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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 Jan 2018Discoveries illuminate highly regulated dynamics of mRNA translation, sequestration, and degradation within the cytoplasm of plants. (Review)
Review
Discoveries illuminate highly regulated dynamics of mRNA translation, sequestration, and degradation within the cytoplasm of plants.
Topics: Cytoplasm; Cytoplasmic Granules; Polyribosomes; RNA Stability; RNA, Messenger; Stress, Physiological
PubMed: 29158329
DOI: 10.1104/pp.17.01468 -
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 -
The New Phytologist Apr 2017This work investigates the extent of translational regulation during seed germination. The polysome occupancy of each gene is determined by genome-wide profiling of...
This work investigates the extent of translational regulation during seed germination. The polysome occupancy of each gene is determined by genome-wide profiling of total mRNA and polysome-associated mRNA. This reveals extensive translational regulation during Arabidopsis thaliana seed germination. The polysome occupancy of thousands of individual mRNAs changes to a large extent during the germination process. Intriguingly, these changes are restricted to two temporal phases (shifts) during germination, seed hydration and germination. Sequence features, such as upstream open reading frame number, transcript length, mRNA stability, secondary structures, and the presence and location of specific motifs correlated with this translational regulation. These features differed significantly between the two shifts, indicating that independent mechanisms regulate translation during seed germination. This study reveals substantial translational dynamics during seed germination and identifies development-dependent sequence features and cis elements that correlate with the translation control, uncovering a novel and important layer of gene regulation during seed germination.
Topics: Arabidopsis; Base Sequence; Gene Expression Regulation, Plant; Germination; Nucleic Acid Conformation; Polyribosomes; Protein Biosynthesis; RNA, Messenger; RNA, Plant; Seeds
PubMed: 27935038
DOI: 10.1111/nph.14355 -
ELife Jan 2016Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human...
Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5' and 3' untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.
Topics: Alternative Splicing; Cell Line; Epithelial Cells; Gene Expression Regulation; Humans; Polyribosomes; Protein Biosynthesis; Protein Isoforms; RNA, Messenger
PubMed: 26735365
DOI: 10.7554/eLife.10921 -
Nucleic Acids Research Jan 2015Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were...
Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20-30 translation rounds during 6-8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed ('juvenile phase'). The progressive loading of the polyribosomes with translating ribosomes induced the opening of the circular polyribosomes and the transformation of a major part of the linear polyribosomes into the dense 3D helices ('transitional phase'). After 2 h from the beginning (about 8-10 rounds of translation) this compact form of polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes ('steady-state phase'). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical polyribosomes.
Topics: Cell-Free System; Cryoelectron Microscopy; Models, Molecular; Polyribosomes; Protein Biosynthesis; RNA, Messenger
PubMed: 25520190
DOI: 10.1093/nar/gku1270 -
MSphere Aug 2018In , mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by...
In , mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3' untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the 3'-UTR probe. A second, homologous protein, Gis2, was identified in the genome of and also bound the 3'-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The 3' UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both and resulted in a modest increase in basal stability of the mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The Δ mutant and Δ Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the Δ and Δ Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis. Stress adaptation is fundamental to the success of as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a element within a G-quadruplex structure that binds two homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.
Topics: 3' Untranslated Regions; Cryptococcus neoformans; Fungal Proteins; Polyribosomes; RNA, Messenger; RNA-Binding Proteins; Ribosomal Proteins; Sensitivity and Specificity; Stress, Physiological
PubMed: 30089646
DOI: 10.1128/mSphere.00201-18 -
Molecular and Biochemical Parasitology Sep 2011In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe...
In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in P. falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.
Topics: Parasitology; Plasmodium falciparum; Polyribosomes; Protein Biosynthesis; RNA, Messenger
PubMed: 21605599
DOI: 10.1016/j.molbiopara.2011.05.003 -
Nucleic Acids Research Sep 2022Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis...
Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. 'molecular sociology', is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.
Topics: Algorithms; Cluster Analysis; Cryoelectron Microscopy; Electron Microscope Tomography; Macromolecular Substances; Polyribosomes
PubMed: 35811088
DOI: 10.1093/nar/gkac547 -
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