ABSTRACT
Eukaryotic mRNAs generally possess a 5' end N7 methyl guanosine (m7G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD+) cap that, in contrast to the m7G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD+ caps, and cocrystal structures of DXO/Rai1 with 3'-NADP+ illuminate the molecular mechanism for how the "deNADding" reaction produces NAD+ and 5' phosphate RNA. Removal of DXO from cells increases NAD+-capped mRNA levels and enables detection of NAD+-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD+ caps can be added to 5'-processed termini. Our findings establish NAD+ as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD+-capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5' end modification distinct from the classical m7G cap that promotes rather than inhibits RNA decay.
Subject(s)
RNA Processing, Post-Transcriptional , RNA Stability , Animals , Endoribonucleases/metabolism , HEK293 Cells , Humans , Mice , NAD/metabolism , Nuclear Proteins/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , RNA, Untranslated/metabolismABSTRACT
Genome-wide association studies (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified noncoding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G-protein-coupled inwardly rectifying potassium channel that regulates neuronal excitability. We studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multielectrode arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21â d of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-induced changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons.
Subject(s)
Ethanol , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Humans , Male , Female , Genome-Wide Association Study , Neurons , RespirationABSTRACT
Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.
Subject(s)
Intellectual Disability , Neurodevelopmental Disorders , Child , Young Adult , Humans , RNA, Messenger/genetics , Phosphoric Monoester Hydrolases/genetics , Neurodevelopmental Disorders/genetics , Intellectual Disability/genetics , Nudix HydrolasesABSTRACT
The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the ß-amyloid (Aß) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aß are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in App gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca2+ reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aß, either fibrillar or oligomeric, has no effect. In culture, APPSwe (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and ßIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.SIGNIFICANCE STATEMENT While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro, modest amounts of excess APP alter the properties of the axon initial segment. The ß-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia.
Subject(s)
Alzheimer Disease , Axon Initial Segment , Male , Female , Mice , Humans , Animals , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Alzheimer Disease/metabolism , Axon Initial Segment/metabolism , Amyloid beta-Peptides/metabolism , Membrane Proteins , Mice, Transgenic , Disease Models, AnimalABSTRACT
Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ("Affected: AF") and four control subjects without variants ("Unaffected: UN"). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single-cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.
Subject(s)
Alcoholism , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Humans , G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Ethanol/pharmacology , Ethanol/metabolism , Neurons/metabolism , Alcoholism/genetics , Alcoholism/metabolism , ElectroencephalographyABSTRACT
Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.
ABSTRACT
Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth, and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.
Subject(s)
Endoribonucleases , Neurogenesis , Animals , Humans , Mice , Neuronal Outgrowth , RNA, MessengerABSTRACT
Specific deletion of the tumor suppressor TRAF3 from B lymphocytes in mice leads to the prolonged survival of mature B cells and expanded B cell compartments in secondary lymphoid organs. In the current study, we investigated the metabolic basis of TRAF3-mediated regulation of B cell survival by employing metabolomic, lipidomic, and transcriptomic analyses. We compared the polar metabolites, lipids, and metabolic enzymes of resting splenic B cells purified from young adult B cell-specific Traf3 -/- and littermate control mice. We found that multiple metabolites, lipids, and enzymes regulated by TRAF3 in B cells are clustered in the choline metabolic pathway. Using stable isotope labeling, we demonstrated that phosphocholine and phosphatidylcholine biosynthesis was markedly elevated in Traf3 -/- mouse B cells and decreased in TRAF3-reconstituted human multiple myeloma cells. Furthermore, pharmacological inhibition of choline kinase α, an enzyme that catalyzes phosphocholine synthesis and was strikingly increased in Traf3 -/- B cells, substantially reversed the survival phenotype of Traf3 -/- B cells both in vitro and in vivo. Taken together, our results indicate that enhanced phosphocholine and phosphatidylcholine synthesis supports the prolonged survival of Traf3 -/- B lymphocytes. Our findings suggest that TRAF3-regulated choline metabolism has diagnostic and therapeutic value for B cell malignancies with TRAF3 deletions or relevant mutations.
