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Cell Sep 2020The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and...
The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.
Topics: Aging; Animals; Circadian Clocks; Colon; Endoplasmic Reticulum, Rough; Enteric Nervous System; Epithelial Cells; Female; Gene Expression Regulation, Developmental; Genetic Predisposition to Disease; Humans; Ileum; Inflammation; Intestinal Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Nervous System Diseases; Neuroglia; Neurons; Nissl Bodies; RNA, Messenger; RNA-Seq; Ribosomes; Single-Cell Analysis; Stromal Cells
PubMed: 32888429
DOI: 10.1016/j.cell.2020.08.003 -
Biochimica Et Biophysica Acta.... Jan 2021Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental... (Review)
Review
Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.
Topics: Cell Compartmentation; Cell Membrane; Cytoplasm; Cytoplasmic Granules; Humans; Liquid Phase Microextraction; Polyribosomes; RNA, Messenger; Ribonucleoproteins; Stress, Physiological
PubMed: 33007331
DOI: 10.1016/j.bbamcr.2020.118876 -
Cell Jul 2020Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular...
Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.
Topics: ATP-Binding Cassette Transporters; Anisomycin; Apoptosis; DNA Damage; Enzyme Activation; Humans; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 14; Phosphorylation; Polyribosomes; Protein Isoforms; Protein Serine-Threonine Kinases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Ribosomes; Signal Transduction; Stress, Physiological; Ultraviolet Rays; eIF-2 Kinase
PubMed: 32610081
DOI: 10.1016/j.cell.2020.06.006 -
Cell Aug 2021Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD synthesis plays an...
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Topics: 3' Untranslated Regions; ADP-Ribosylation; Animals; Base Sequence; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Stress; Fallopian Tubes; Female; Humans; Mice, Inbred NOD; Mice, SCID; NAD; Nicotinamide-Nucleotide Adenylyltransferase; Nucleic Acid Conformation; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Polyribosomes; Protein Biosynthesis; Proteostasis; RNA, Messenger; RNA, Small Interfering; Ribosomal Proteins; Ribosomes; Mice
PubMed: 34314702
DOI: 10.1016/j.cell.2021.07.005 -
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 -
Molecular Cell Nov 2021Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel...
Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.
Topics: Animals; Disease Progression; Eukaryotic Initiation Factor-4F; Eukaryotic Initiation Factor-4G; Genome, Human; HEK293 Cells; Hematologic Neoplasms; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Open Reading Frames; Peptides; Polyribosomes; Protein Biosynthesis; RNA, Messenger; RNA, Untranslated; RNA-Binding Proteins; Ribosomes; Sensitivity and Specificity; Treatment Outcome
PubMed: 34555354
DOI: 10.1016/j.molcel.2021.08.033 -
Cell Oct 2016tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact...
tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N-methyladenosine (mA) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.
Topics: Adenosine; AlkB Homolog 1, Histone H2a Dioxygenase; Gene Expression Regulation; Glucose; HeLa Cells; Humans; Methylation; Polyribosomes; Protein Biosynthesis; RNA, Transfer
PubMed: 27745969
DOI: 10.1016/j.cell.2016.09.038 -
Bio-protocol Mar 2017Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By...
Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription - PCR to investigate the translational state of the mRNA.
PubMed: 28603752
DOI: 10.21769/BioProtoc.2192 -
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 Sep 2018N-methyladenosine (mA) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and...
N-methyladenosine (mA) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered mA homeostasis is linked to cancer. mA modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon. METTL3 can promote translation but the mechanism and relevance of this process remain unknown. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also mA-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.
Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cyclization; Eukaryotic Initiation Factor-3; Female; Humans; Lung Neoplasms; Methyltransferases; Mice; Mice, Nude; Nucleic Acid Conformation; Polyribosomes; Protein Binding; Protein Biosynthesis; RNA, Messenger
PubMed: 30232453
DOI: 10.1038/s41586-018-0538-8