ABSTRACT
Recent studies on the regulatory networks implicated in Alzheimer's disease (AD) evince long non-coding RNAs (lncRNAs) as crucial regulatory players, albeit a poor understanding of the mechanism. Analyzing differential gene expression in the RNA-seq data from the post-mortem AD brain hippocampus, we categorized a list of AD-dysregulated lncRNA transcripts into functionally similar communities based on their k-mer profiles. Using machine-learning-based algorithms, their subcellular localizations were mapped. We further explored the functional relevance of each community through AD-dysregulated miRNA, RNA-binding protein (RBP) interactors, and pathway enrichment analyses. Further investigation of the miRNA-lncRNA and RBP-lncRNA networks from each community revealed the top RBPs, miRNAs, and lncRNAs for each cluster. The experimental validation community yielded ELAVL4 and miR-16-5p as the predominant RBP and miRNA, respectively. Five lncRNAs emerged as the top-ranking candidates from the RBP/miRNA-lncRNA networks. Further analyses of these networks revealed the presence of multiple regulatory triads where the RBP-lncRNA interactions could be augmented by the enhanced miRNA-lncRNA interactions. Our results advance the understanding of the mechanism of lncRNA-mediated AD regulation through their interacting partners and demonstrate how these functionally segregated but overlapping regulatory networks can modulate the disease holistically.
Subject(s)
Alzheimer Disease , Gene Regulatory Networks , MicroRNAs , RNA, Long Noncoding , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , ELAV-Like Protein 4/metabolism , ELAV-Like Protein 4/genetics , Hippocampus/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/geneticsABSTRACT
The RNA-binding protein HuD/ELAVL4 is essential for neuronal development and synaptic plasticity by governing various post-transcriptional processes of target mRNAs, including stability, translation, and localization. We previously showed that the linker region and poly(A)-binding domain of HuD play a pivotal role in promoting translation and inducing neurite outgrowth. In addition, we found that HuD interacts exclusively with the active form of Akt1, through the linker region. Although this interaction is essential for neurite outgrowth, HuD is not a substrate for Akt1, raising questions about the dynamics between HuD-mediated translational stimulation and its association with active Akt1. Here, we demonstrate that active Akt1 interacts with the cap-binding complex via HuD. We identify key amino acids in linker region of HuD responsible for Akt1 interaction, leading to the generation of two point-mutated HuD variants: one that is incapable of binding to Akt1 and another that can interact with Akt1 regardless of its phosphorylation status. In vitro translation assays using these mutants reveal that HuD-mediated translation stimulation is independent of its binding to Akt1. In addition, it is evident that the interaction between HuD and active Akt1 is essential for HuD-induced neurite outgrowth, whereas a HuD mutant capable of binding to any form of Akt1 leads to aberrant neurite development. Collectively, our results revisit the understanding of the HuD-Akt1 interaction in translation and suggest that this interaction contributes to HuD-mediated neurite outgrowth via a unique molecular mechanism distinct from translation regulation.
Subject(s)
ELAV-Like Protein 4 , Neurons , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-akt/metabolism , ELAV-Like Protein 4/metabolism , ELAV-Like Protein 4/genetics , Humans , Animals , Neurons/metabolism , Neurons/cytology , Cell Differentiation , HEK293 Cells , Protein Binding , Phosphorylation , Mice , Neurogenesis , Rats , Neurites/metabolismABSTRACT
The RNA-binding protein HuD has been shown to play a crucial role in gene regulation in the nervous system and is involved in various neurological and psychiatric diseases. In this study, through the creation of an interaction network on HuD and its potential targets, we identified a strong association between HuD and several diseases of the nervous system. Specifically, we focused on the relationship between HuD and the brain-derived neurotrophic factor (BDNF), whose protein is implicated in several neuronal diseases and is involved in the regulation of neuronal development, survival, and function. To better investigate this relationship and given that we previously demonstrated that folic acid (FA) is able to directly bind HuD itself, we performed in vitro experiments in neuron-like human SH-SY5Y cells in the presence of FA, also known to be a pivotal environmental factor influencing the nervous system development. Our findings show that FA exposure results in a significant increase in both HuD and BDNF transcripts and proteins after 2 and 4 h of treatment, respectively. Similar data were obtained after 2 h of FA incubation followed by 2 h of washout. This increase was no longer detected upon 24 h of FA exposure, probably due to a signaling shutdown mechanism. Indeed, we observed that following 24 h of FA exposure HuD is methylated. These findings indicate that FA regulates BDNF expression via HuD and suggest that FA can behave as an epigenetic modulator of HuD in the nervous system acting via short- and long-term mechanisms. Finally, the present results also highlight the potential of BDNF as a therapeutic target for specific neurological and psychiatric diseases.