Subject(s)
B-Lymphocytes/physiology , Choline Kinase/metabolism , Choline/metabolism , Multiple Myeloma/metabolism , TNF Receptor-Associated Factor 3/metabolism , Animals , Cell Line , Cell Survival , Choline Kinase/genetics , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Knockout , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Mutation/genetics , Phosphorylcholine/metabolism , Signal Transduction , TNF Receptor-Associated Factor 3/geneticsABSTRACT
We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Å resolution provides insights into the molecular basis of the deNADding activity in the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells, and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism.
Subject(s)
NAD/metabolism , Pyrophosphatases/metabolism , RNA, Messenger/metabolism , Humans , NAD/chemistry , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , RNA, Messenger/chemistryABSTRACT
The OPRM1 A118G single nucleotide polymorphism (SNP rs1799971) gene variant encoding the N40D µ-opioid receptor (MOR) has been associated with dependence on opiates and other drugs of abuse but its mechanism is unknown. The frequency of G-allele carriers is ~40% in Asians, ~16% in Europeans, and ~3% in African-Americans. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Here we generated homozygous N40D subject-specific induced inhibitory neuronal cells (iNs) from seven human-induced pluripotent stem (iPS) cell lines from subjects of European descent (both male and female) and probed the impact of N40D MOR regulation on synaptic transmission. We found that D40 iNs exhibit consistently stronger suppression (versus N40) of spontaneous inhibitory postsynaptic currents (sIPSCs) across multiple subjects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were recapitulated in two sets of independently engineered isogenic N40D iNs. In addition, we employed biochemical analysis and observed differential N-linked glycosylation of human MOR N40D. This study identifies neurophysiological and molecular differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.
Subject(s)
Neurons/metabolism , Opioid-Related Disorders/genetics , Polymorphism, Single Nucleotide/genetics , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Synapses/metabolism , Cell Line , Female , Humans , Inhibitory Postsynaptic Potentials , Male , Neurons/cytology , Receptors, Opioid, mu/chemistryABSTRACT
African Americans (AA) have lower prevalence of alcohol dependence and higher subjective response to alcohol than European Americans. Genome-wide association studies (GWAS) have identified genes/variants associated with alcohol dependence specifically in AA; however, the sample sizes are still not large enough to detect variants with small effects. Admixture mapping is an alternative way to identify alcohol dependence genes/variants that may be unique to AA. In this study, we performed the first admixture mapping of DSM-IV alcohol dependence diagnosis, DSM-IV alcohol dependence criterion count, and two scores from the self-rating of effects of ethanol (SRE) as measures of response to alcohol: the first five times of using alcohol (SRE-5) and average of SRE across three times (SRE-T). Findings revealed a region on chromosome 4 that was genome-wide significant for SRE-5 (p value = 4.18E-05). Fine mapping did not identify a single causal variant to be associated with SRE-5; instead, conditional analysis concluded that multiple variants collectively explained the admixture mapping signal. PPARGC1A, a gene that has been linked to alcohol consumption in previous studies, is located in this region. Our finding suggests that admixture mapping is a useful tool to identify genes/variants that may have been missed by current GWAS approaches in admixed populations.
