ABSTRACT
Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Lyme Disease/drug therapy , Animals , Borrelia burgdorferi/drug effects , Calibration , Cinnamates/chemistry , Cinnamates/pharmacology , Cinnamates/therapeutic use , Drug Evaluation, Preclinical , Feces/microbiology , Female , HEK293 Cells , Hep G2 Cells , Humans , Hygromycin B/analogs & derivatives , Hygromycin B/chemistry , Hygromycin B/pharmacology , Hygromycin B/therapeutic use , Lyme Disease/microbiology , Mice , Microbial Sensitivity Tests , Microbiota/drug effectsABSTRACT
Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.
Subject(s)
Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Adenine/metabolism , Adenosine/metabolism , Adenosine Deaminase/metabolism , Chromatography, Liquid/methods , HEK293 Cells , Hep G2 Cells , Humans , Intracellular Signaling Peptides and Proteins/physiology , Mass Spectrometry/methods , Multifunctional Enzymes/genetics , Phosphorylation , Proteins/genetics , Purine Nucleotides/metabolism , Purines/metabolismABSTRACT
Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.
Subject(s)
Endoplasmic Reticulum/metabolism , Lactic Acid/metabolism , Magnesium/metabolism , Animals , COS Cells , Calcium/metabolism , Calcium Signaling/physiology , Chlorocebus aethiops , Endoplasmic Reticulum/physiology , Female , HeLa Cells , Hep G2 Cells , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolismABSTRACT
Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.
Subject(s)
Databases, Protein , Gene-Environment Interaction , Genotype , Sex Characteristics , A549 Cells , Animals , Female , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Jurkat Cells , K562 Cells , MCF-7 Cells , Male , Mice , ProteomicsABSTRACT
We performed the first proteogenomic characterization of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using paired tumor and adjacent liver tissues from 159 patients. Integrated proteogenomic analyses revealed consistency and discordance among multi-omics, activation status of key signaling pathways, and liver-specific metabolic reprogramming in HBV-related HCC. Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival, tumor thrombus, genetic profile, and the liver-specific proteome. These proteomic subgroups have distinct features in metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutics. Two prognostic biomarkers, PYCR2 and ADH1A, related to proteomic subgrouping and involved in HCC metabolic reprogramming, were identified. CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed, among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation. Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Fructose-Bisphosphate Aldolase/genetics , Hepatitis B virus , Hepatitis B, Chronic/complications , Liver Neoplasms/genetics , Liver Neoplasms/virology , Proteogenomics/methods , beta Catenin/genetics , Animals , Cell Proliferation , Cohort Studies , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Hep G2 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Tumor Microenvironment/geneticsABSTRACT
Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.
Subject(s)
Chromatin/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , Transcription, Genetic/genetics , YY1 Transcription Factor/metabolism , Binding Sites , Gene Expression Regulation , Genome, Human/genetics , Hep G2 Cells , Humans , K562 Cells , Nuclear Proteins , Promoter Regions, Genetic/genetics , Protein Binding , RNA-Binding Proteins/genetics , RNA-Seq , Transcriptome , YY1 Transcription Factor/geneticsABSTRACT
The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , NF-E2-Related Factor 2/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Carcinoma, Hepatocellular/pathology , Female , Gene Knockdown Techniques , Glucose/metabolism , Glycosylation , HEK293 Cells , Hep G2 Cells , Heterografts , Humans , Kelch-Like ECH-Associated Protein 1/metabolism , Liver Neoplasms/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Phosphotransferases (Alcohol Group Acceptor)/genetics , Proto-Oncogene Proteins c-myc/metabolism , Transduction, GeneticABSTRACT
In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.
Subject(s)
Antigens, Differentiation/biosynthesis , Cell Culture Techniques , Cell Proliferation/drug effects , Hepatocytes/metabolism , Spheroids, Cellular/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Line, Transformed , Hep G2 Cells , Hepatocytes/transplantation , Human Umbilical Vein Endothelial Cells , Humans , Liver/injuries , Liver/metabolism , Mice, Knockout , Spheroids, Cellular/transplantation , Time FactorsABSTRACT
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Subject(s)
Antibiotics, Antitubercular/pharmacology , Congenital Disorders of Glycosylation/metabolism , Enzyme Inhibitors/pharmacology , N-Acetylglucosaminyltransferases/chemistry , Animals , Antibiotics, Antitubercular/chemistry , Binding Sites , Congenital Disorders of Glycosylation/genetics , Enzyme Inhibitors/chemistry , Female , HEK293 Cells , Hep G2 Cells , Humans , Lipid Metabolism , Mice , Molecular Docking Simulation , Mutation , N-Acetylglucosaminyltransferases/antagonists & inhibitors , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Protein Binding , Sf9 Cells , Spodoptera , Tunicamycin/chemistry , Tunicamycin/pharmacology , Uridine Diphosphate Glucuronic Acid/chemistry , Uridine Diphosphate Glucuronic Acid/metabolismABSTRACT
Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.