Subject(s)
Brain-Derived Neurotrophic Factor , Neuroblastoma , Humans , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , ELAV Proteins/genetics , ELAV Proteins/metabolism , ELAV-Like Protein 4/genetics , Neuroblastoma/metabolism , Neurons/metabolismABSTRACT
The main goal of this review is to provide an updated overview of the involvement of the RNA-binding protein (RBP) HuD, encoded by the ELAVL4 gene, in nervous system development, maintenance, and function, and its emerging role in nervous system diseases. A particular focus is on recent studies reporting altered HuD levels, or activity, in disease models and patients. Substantial evidence suggests HuD involvement in Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Interestingly, while possible disease-causing mutations in the ELAVL4 gene remain elusive, a common theme in these diseases seems to be the altered regulation of HuD at multiple steps, including post-transcriptional and post-translational levels. In turn, the changed activity of HuD can have profound implications for its target transcripts, which are overly stabilized in case of HuD gain of function (as proposed in PD and ALS) or reduced in case of decreased HuD binding (as suggested by some studies in AD). Moreover, the recent discovery that HuD is a component of pathological cytoplasmic inclusion in both familial and sporadic ALS patients might help uncover the common molecular mechanisms underlying such complex diseases. We believe that deepening our understanding of the involvement of HuD in neurodegeneration could help developing new diagnostic and therapeutic tools.
Subject(s)
Alzheimer Disease , Amyotrophic Lateral Sclerosis , ELAV-Like Protein 4 , Parkinson Disease , Humans , Alzheimer Disease/genetics , Amyotrophic Lateral Sclerosis/genetics , ELAV-Like Protein 4/genetics , RNA-Binding Proteins/genetics , Parkinson Disease/geneticsABSTRACT
RNA binding protein HuD plays essential roles in gene expression by regulating RNA metabolism, and its dysregulation is involved in the pathogenesis of several diseases, including tumors, neurodegenerative diseases, and diabetes. Here, we explored HuD-mediated differential expression of secretory proteins in mouse insulinoma ßTC6 cells using a cytokine array. Endostatin and Serpin E1 that play anti-angiogenic roles were identified as differentially expressed proteins by HuD. HuD knockdown increased the expression of α chain of collagen XVIII (Col18a1), a precursor form of endostatin, and Serpin E1 by associating with the 3'-untranslated regions (UTRs) of Col18a1 and Serpin E1 mRNAs. Reporter analysis revealed that HuD knockdown increased the translation of EGFP reporters containing 3'UTRs of Col18a1 and Serpin E1 mRNAs, which suggests the role of HuD as a translational repressor. Co-cultures of ßTC6 cells and pancreatic islet endothelial MS1 cells were used to assess the crosstalk between ß cells and islet endothelial cells, and the results showed that HuD downregulation in ßTC6 cells inhibited the growth and migration of MS1 cells. Ectopic expression of HuD decreased Col18a1 and Serpin E1 expression, while increasing the markers of islet vascular cells in the pancreas of db/db mice. Taken together, these results suggest that HuD has the potential to regulate the crosstalk between ß cells and islet endothelial cells by regulating Endostatin and Serpin E1 expression, thereby contributing to the maintenance of homeostasis in the islet microenvironment.