Subject(s)
Alcoholism/genetics , Black or African American/genetics , Ethanol/pharmacology , Genetic Predisposition to Disease , Genome-Wide Association Study , Polymorphism, Single Nucleotide , Self Report , Alcoholism/etiology , Alcoholism/physiopathology , Case-Control Studies , Diagnostic and Statistical Manual of Mental Disorders , Humans , Retrospective Studies , White PeopleABSTRACT
Neuropsychiatric disorders have traditionally been difficult to study due to the complexity of the human brain and limited availability of human tissue. Induced pluripotent stem (iPS) cells provide a promising avenue to further our understanding of human disease mechanisms, but traditional 2D cell cultures can only provide a limited view of the neural circuits. To better model complex brain neurocircuitry, compartmentalized culturing systems and 3D organoids have been developed. Early compartmentalized devices demonstrated how neuronal cell bodies can be isolated both physically and chemically from neurites. Soft lithographic approaches have advanced this approach and offer the tools to construct novel model platforms, enabling circuit-level studies of disease, which can accelerate mechanistic studies and drug candidate screening. In this review, we describe some of the common technologies used to develop such systems and discuss how these lithographic techniques have been used to advance our understanding of neuropsychiatric disease. Finally, we address other in vitro model platforms such as 3D culture systems and organoids and compare these models with compartmentalized models. We ask important questions regarding how we can further harness iPS cells in these engineered culture systems for the development of improved in vitro models. Developmental Dynamics 248:65-77, 2019. © 2018 Wiley Periodicals, Inc.
Subject(s)
Cell Culture Techniques/methods , Models, Biological , Organoids/cytology , Animals , Brain/anatomy & histology , Cell Compartmentation , Humans , Induced Pluripotent Stem Cells/cytology , Neurons , Organoids/innervation , Printing, Three-DimensionalABSTRACT
The functional state of denervated muscle is a critical factor in the ability to restore movement after injury- or disease-related paralysis. Here we used peripheral optogenetic stimulation and transcriptome profiling in the mouse whisker system to investigate the time course of changes in neuromuscular function following complete unilateral facial nerve transection. While most skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after denervation. RNA-seq analysis showed distinct regulation of multiple gene families in denervated whisker pad muscles compared with the atrophy-prone soleus, including prominent changes in ion channels and contractile fibers. Together, our results define the unique functional and transcriptomic landscape of denervated facial muscles and have general implications for restoring movement after neuromuscular injury or disease. NEW & NOTEWORTHY Optogenetic activation of muscle can be used to noninvasively induce movements and probe muscle function. We used this technique in mice to investigate changes in whisker movements following facial nerve transection. We found unexpectedly enhanced functional properties of whisker pad muscle following denervation, accompanied by unique transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for investigating the restoration of movement after paralysis.
Subject(s)
Muscle, Skeletal/metabolism , Transcriptome , Vibrissae/metabolism , Animals , Contractile Proteins/genetics , Contractile Proteins/metabolism , Female , Ion Channels/genetics , Ion Channels/metabolism , Male , Mice , Muscle Denervation , Muscle Fatigue , Muscle, Skeletal/innervation , Muscle, Skeletal/physiology , Optogenetics , Vibrissae/innervation , Vibrissae/physiologyABSTRACT
Tuberous Sclerosis Complex (TSC) is a disease caused by autosomal dominant mutations in the TSC1 or TSC2 genes, and is characterized by tumor susceptibility, brain lesions, seizures and behavioral impairments. The TSC1 and TSC2 genes encode proteins forming a complex (TSC), which is a major regulator and suppressor of mammalian target of rapamycin complex 1 (mTORC1), a signaling complex that promotes cell growth and proliferation. TSC1/2 loss of heterozygosity (LOH) and the subsequent complete loss of TSC regulatory activity in null cells causes mTORC1 dysregulation and TSC-associated brain lesions or other tissue tumors. However, it is not clear whether TSC1/2 heterozygous brain cells are abnormal and contribute to TSC neuropathology. To investigate this issue, we generated induced pluripotent stem cells (iPSCs) from TSC patients and unaffected controls, and utilized these to obtain neural progenitor cells (NPCs) and differentiated neurons in vitro. These patient-derived TSC2 heterozygous NPCs were delayed in their ability to differentiate into neurons. Patient-derived progenitor cells also exhibited a modest activation of mTORC1 signaling downstream of TSC, and a marked attenuation of upstream PI3K/AKT signaling. We further show that pharmacologic PI3K or AKT inhibition, but not mTORC1 inhibition, causes a neuronal differentiation delay, mimicking the patient phenotype. Together these data suggest that heterozygous TSC2 mutations disrupt neuronal development, potentially contributing to the disease neuropathology, and that this defect may result from dysregulated PI3K/AKT signaling in neural progenitor cells.