Subject(s)
Disease Progression , Erythroblasts/cytology , Nerve Tissue Proteins/blood , Spleen/cytology , Transforming Growth Factor beta/metabolism , Animals , Apoptosis , Carcinoma, Hepatocellular/metabolism , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Hep G2 Cells , Humans , Leukocyte Common Antigens/metabolism , Leukocytes/cytology , Liver Neoplasms/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Invasiveness/genetics , Signal TransductionABSTRACT
The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.
Subject(s)
Alphavirus Infections/immunology , Interferons/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , Repressor Proteins/metabolism , Sindbis Virus/physiology , Alphavirus Infections/genetics , Feedback, Physiological , HEK293 Cells , Hep G2 Cells , Homeostasis , Humans , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Protein Binding , Protein Isoforms/genetics , RNA/genetics , RNA, Small Interfering/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Virus ReplicationABSTRACT
Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.
Subject(s)
Gene Expression Regulation , Genes, Reporter , Genetic Diseases, Inborn/genetics , Genetic Techniques , Genetic Variation , Alleles , Gene Library , Hep G2 Cells , Humans , Quantitative Trait Loci , Sensitivity and Specificity , Spondylitis, Ankylosing/geneticsABSTRACT
Insulin signaling regulates many facets of animal physiology. Its dysregulation causes diabetes and other metabolic disorders. The spindle checkpoint proteins MAD2 and BUBR1 prevent precocious chromosome segregation and suppress aneuploidy. The MAD2 inhibitory protein p31(comet) promotes checkpoint inactivation and timely chromosome segregation. Here, we show that whole-body p31(comet) knockout mice die soon after birth and have reduced hepatic glycogen. Liver-specific ablation of p31(comet) causes insulin resistance, hyperinsulinemia, glucose intolerance, and hyperglycemia and diminishes the plasma membrane localization of the insulin receptor (IR) in hepatocytes. MAD2 directly binds to IR and facilitates BUBR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31(comet) blocks the MAD2-BUBR1 interaction and prevents spontaneous clathrin-mediated IR endocytosis. BUBR1 deficiency enhances insulin sensitivity in mice. BUBR1 depletion in hepatocytes or the expression of MAD2-binding-deficient IR suppresses the metabolic phenotypes of p31(comet) ablation. Our findings establish a major IR regulatory mechanism and link guardians of chromosome stability to nutrient metabolism.
Subject(s)
Cell Cycle Checkpoints , Insulin/metabolism , Mitosis , Signal Transduction , Adaptor Protein Complex 2/metabolism , Adaptor Proteins, Signal Transducing , Aneuploidy , Animals , Cell Cycle Proteins/metabolism , Cells, Cultured , Chromosome Segregation , Clathrin/metabolism , Endocytosis , Hep G2 Cells , Homeostasis , Humans , Insulin Resistance , Liver/metabolism , Mad2 Proteins/metabolism , Mice , Mice, Knockout , Nuclear Proteins , Protein Serine-Threonine Kinases/metabolism , Receptor, Insulin/metabolismABSTRACT
Interleukin-22 (IL-22) acts on epithelial cells to promote tissue protection and regeneration, but can also elicit pro-inflammatory effects, contributing to disease pathology. Here, we engineered a high-affinity IL-22 super-agonist that enabled the structure determination of the IL-22-IL-22Rα-IL-10Rß ternary complex to a resolution of 2.6 Å. Using structure-based design, we systematically destabilized the IL-22-IL-10Rß binding interface to create partial agonist analogs that decoupled downstream STAT1 and STAT3 signaling. The extent of STAT bias elicited by a single ligand varied across tissues, ranging from full STAT3-biased agonism to STAT1/3 antagonism, correlating with IL-10Rß expression levels. In vivo, this tissue-selective signaling drove tissue protection in the pancreas and gastrointestinal tract without inducing local or systemic inflammation, thereby uncoupling these opposing effects of IL-22 signaling. Our findings provide insight into the mechanisms underlying the cytokine pleiotropy and illustrate how differential receptor expression levels and STAT response thresholds can be synthetically exploited to endow pleiotropic cytokines with enhanced functional specificity.
