RESUMO
The causes of Alzheimer's disease (AD) are poorly understood, although many genes are known to be involved in this pathology. To gain insights into the underlying molecular mechanisms, it is essential to identify the relationships between individual AD genes. Previous work has shown that the splice variant E of KLC1 (KLC1_vE) promotes AD, and that the CELF1 gene, which encodes an RNA-binding protein involved in splicing regulation, is at a risk locus for AD. Here, we identified a functional link between CELF1 and KLC1 in AD pathogenesis. Transcriptomic data from human samples from different ethnic groups revealed that CELF1 mRNA levels are low in AD brains, and the splicing pattern of KLC1 is strongly correlated with CELF1 expression levels. Specifically, KLC1_vE is negatively correlated with CELF1. Depletion and overexpression experiments in cultured cells demonstrated that the CELF1 protein down-regulates KLC1_vE. In a cross-linking and immunoprecipitation sequencing (CLIP-seq) database, CELF1 directly binds to KLC1 RNA, following which it likely modulates terminal exon usage, hence KLC1_vE formation. These findings reveal a new pathogenic pathway where a risk allele of CELF1 is associated with reduced CELF1 expression, which up-regulates KLC1_vE to promote AD.
Assuntos
Processamento Alternativo , Doença de Alzheimer , Proteínas CELF1 , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteínas CELF1/metabolismo , Proteínas CELF1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1cKO mouse lenses at postnatal day (P) 0. Celf1cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts.
Assuntos
Proteínas CELF1 , Catarata , Cristalino , Animais , Humanos , Camundongos , Catarata/metabolismo , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Cristalino/metabolismo , Camundongos Knockout , RNA/metabolismo , Transcriptoma/genéticaRESUMO
Previous studies have shown that miR-322 regulates the functions of various stem cells. However, the role and mechanism of embryonic stem cell (ESCs) differentiation into cardiomyocytes remains unknown. Celf1 plays a vital role in stem cell differentiation and may be a potential target of miR-322 in ESCs' differentiation. We studied the function of miR-322An using mESCs transfected with lentivirus-mediated miR-322. RT-PCR results indicated that miR-322 increased NKX-2.5, MLC2V, and α-MHC mRNA expression, signifying that miR-322 might promote the differentiation of ESCs toward cardiomyocytes in vitro. The western blotting and immunofluorescence results confirmed this conclusion. In addition, the knockdown of miR-322 expression inhibited ESCs' differentiation toward cardiomyocytes in cultured ESCs in vitro. Western blotting results showed that miR-322 suppressed celf1 protein expression. Furthermore, Western blotting, RT-PCR, and immunofluorescence results showed that celf1 may inhibit ESCs' differentiation toward cardiomyocytes in vitro. Overall, the results indicate that miR-322 might promote ESCs' differentiation toward cardiomyocytes by regulating celf1 expression.
Assuntos
Proteínas CELF1 , MicroRNAs , Células-Tronco Embrionárias Murinas , Miócitos Cardíacos , Diferenciação Celular/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Camundongos , Proteínas CELF1/genéticaRESUMO
Cataract is a global eye disease caused by the opacification of lens, while its underlying molecular pathogenesis is not clear, making it difficult for prevention. CELF1, an RNA binding protein, mediates Alternative Splicing (AS) of genes involved in diverse diseases and regulates development or defects of lens. Utilizing transcriptome-wide approaches, we analyzed and compared AS patterns between human lens epithelial cells (SRA01/04) with CELF1 overexpression (CELF1-OE) and control cells. Extensive changes in AS patterns upon CELF1-OE were identified in SRA01/04 cells. We finally identified 840 CELF1-regulated AS events (RASEs) and found that CELF1-OE preferred to repress exon skipping events in SRA01/04 cells. CELF1-regulated AS genes were enriched in the regulation of DNA repair, cellular response to DNA damage stimulus, and apoptosis pathways (including HMGA2, CSNK1E, and YAP1). These biological functions and pathways have been reported to be associated with lens development or other eye diseases. To further explore the mechanisms of CELF1 in regulating AS genes, we downloaded and re-analyzed a set of CELF1-RNA interactome data. We found that 194 genes were bound and regulated by CELF1 at the AS level. 10 genes involved in DNA repair-related pathways were also bound by CELF1. Motif analysis for CELF1-bound peaks and splicing sites of RASEs showed that CELF1 regulates AS by binding to the AGGU[AG]AG motif in SRA01/04 cells. CELF1 could mediate AS of DNA repair-related genes through directly binding to their transcripts with distinct motif bias. The functional mechanism of CELF1 may ultimately participate in cataract formation and lens development.