Subject(s)
ELAV-Like Protein 4 , Endostatins , Insulin-Secreting Cells , Plasminogen Activator Inhibitor 1 , Animals , Mice , 3' Untranslated Regions/genetics , Endostatins/genetics , Endostatins/metabolism , Endothelial Cells/metabolism , Insulin-Secreting Cells/metabolism , Plasminogen Activator Inhibitor 1/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , ELAV-Like Protein 4/genetics , ELAV-Like Protein 4/metabolismABSTRACT
BACKGROUND: Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied. METHODS: RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells. RESULTS: In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts. CONCLUSIONS: HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , ELAV-Like Protein 4/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Membrane Proteins/metabolism , RNA-Binding Proteins/genetics , Animals , Autophagy , Humans , Male , Mice , Mice, Nude , TransfectionABSTRACT
The dysregulation of glycolysis regardless of oxygen availability is one of the major characteristics of cancer cells. While the drug resistance of ovarian cancer cells has been extensively studied, the molecular mechanism of anticancer drug resistance under low-glucose conditions remains unknown. In this study, we investigated the pathway mediating drug resistance under low-glucose conditions by examining the relationship between embryonic lethal abnormal vision Drosophila homolog-like (ELAVL) protein and glycolysis-related enzymes. Ovarian cancer cells resistant to 2.5 nM paclitaxel were exposed to low-glucose media for 2 weeks, and the expression levels of ELAVL2, ELAVL4, glycolytic enzymes, and drug resistance-related proteins were elevated to levels comparable to those in cells resistant to 100 nM paclitaxel. Gene silencing of ELAVL2/4 using small interfering RNA prevented the upregulation of glycolysis-related enzymes, reduced lactate production, and sensitized 2.5 nM paclitaxel-resistant ovarian cancer cells to anticancer agents under hypoglycemic conditions. Furthermore, pharmacological inhibition of glycolytic enzymes with 2-deoxyglucose, a specific inhibitor of glycolysis, triggered caspase-dependent apoptosis, reduced lactate generation, and blocked the expression of drug resistance-related proteins under low-glucose conditions. These results suggest that the level of ELAVL2/4 is responsible for the development of chemoresistance through activation of the glycolysis pathway under glucose deprivation conditions.
Subject(s)
Drug Resistance, Neoplasm/genetics , ELAV-Like Protein 2/genetics , ELAV-Like Protein 4/genetics , Glycolysis/genetics , Ovarian Neoplasms/genetics , Antineoplastic Agents/pharmacology , Caspases/metabolism , Cell Line, Tumor , Female , Glucose/metabolism , Humans , Lactic Acid/metabolism , Ovarian Neoplasms/pathology , Paclitaxel/pharmacologyABSTRACT
Mutations in the RNA-binding protein (RBP) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3'UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.
Subject(s)
Axons/metabolism , ELAV-Like Protein 4/genetics , Fragile X Mental Retardation Protein/genetics , RNA-Binding Protein FUS/genetics , Animals , Cell Line , ELAV-Like Protein 4/metabolism , Fragile X Mental Retardation Protein/metabolism , Humans , Mice , Motor Neurons/metabolism , Mutation , RNA-Binding Protein FUS/metabolismABSTRACT
Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.
Subject(s)
Argonaute Proteins/genetics , Eukaryotic Initiation Factor-4F/genetics , Peptide Chain Initiation, Translational , Poly(A)-Binding Proteins/genetics , RNA Caps/genetics , Transcription Factors/genetics , Animals , Argonaute Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , ELAV-Like Protein 4/genetics , ELAV-Like Protein 4/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4F/metabolism , Humans , Mammals , MicroRNAs/genetics , MicroRNAs/metabolism , Poly A/genetics , Poly A/metabolism , Poly(A)-Binding Proteins/metabolism , Protein Binding , RNA Caps/chemistry , RNA Caps/metabolism , RNA Stability , RNA-Induced Silencing Complex/genetics , RNA-Induced Silencing Complex/metabolism , Transcription Factors/metabolismABSTRACT
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.
Subject(s)
Cerebrum/pathology , ELAV-Like Protein 4/genetics , Glutamic Acid/metabolism , Mutation/genetics , Neurons/pathology , Organoids/metabolism , RNA Splicing/genetics , tau Proteins/genetics , Autophagy/drug effects , Autophagy/genetics , Biomarkers/metabolism , Body Patterning/drug effects , Body Patterning/genetics , Cell Death/drug effects , Cell Line , Humans , Hydrazones/pharmacology , Lysosomes/drug effects , Lysosomes/metabolism , Morpholines/pharmacology , Neurons/drug effects , Neurons/metabolism , Organoids/drug effects , Organoids/ultrastructure , Phosphorylation/drug effects , Pyrimidines/pharmacology , RNA Splicing/drug effects , Signal Transduction/drug effects , Stress Granules/drug effects , Stress Granules/metabolism , Synapses/metabolism , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3'-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4-/- (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3'-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.
Subject(s)
Alternative Splicing/genetics , ELAV-Like Protein 4/genetics , Neocortex/metabolism , Polyadenylation/genetics , Animals , ELAV-Like Protein 4/metabolism , Exons/genetics , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.