Subject(s)
Inflammation/metabolism , Interleukins/metabolism , Animals , Binding Sites/physiology , Cell Line , Cell Line, Tumor , Cytokines/metabolism , Female , HEK293 Cells , HT29 Cells , Hep G2 Cells , Humans , Mice, Inbred C57BL , Protein Binding/physiology , Signal Transduction/physiology , Interleukin-22ABSTRACT
Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here, we report that tetracycline antibiotics, which target the mitoribosome, protected against sepsis without affecting the pathogen load. Mechanistically, we found that mitochondrial inhibition of protein synthesis perturbed the electron transport chain (ETC) decreasing tissue damage in the lung and increasing fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a liver-specific partial and acute deletion of Crif1, a critical mitoribosomal component for protein synthesis, we found that mice were protected against sepsis, an observation that was phenocopied by the transient inhibition of complex I of the ETC by phenformin. Together, we demonstrate that mitoribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a mechanism for the induction of disease tolerance.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Doxycycline/therapeutic use , Liver/immunology , Lung/immunology , Mitochondria/metabolism , Sepsis/drug therapy , Tetracycline/therapeutic use , Animals , Cell Cycle Proteins/genetics , Disease Models, Animal , Electron Transport , Hep G2 Cells , Humans , Lipid Metabolism , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.
Subject(s)
Acyl Coenzyme A/metabolism , Cell Compartmentation , Cell Nucleus/metabolism , Energy Metabolism , Histones/metabolism , Metabolomics , Protein Processing, Post-Translational , Animals , Cell Differentiation , Chromatography, Liquid , Cytosol/metabolism , Epigenesis, Genetic , Hep G2 Cells , Humans , Isoleucine , Metabolome , Mice , Mitochondria/metabolism , Oxygen/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.
Subject(s)
Alternative Splicing , Intramolecular Transferases/metabolism , RNA 3' End Processing , RNA Precursors/metabolism , RNA, Messenger/metabolism , Transcription, Genetic , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , HEK293 Cells , Hep G2 Cells , Humans , Intramolecular Transferases/genetics , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , RNA Precursors/genetics , RNA, Messenger/geneticsABSTRACT
p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a â¼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.
Subject(s)
Apoptosis , Caspase 3/metabolism , Colorectal Neoplasms/enzymology , DNA Breaks, Double-Stranded , DNA Breaks, Single-Stranded , Poly (ADP-Ribose) Polymerase-1/metabolism , RNA, Long Noncoding/metabolism , Tumor Suppressor Protein p53/metabolism , A549 Cells , Animals , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DNA Repair , Gene Expression Regulation, Neoplastic , HCT116 Cells , HEK293 Cells , Hep G2 Cells , Humans , Male , Mice, Nude , MicroRNAs/genetics , MicroRNAs/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , RNA, Long Noncoding/genetics , Signal Transduction , Tumor Suppressor Protein p53/geneticsABSTRACT
Non-covalent complexes of glycolytic enzymes, called metabolons, were postulated in the 1970s, but the concept has been controversial. Here we show that a c-Myc-responsive long noncoding RNA (lncRNA) that we call glycoLINC (gLINC) acts as a backbone for metabolon formation between all four glycolytic payoff phase enzymes (PGK1, PGAM1, ENO1, and PKM2) along with lactate dehydrogenase A (LDHA). The gLINC metabolon enhances glycolytic flux, increases ATP production, and enables cell survival under serine deprivation. Furthermore, gLINC overexpression in cancer cells promotes xenograft growth in mice fed a diet deprived of serine, suggesting that cancer cells employ gLINC during metabolic reprogramming. We propose that gLINC makes a functional contribution to cancer cell adaptation and provide the first example of a lncRNA-facilitated metabolon.
Subject(s)
Biomarkers, Tumor/metabolism , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Glycolysis , Membrane Proteins/metabolism , Neoplasms/enzymology , Phosphoglycerate Kinase/metabolism , Phosphoglycerate Mutase/metabolism , Phosphopyruvate Hydratase/metabolism , RNA, Long Noncoding/metabolism , Thyroid Hormones/metabolism , Tumor Suppressor Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Biomarkers, Tumor/genetics , Carrier Proteins/genetics , Cell Proliferation , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , L-Lactate Dehydrogenase/genetics , L-Lactate Dehydrogenase/metabolism , Membrane Proteins/genetics , Mice, Nude , Multienzyme Complexes , Neoplasms/genetics , Neoplasms/pathology , Phosphoglycerate Kinase/genetics , Phosphoglycerate Mutase/genetics , Phosphopyruvate Hydratase/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Long Noncoding/genetics , Serine/deficiency , Thyroid Hormones/genetics , Tumor Burden , Tumor Suppressor Proteins/genetics , Thyroid Hormone-Binding ProteinsABSTRACT
The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.