Assuntos
Processamento Alternativo , Catarata , Humanos , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Linhagem Celular , Catarata/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
In sex determination of the crustacean Daphnia magna, male-specific expression of DM-domain transcription factor Doublesex1 (Dsx1) orchestrates the male developmental program triggered by environmental stimuli. We previously identified the CELF1 ortholog as a candidate of proteins associated with the 5' UTR of the Dsx1α isoform. Here we report the CELF1-dependent suppression of Dsx1 expression in D. magna. During embryogenesis, CELF1 expression was not sexually dimorphic. Silencing of CELF1 led to the activation of Dsx1 expression both in female and male embryos. Overexpression of CELF1 in male embryos resulted in a reduction of Dsx1 expression. By these manipulations of CELF1 expression, the Dsx1 transcript level was not significantly changed. To investigate whether the CELF1 controls Dsx1 expression via its 5' UTR, we injected the GFP reporter mRNA having intact Dsx1α 5' UTR or mutated one lacking the GU-rich element (GRE) that is known as a binding site of the CELF1 ortholog. We found that deletion of the GRE significantly increased the reporter gene expression. These results indicate that CELF1 suppresses Dsx1 expression both in females and males, possibly at the post-transcriptional level. We speculate that CELF1 may avoid unintended Dsx1 expression and generation of sexual ambiguity by setting a threshold of Dsx1 expression.
Assuntos
Daphnia , Regulação da Expressão Gênica , Regiões 5' não Traduzidas/genética , Animais , Proteínas CELF1/genética , Daphnia/fisiologia , Feminino , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Intestinal epithelial integrity is commonly disrupted in patients with critical disorders, but the exact underlying mechanisms are unclear. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control different cell functions and are involved in pathologies. Here, we investigated the role of T-UCRs in intestinal epithelial homeostasis and identified T-UCR uc.230 as a major regulator of epithelial renewal, apoptosis, and barrier function. Compared with controls, intestinal mucosal tissues from patients with ulcerative colitis and from mice with colitis or fasted for 48 hours had increased levels of uc.230. Silencing uc.230 inhibited the growth of intestinal epithelial cells (IECs) and organoids and caused epithelial barrier dysfunction. Silencing uc.230 also increased IEC vulnerability to apoptosis, whereas increasing uc.230 levels protected IECs against cell death. In mice with colitis, reduced uc.230 levels enhanced mucosal inflammatory injury and delayed recovery. Mechanistic studies revealed that uc.230 increased CUG-binding protein 1 (CUGBP1) by acting as a natural decoy RNA for miR-503, which interacts with Cugbp1 mRNA and represses its translation. These findings indicate that uc.230 sustains intestinal mucosal homeostasis by promoting epithelial renewal and barrier function and that it protects IECs against apoptosis by serving as a natural sponge for miR-503, thereby preserving CUGBP1 expression.