Subject(s)
Neural Stem Cells/metabolism , Neurogenesis , Signal Transduction , Tuberous Sclerosis/metabolism , Adolescent , Adult , Cell Line , Cells, Cultured , Female , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Insulin Receptor Substrate Proteins/metabolism , Male , Neural Stem Cells/cytology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Tuberous Sclerosis/pathologyABSTRACT
Inflammation is highly associated with colon carcinogenesis. Epigenetic mechanisms could play an important role in the initiation and progression of colon cancer. Curcumin, a dietary phytochemical, shows promising effects in suppressing colitis-associated colon cancer in azoxymethane-dextran sulfate sodium (AOM-DSS) mice. However, the potential epigenetic mechanisms of curcumin in colon cancer remain unknown. In this study, the anticancer effect of curcumin in suppressing colon cancer in an 18-week AOM-DSS colon cancer mouse model was confirmed. We identified lists of differentially expressed and differentially methylated genes in pairwise comparisons and several pathways involved in the potential anticancer effect of curcumin. These pathways include LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, production of NO and ROS in macrophages and IL-6 signaling. Among these genes, Tnf stood out with decreased DNA CpG methylation of Tnf in the AOM-DSS group and reversal of the AOM-DSS induced Tnf demethylation by curcumin. These observations in Tnf methylation correlated with increased and decreased Tnf expression in RNA-seq. The functional role of DNA methylation of Tnf was further confirmed by in vitro luciferase transcriptional activity assay. In addition, the DNA methylation level in a group of inflammatory genes was decreased in the AOM+DSS group but restored by curcumin and was validated by pyrosequencing. This study shows for the first time epigenomic changes in DNA CpG methylation in the inflammatory response from colitis-associated colon cancer and the reversal of their CpG methylation changes by curcumin. Future clinical epigenetic studies with curcumin in inflammation-associated colon cancer would be warranted.
Subject(s)
Colitis/complications , Colonic Neoplasms/etiology , Colonic Neoplasms/prevention & control , Curcumin/pharmacology , DNA Methylation/drug effects , Transcriptome/drug effects , Animals , Azoxymethane/pharmacology , Colon/drug effects , Dextran Sulfate/pharmacology , Disease Models, Animal , Epigenesis, Genetic/drug effects , Inflammation/prevention & control , Male , Mice , Mice, Inbred C57BL , Oxidative Stress/drug effectsABSTRACT
Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin ß6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin ß6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin ß6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.
Subject(s)
Acid Anhydride Hydrolases/physiology , Cell Movement/physiology , Acid Anhydride Hydrolases/genetics , Down-Regulation , Endoribonucleases , Fibronectins/metabolism , Gene Knockdown Techniques , Humans , Integrin beta Chains/metabolism , MCF-7 CellsABSTRACT
Alcohol use disorder (AUD) affects millions of people and costs nearly 250 billion dollars annually. Few effective FDA-approved treatments exist, and more are needed. AUDs have a strong heritability, but only a few genes have been identified with a large effect size on disease phenotype. Genomewide association studies (GWASs) have identified common variants with low effect sizes, most of which are in noncoding regions of the genome. Animal models frequently fail to recapitulate key molecular features of neuropsychiatric disease due to the polygenic nature of the disease, partial conservation of coding regions, and significant disparity in noncoding regions. By contrast, human induced pluripotent stem cells (hiPSCs) derived from patients provide a powerful platform for evaluating genes identified by GWAS and modeling complex interactions in the human genome. hiPSCs can be differentiated into a wide variety of human cells, including neurons, glia, and hepatic cells, which are compatible with numerous functional assays and genome editing techniques. In this review, we focus on current applications and future directions of patient hiPSC-derived central nervous system cells for modeling AUDs in addition to highlighting successful applications of hiPSCs in polygenic neuropsychiatric diseases.
