RESUMO
BACKGROUND: The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the high costs of full-length mRNA sequencing have posed a limit on the accessibility and scalability of the technology. To address this, we developed 3'Pool-seq: a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3'-end of mRNA. We drew from aspects of SMART-seq, Drop-seq, and TruSeq to implement an easy workflow, and optimized parameters such as input RNA concentrations, tagmentation conditions, and read depth specifically for bulk-RNA. RESULTS: Thorough optimization resulted in a protocol that takes less than 12 h to perform, does not require custom sequencing primers or instrumentation, and cuts over 90% of the costs associated with TruSeq, while still achieving accurate gene expression quantification (Pearson's correlation coefficient with ERCC theoretical concentration r = 0.96) and differential gene detection (ROC analysis of 3'Pool-seq compared to TruSeq AUC = 0.921). The 3'Pool-seq dual indexing scheme was further adapted for a 96-well plate format, and ERCC spike-ins were used to correct for potential row or column pooling effects. Transcriptional profiling of troglitazone and pioglitazone treatments at multiple doses and time points in HepG2 cells was then used to show how 3'Pool-seq could distinguish the two molecules based on their molecular signatures. CONCLUSIONS: 3'Pool-seq can accurately detect gene expression at a level that is on par with TruSeq, at one tenth of the total cost. Furthermore, its unprecedented TruSeq/Nextera hybrid indexing scheme and streamlined workflow can be applied in several different formats, including 96-well plates, which allows users to thoroughly evaluate biological systems under several conditions and timepoints. Care must be taken regarding experimental design and plate layout such that potential pooling effects can be accounted for and corrected. Lastly, further studies using multiple sets of ERCC spike-ins may be used to simulate differential gene expression in a system with known ground-state values.
Assuntos
RNA-Seq/métodos , Animais , Análise Custo-Benefício , Células Hep G2 , Humanos , Camundongos , Pioglitazona/farmacologia , RNA-Seq/economia , Transcriptoma/efeitos dos fármacos , Troglitazona/farmacologiaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered rapid widespread changes in protein phosphorylation following DUX4 induction, indicating that alterations in kinase signaling might play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover that the JNK pathway is involved in DUX4-mediated cell death and provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.
Assuntos
Distrofia Muscular Facioescapuloumeral , Humanos , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mioblastos/metabolismo , Regulação da Expressão Gênica , Músculo Esquelético/metabolismoRESUMO
Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wildtype PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.
Assuntos
Distrofia Muscular de Duchenne , Células-Tronco Pluripotentes , Humanos , Distrofina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/patologia , Músculo Esquelético/patologia , Fibras Musculares Esqueléticas/patologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologiaRESUMO
The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminopiridinas/farmacologia , Ativadores de Enzimas/farmacologia , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Indóis/farmacologia , Aminopiridinas/uso terapêutico , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/uso terapêutico , Feminino , Hipoglicemiantes/uso terapêutico , Indóis/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismoRESUMO
Local protein synthesis is required for long-lasting synapse-specific plasticity in cultured Aplysia sensorimotor synapses. To identify synaptically localized mRNAs, we prepared a cDNA library from isolated sensory neurites. By sequence analysis, we estimate that the library contains 263 distinct mRNAs, with 98 of these mRNAs constituting 70% of all clones. The localized transcripts are enriched for mRNAs encoding cytoskeletal elements and components of the translational machinery. In situ hybridization confirms that the mRNAs for at least eight of these transcripts are present in distal neurites. Immunocytochemistry reveals that serotonin regulates the translation of one of the localized mRNAs, that encoding alpha1-tubulin. Our identification of mRNAs encoding cytoskeletal elements suggests that local protein synthesis is required for the growth of new synaptic connections associated with persistent synaptic strengthening. Our finding of mRNAs encoding components of the translational machinery suggests that local protein synthesis serves to increase the translational capacity of synapses.