Assuntos
Proteínas CELF1 , Colite , Homeostase , Mucosa Intestinal , RNA Longo não Codificante , Cicatrização , Animais , Apoptose , Proteínas CELF1/genética , Proteínas CELF1/imunologia , Colite/genética , Colite/imunologia , Homeostase/genética , Homeostase/imunologia , Mucosa Intestinal/imunologia , Camundongos , MicroRNAs/genética , MicroRNAs/imunologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Cicatrização/genética , Cicatrização/imunologia , Ferimentos e Lesões/genética , Ferimentos e Lesões/imunologiaRESUMO
BACKGROUND: RNA binding proteins (RBPs)-mediated regulation plays important roles in many eye diseases, including the canonical RBP CELF1 in cataract. While the definite molecular regulatory mechanisms of CELF1 on cataract still remain elusive. METHODS: In this study, we overexpressed CELF1 in human cultured lens epithelial SRA01/04 cells and applied whole transcriptome sequencing (RNA-seq) method to analyze the global differences mediated by CELF1. We then analyzed public RNA-seq and CELF1-RNA interactome data to decipher the underlying mechanisms. RESULTS: The results showed that transcriptome profile was globally changed by CELF1 overexpression (CELF1-OE). Functional analysis revealed CELF1 specifically increased the expression of genes in extracellular matrix disassembly, extracellular matrix organization, and proteolysis, which could be classified into matrix metalloproteinases (MMPs) family. This finding was also validated by RT-qPCR and public mouse early embryonic lens data. Integrating analysis with public CELF1-RNA interactome data revealed that no obvious CELF1-binding peak was found on the transcripts of these genes, indicating an indirectly regulatory role of CELF1 in lens epithelial cells. CONCLUSIONS: Our study demonstrated that CELF1-OE promotes transcriptional level of MMP genes; and this regulation may be completed by other ways except for binding to RNA targets. These results suggest that CELF1-OE is implicated in the development of lens, which is associated with cataract and expands our understanding of CELF1 regulatory roles as an RNA binding protein.
Assuntos
Células Epiteliais , Proteínas de Ligação a RNA , Animais , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Células Epiteliais/metabolismo , Metaloproteinases da Matriz/genética , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , TranscriptomaRESUMO
CUG-binding protein, ELAV-like Family Member 1 (CELF1) plays an important role during the development of different tissues, such as striated muscle and brain tissue. CELF1 is an RNA-binding protein that regulates RNA metabolism processes, e.g., alternative splicing, and antagonizes other RNA-binding proteins, such as Muscleblind-like proteins (MBNLs). Abnormal activity of both classes of proteins plays a crucial role in the pathogenesis of myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults. In this work, we show that alternative splicing of exons forming both the 5' and 3' untranslated regions (UTRs) of CELF1 mRNA is efficiently regulated during development and tissue differentiation and is disrupted in skeletal muscles in the context of DM1. Alternative splicing of the CELF1 5'UTR leads to translation of two potential protein isoforms that differ in the lengths of their N-terminal domains. We also show that the MBNL and CELF proteins regulate the distribution of mRNA splicing isoforms with different 5'UTRs and 3'UTRs and affect the CELF1 expression by changing its sensitivity to specific microRNAs or RNA-binding proteins. Together, our findings show the existence of different mechanisms of regulation of CELF1 expression through the distribution of various 5' and 3' UTR isoforms within CELF1 mRNA.