Subject(s)
Alcoholism/diagnosis , Alcoholism/genetics , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study/trends , Induced Pluripotent Stem Cells/physiology , Alcoholism/therapy , Animals , Cells, Cultured , Genome-Wide Association Study/methods , HumansABSTRACT
BACKGROUND: DNA methylation is an epigenetic modification that is studied at a single-base resolution with bisulfite treatment followed by high-throughput sequencing. After alignment of the sequence reads to a reference genome, methylation counts are analyzed to determine genomic regions that are differentially methylated between two or more biological conditions. Even though a variety of software packages is available for different aspects of the bioinformatics analysis, they often produce results that are biased or require excessive computational requirements. RESULTS: DMRfinder is a novel computational pipeline that identifies differentially methylated regions efficiently. Following alignment, DMRfinder extracts methylation counts and performs a modified single-linkage clustering of methylation sites into genomic regions. It then compares methylation levels using beta-binomial hierarchical modeling and Wald tests. Among its innovative attributes are the analyses of novel methylation sites and methylation linkage, as well as the simultaneous statistical analysis of multiple sample groups. To demonstrate its efficiency, DMRfinder is benchmarked against other computational approaches using a large published dataset. Contrasting two replicates of the same sample yielded minimal genomic regions with DMRfinder, whereas two alternative software packages reported a substantial number of false positives. Further analyses of biological samples revealed fundamental differences between DMRfinder and another software package, despite the fact that they utilize the same underlying statistical basis. For each step, DMRfinder completed the analysis in a fraction of the time required by other software. CONCLUSIONS: Among the computational approaches for identifying differentially methylated regions from high-throughput bisulfite sequencing datasets, DMRfinder is the first that integrates all the post-alignment steps in a single package. Compared to other software, DMRfinder is extremely efficient and unbiased in this process. DMRfinder is free and open-source software, available on GitHub ( github.com/jsh58/DMRfinder ); it is written in Python and R, and is supported on Linux.
Subject(s)
DNA Methylation , Software , Cell Line , Cluster Analysis , CpG Islands , High-Throughput Nucleotide Sequencing/methods , Humans , Sequence Analysis, DNA/methods , User-Computer InterfaceABSTRACT
Topoisomerase II beta (Top2b) is an enzyme that alters the topologic states of DNA during transcription. Top2b deletion in early retinal progenitor cells causes severe defects in neural differentiation and affects cell survival in all retinal cell types. However, it is unclear whether the observed severe phenotypes are the result of cell-autonomous/primary defects or non-cell-autonomous/secondary defects caused by alterations of other retinal cells. Using photoreceptor cells as a model, we first characterized the phenotypes in Top2b conditional knockout. Top2b deletion leads to malformation of photoreceptor outer segments (OSs) and synapses accompanied by dramatic cell loss at late-stage photoreceptor differentiation. Then, we performed mosaic analysis with shRNA-mediated Top2b knockdown in neonatal retina using in vivo electroportation to target rod photoreceptors in neonatal retina. Top2b knockdown causes defective OS without causing a dramatic cell loss, suggesting a Top2b cell-autonomous function. Furthermore, RNA-seq analysis reveals that Top2b controls the expression of key genes in the photoreceptor gene-regulatory network (e.g., Crx, Nr2e3, Opn1sw, Vsx2) and retinopathy-related genes (e.g., Abca4, Bbs7, Pde6b). Together, our data establish a combinatorial cell-autonomous and non-cell-autonomous role for Top2b in the late stage of photoreceptor differentiation and maturation. © 2017 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.
Subject(s)
DNA Topoisomerases, Type II/metabolism , Gene Expression Regulation, Developmental/genetics , Gene Regulatory Networks/genetics , Photoreceptor Cells/cytology , Poly-ADP-Ribose Binding Proteins/metabolism , Retina/embryology , Animals , Cell Differentiation/genetics , Female , Male , Mice , Mice, Knockout , Retina/growth & development , Synapses/genetics , Synapses/metabolism , Transcription, GeneticABSTRACT
Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.