome fraction, the reads mapping to cellular genes were reduced. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)-encoded by 2 such mRNAs-support viral replication. Correspondingly, regulated in development and DNA damage 1 (Redd1) encoded by a host mRNA with reduced polysome association inhibits viral infection. These data underscore the importance of viral mRNA abundance in the shut-off of host translation in VSV infected cells and link the differential translatability of some cellular mRNAs with pro- or antiviral function.
Topics: HeLa Cells; Humans; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Vesicular Stomatitis; Vesiculovirus; Virus Replication
PubMed: 31226162
DOI: 10.1371/journal.ppat.1007875 -
Scientific Data Dec 2018The regulation of gene expression acts at numerous complementary levels to control and refine protein abundance. The analysis of mRNAs associated with polysomes, called...
The regulation of gene expression acts at numerous complementary levels to control and refine protein abundance. The analysis of mRNAs associated with polysomes, called polysome profiling, has been used to investigate the post-transcriptional mechanisms that are involved in different biological processes. Pluripotent stem cells are able to differentiate into a variety of cell lineages, and the cell commitment progression is carefully orchestrated. Genome-wide expression profiling has provided the possibility to investigate transcriptional changes during cardiomyogenesis; however, a more accurate study regarding post-transcriptional regulation is required. In the present work, we isolated and high-throughput sequenced ribosome-free and polysome-bound RNAs from NKX2-5 HES3 undifferentiated pluripotent stem cells at the subsequent differentiation stages of cardiomyogenesis: embryoid body aggregation, mesoderm, cardiac progenitor and cardiomyocyte. The expression of developmental markers was followed by flow cytometry, and quality analyses were performed as technical controls to ensure high quality data. Our dataset provides valuable information about hESC cardiac differentiation and can be used to investigate genes potentially controlled by post-transcriptional mechanisms.
Topics: Cell Differentiation; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Human Embryonic Stem Cells; Humans; Myocytes, Cardiac; Polyribosomes
PubMed: 30512016
DOI: 10.1038/sdata.2018.287 -
Plant Physiology Jul 2012Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression...
Posttranscriptional control of photosynthetic mRNA decay under stress conditions requires 3' and 5' untranslated regions and correlates with differential polysome association in rice.
Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3' and 5' untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3' and 5' untranslated regions and correlates with differential polysome association.
Topics: 3' Untranslated Regions; 5' Untranslated Regions; Cluster Analysis; Cold Temperature; Droughts; Gene Expression Regulation, Plant; Genes, Plant; Half-Life; Oligonucleotide Array Sequence Analysis; Oryza; Photosynthesis; Plant Proteins; Plants, Genetically Modified; Polyribosomes; RNA Stability; Sodium Chloride; Stress, Physiological; Transcription, Genetic; Untranslated Regions
PubMed: 22566494
DOI: 10.1104/pp.112.194928 -
International Journal of Molecular... Jan 2021Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel...
Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation.
Topics: Cell Differentiation; Cell Line; Energy Metabolism; Human Embryonic Stem Cells; Humans; Lipid Metabolism; Myocytes, Cardiac; Polyribosomes; RNA, Messenger; Signal Transduction; Transcriptome
PubMed: 33572750
DOI: 10.3390/ijms22031330