Assuntos
Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Processamento Alternativo , Proteínas CELF1/biossíntese , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , RNA Mensageiro/metabolismo , Adulto , Idoso , Animais , Sítios de Ligação , Proteínas CELF1/genética , Células COS , Chlorocebus aethiops , Éxons , Feminino , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Distrofias Musculares/genética , Biossíntese de Proteínas , Isoformas de Proteínas , RNA Mensageiro/genética , Adulto JovemRESUMO
Cisplatin (CDDP) based chemotherapy is widely used as the first-line strategy in treating non-small cell lung cancer (NSCLC), especially lung squamous cell carcinoma (LUSC). However, secondary cisplatin resistance majorly undermines the cisplatin efficacy leading to a worse prognosis. In this respect, we have identified the role of the DLX6-AS1/miR-181a-5p/miR-382-5p/CELF1 axis in regulating cisplatin resistance of LUSC. qRT-PCR and Western blot analysis were applied to detect gene expression. Transwell assay was used to evaluate the migration and invasion ability of LUSC cells. CCK-8 assay was used to investigate the IC50 of LUSC cells. Flow cytometry was used to test cell apoptosis rate. RNA pull-down and Dual luciferase reporter gene assay were performed to evaluate the crosstalk. DLX6-AS1 was aberrantly high expressed in LUSC tissues and cell lines, and negatively correlated with miR-181a-5p and miR-382-5p expression. DLX6-AS1 expression was enhanced by H3K4me1 in cisplatin resistant LUSC cells. Besides, DLX6-AS1 knockdown led to impaired IC50 of cisplatin resistant LUSC cells. Furthermore, DLX6-AS1 interacted with miR-181a-5p and miR-382-5p to regulate CELF1 expression and thereby mediated the cisplatin sensitivity of cisplatin resistant LUSC cells. DLX6-AS1 induced by H3K4me1 played an important role in promoting secondary cisplatin resistance of LUSC through regulating the miR-181a-5p/miR-382-5p/CELF1 axis. Therefore, targeting DLX6-AS1 might be a novel way of reversing secondary cisplatin resistance in LUSC.
Assuntos
Proteínas CELF1/genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Interferência de RNARESUMO
Chronic Kidney Disease (CKD) is a serious threat to human health. In addition, kidney fibrosis is a key pathogenic intermediate for the progression of CDK. Moreover, excessive activation of fibroblasts is key to the development of kidney fibrosis and this process is difficult to control. Notably, fraxinellone is a natural compound isolated from Dictamnus dasycarpus and has a variety of pharmacological activities, including hepatoprotective, anti-inflammatory and anti-cancer effects. However, the effect of fraxinellone on kidney fibrosis is largely unknown. The present study showed that fraxinellone could alleviate folic acid-induced kidney fibrosis in mice in a dose dependent manner. Additionally, the results revealed that fraxinellone could effectively down-regulate the expression of CUGBP1, which was highly up-regulated in human and murine fibrotic renal tissues. Furthermore, expression of CUGBP1 was selectively induced by the Transforming Growth Factor-beta (TGF-ß) through p38 and JNK signaling in kidney fibroblasts. On the other hand, downregulating the expression of CUGBP1 significantly inhibited the activation of kidney fibroblasts. In conclusion, these findings demonstrated that fraxinellone might be a new drug candidate and CUGBP1 could be a promising target for the treatment of kidney fibrosis.
Assuntos
Benzofuranos/farmacologia , Proteínas CELF1/metabolismo , Fibroblastos/efeitos dos fármacos , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Animais , Proteínas CELF1/genética , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Ácido Fólico , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Rim/metabolismo , Rim/patologia , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Ratos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Vascular rarefaction due to impaired angiogenesis is associated with contractile dysfunction and the transition from compensation to decompensation and heart failure. The regulatory mechanism controlling vascular rarefaction during the transition remains elusive. Increased expression of a nuclear RNA-binding protein CUGBP Elav-like family member 1 (CELF1) in the adult heart is associated with the transition from compensated hypertrophy to decompensated heart failure. Elevated CELF1 level resulted in degradation of the major cardiac gap junction protein, connexin 43, in dilated cardiomyopathy (DCM), the most common cause of heart failure. In the present study, we investigated the role of increased CELF1 expression in causing vascular rarefaction in DCM. CELF1 overexpression (CELF1-OE) in cardiomyocytes resulted in reduced capillary density. CELF1-OE mice administered hypoxyprobe showed immunoreactivity and increased mRNA levels of HIF1α, Glut-1, and Pdk-1, which suggested the association of a reduced capillary density-induced hypoxic condition with CELF1 overexpression. Vegfa mRNA level was downregulated in mouse hearts exhibiting DCM, including CELF1-OE and infarcted hearts. Vegfa mRNA level was also downregulated to a similar extent in cardiomyocytes isolated from infarcted hearts by Langendorff preparation, which suggested cardiomyocyte-derived Vegfa expression mediated by CELF1. Cardiomyocyte-specific depletion of CELF1 preserved the capillary density and Vegfa mRNA level in infarcted mouse hearts. Also, CELF1 bound to Vegfa mRNA and regulated Vegfa mRNA stability via the 3' untranslated region. These results suggest that elevated CELF1 level has dual effects on impairing the functions of cardiomyocytes and microvasculature in DCM.