Subject(s)
3' Untranslated Regions , Axons/enzymology , Cell Body/enzymology , Phosphoric Monoester Hydrolases/metabolism , RNA, Messenger/metabolism , Superior Cervical Ganglion/enzymology , Transcription, Genetic , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , ELAV-Like Protein 4/genetics , ELAV-Like Protein 4/metabolism , Female , Gene Expression Regulation, Enzymologic , Male , PC12 Cells , Phosphoric Monoester Hydrolases/genetics , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Polyadenylation , Protein Biosynthesis , Protein Isoforms , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Superior Cervical Ganglion/cytology , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.
Subject(s)
Insulin-Like Growth Factor Binding Proteins/genetics , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor I/genetics , Organoids/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Pluripotent Stem Cells/metabolism , Catechols/pharmacology , Cell Differentiation/drug effects , ELAV-Like Protein 3/genetics , ELAV-Like Protein 3/metabolism , ELAV-Like Protein 4/genetics , ELAV-Like Protein 4/metabolism , Gene Expression Regulation , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Insulin-Like Growth Factor Binding Proteins/antagonists & inhibitors , Insulin-Like Growth Factor Binding Proteins/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor II/metabolism , Isoquinolines/pharmacology , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Organoids/cytology , Organoids/drug effects , Photoreceptor Cells, Vertebrate/cytology , Photoreceptor Cells, Vertebrate/drug effects , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Recoverin/genetics , Recoverin/metabolism , Signal Transduction , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , gamma-Synuclein/genetics , gamma-Synuclein/metabolismABSTRACT
The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3' untranslated region (3'UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , ELAV-Like Protein 4/genetics , Gene Expression Regulation/genetics , Motor Cortex/metabolism , Neuroblastoma/metabolism , Neurons/metabolism , Oxidative Stress/genetics , Superoxide Dismutase-1/genetics , Amyotrophic Lateral Sclerosis/metabolism , Cell Line, Tumor , ELAV-Like Protein 4/metabolism , Humans , RNA, Messenger/metabolism , Superoxide Dismutase-1/metabolismABSTRACT
Glucagon is a peptide hormone generated by pancreatic α cells. It is the counterpart of insulin and plays an essential role in the regulation of blood glucose level. Therefore, a tight regulation of glucagon levels is pivotal to maintain homeostasis of blood glucose. However, little is known about the mechanisms regulating glucagon biosynthesis. In this study, we demonstrate that the RNA-binding protein HuD regulates glucagon expression in pancreatic α cells. HuD was found in α cells from mouse pancreatic islet and mouse glucagonoma αTC1 cell line. Ribonucleoprotein immunoprecipitation analysis, followed by RT-qPCR showed the association of HuD with glucagon mRNA. Knockdown of HuD resulted in a reduction in both proglucagon expression and cellular glucagon level by decreasing its de novo synthesis. Reporter analysis using the EGFP reporter containing 3' untranslated region (3'UTR) of glucagon mRNA showed that HuD regulates proglucagon expression via its 3'UTR. In addition, the relative level of glucagon in the islets and plasma was lower in HuD knockout (KO) mice compared to age-matched control mice. Taken together, these results suggest that HuD is a novel factor regulating the biosynthesis of proglucagon in pancreatic α cells.
Subject(s)
ELAV-Like Protein 4/metabolism , Glucagon-Secreting Cells/metabolism , Proglucagon/metabolism , Animals , Biosynthetic Pathways , Cell Line , Cell Line, Tumor , Down-Regulation , ELAV-Like Protein 4/genetics , Gene Knockdown Techniques , Glucagon-Secreting Cells/cytology , Mice , Proglucagon/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.
Subject(s)
CELF1 Protein/metabolism , ELAV-Like Protein 4/genetics , Gene Expression Regulation, Developmental , Neocortex/growth & development , Neurogenesis/genetics , 5' Untranslated Regions/genetics , Alternative Splicing , Animals , Cell Line, Tumor , Female , Glutamic Acid/metabolism , Male , Mice , Mice, Transgenic , Neocortex/cytology , Neural Stem Cells/metabolism , Neuroglia/metabolism , Neurons/metabolism , Polyribosomes/metabolism , Primary Cell Culture , Protein Biosynthesis/genetics , RNA Isoforms/genetics , RNA-SeqABSTRACT
Amyotrophic lateral sclerosis (ALS) has been genetically linked to mutations in RNA-binding proteins (RBPs), including FUS. Here, we report the RNA interactome of wild-type and mutant FUS in human motor neurons (MNs). This analysis identified a number of RNA targets. Whereas the wild-type protein preferentially binds introns, the ALS mutation causes a shift toward 3' UTRs. Neural ELAV-like RBPs are among mutant FUS targets. As a result, ELAVL4 protein levels are increased in mutant MNs. ELAVL4 and mutant FUS interact and co-localize in cytoplasmic speckles with altered biomechanical properties. Upon oxidative stress, ELAVL4 and mutant FUS are engaged in stress granules. In the spinal cord of FUS ALS patients, ELAVL4 represents a neural-specific component of FUS-positive cytoplasmic aggregates, whereas in sporadic patients it co-localizes with phosphorylated TDP-43-positive inclusions. We propose that pathological mutations in FUS trigger an aberrant crosstalk with ELAVL4 with implications for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Cytoplasm/metabolism , ELAV-Like Protein 4/metabolism , Mutation , RNA-Binding Protein FUS/metabolism , 3' Untranslated Regions , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Cytoplasm/genetics , Cytoplasm/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , ELAV-Like Protein 4/genetics , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Oxidative Stress/genetics , RNA-Binding Protein FUS/geneticsABSTRACT
Certain patients with anorectal malforma-tions (ARMs) continue to suffer from postoperative dysphoria. The enteric nervous system (ENS) is closely associated with defecation. The purinergic receptor P2Y2 (P2Y2) and Hu antigen D (HuD) proteins contain multiple motifs that enable their activation and direct coupling to integrin and growth factor receptor signaling pathways; thus, they may serve as key points in ENS development. The aim of the present study was to investigate the expression pattern of P2Y2 and HuD proteins during anorectal development in ARM embryos. The embryogenesis of ARM in rats was induced by ethylenethiourea (ETU) on the 10th gestational day. The expression patterns of P2Y2 and HuD proteins were evaluated by immunohistochemistry and western blot analysis in normal, ETU and ARM rat embryos on embryonic days E17, E19 and E21; their mRNA levels were assessed via reverse transcriptionquantitative polymerase chain reaction (RTqPCR) of the distal rectum of fetal rats. Immunohistochemistry of the distal rectum demonstrated that on E17, the expression levels of the two proteins were not different between the three groups. On E19, the expression of HuD was significantly decreased in the ARM group. On E21, the two proteins were significantly decreased in the ARM group. Additionally, the expression levels of the two proteins on E17 were significantly lower than on E21 in the ARM group. Western blotting and RTqPCR also revealed that the P2Y2 and HuD proteins and mRNA expression levels were significantly decreased in the ARM groups when compared with the normal group on E17 and E21 (P<0.01). Thus, the present study demonstrated that downregulation of P2Y2 and HuD may partly be related to the development of the ENS in ARM embryos.
Subject(s)
Anorectal Malformations/embryology , Anorectal Malformations/genetics , Down-Regulation/genetics , ELAV-Like Protein 4/genetics , Enteric Nervous System/embryology , Fetus/metabolism , Gene Expression Regulation, Developmental , Receptors, Purinergic P2Y2/genetics , Animals , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Receptors, Purinergic P2Y2/metabolismABSTRACT
The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.
Subject(s)
ELAV-Like Protein 2/genetics , ELAV-Like Protein 3/genetics , ELAV-Like Protein 4/genetics , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Gene Expression Regulation , Sensory Receptor Cells/metabolism , Animals , Biomarkers , Blood Glucose , Body Weight , Diabetes Mellitus, Experimental , ELAV-Like Protein 2/metabolism , ELAV-Like Protein 3/metabolism , ELAV-Like Protein 4/metabolism , Ganglia, Spinal/physiopathology , Immunohistochemistry , Mice , RNA-Binding ProteinsABSTRACT
Detecting the association between a set of variants and a phenotype of interest is the first and important step in genetic and genomic studies. Although it attracted a large amount of attention in the scientific community and several related statistical approaches have been proposed in the literature, powerful and robust statistical tests are still highly desired and yet to be developed in this area. In this paper, we propose a powerful and robust association test, which combines information from each individual single-nucleotide polymorphisms based on sequential independent burden tests. We compare the proposed approach with some popular tests through a comprehensive simulation study and real data application. Our results show that, in general, the new test is more powerful; the gain in detecting power can be substantial in many situations, compared to other methods.