Assuntos
Proteínas CELF1/metabolismo , Insuficiência Cardíaca/patologia , Microvasos/patologia , Proteólise , Estabilidade de RNA , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteínas CELF1/genética , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3' regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fígado/metabolismo , Estabilidade de RNA , Animais , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Feminino , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Fígado/embriologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/genética , Transativadores/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is the most common type of adult muscular dystrophy caused by the expanded triple-nucleotides (CUG) repeats. Myoblast in DM1 displayed many defects, including defective myoblast differentiation, ribonuclear foci, and aberrant alternative splicing. Despite many were revealed to function in DM1, microRNAs that regulated DM1 via directly targeting the expanded CUG repeats were rarely reported. Here we discovered that miR-322/-503 rescued myoblast defects in DM1 cell model by targeting the expanded CUG repeats. First, we studied the function of miR-322/-503 in normal C2C12 myoblast cells. Downregulation of miR-322/-503 significantly hindered the myoblast differentiation, while miR-322/-503 overexpression promoted the process. Next, we examined the role of miR-322/-503 in the DM1 C2C12 cell model. miR-322/-503 was downregulated in the differentiation of DM1 C2C12 cells. When we introduced ectopic miR-322/-503 expression into DM1 C2C12 cells, myoblast defects were almost fully rescued, marked by significant improvements of myoblast differentiation and repressions of ribonuclear foci formation and aberrant alternative splicing. Then we investigated the downstream mechanism of miR-322/-503 in DM1. Agreeing with our previous work, Celf1 was proven to be miR-322/-503's target. Celf1 knockdown partially reproduced miR-322/-503's function in rescuing DM1 C2C12 differentiation but was unable to repress ribonuclear foci, suggesting other targets of miR-322/-503 existed in the DM1 C2C12 cells. As the seed regions of miR-322 and miR-503 were complementary to the CUG repeats, we hypothesized that the CUG repeats were the target of miR-322/-503. Through expression tests, reporter assays, and colocalization staining, miR-322/-503 was proved to directly and specifically target the expanded CUG repeats in the DM1 cell model rather than the shorter ones in normal cells. Those results implied a potential therapeutic function of miR-322/-503 on DM1, which needed further investigations in the future.
Assuntos
Proteínas CELF1/genética , MicroRNAs/fisiologia , Mioblastos/fisiologia , Distrofia Miotônica/genética , Processamento Alternativo , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células HEK293 , HumanosRESUMO
The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells-directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3' UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.
Assuntos
Proteínas CELF1/genética , Catarata/genética , Proteínas de Homeodomínio/genética , Cristalino/metabolismo , Fator de Transcrição PAX6/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas CELF1/antagonistas & inibidores , Proteínas CELF1/deficiência , Catarata/patologia , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Cristalino/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genéticaRESUMO
mRNA processing is highly regulated during development through changes in RNA-binding protein (RBP) activities. CUG-BP, Elav-like family member 1 (CELF1, also called CUGBP1) is an RBP, the expression of which decreases in skeletal muscle soon after birth. CELF1 regulates multiple nuclear and cytoplasmic RNA processing events. In the nucleus, CELF1 regulates networks of postnatal alternative splicing (AS) transitions, while in the cytoplasm, CELF1 regulates mRNA stability and translation. Stabilization and misregulation of CELF1 has been implicated in human diseases including myotonic dystrophy type 1, Alzheimer's disease and multiple cancers. To understand the contribution of nuclear and cytoplasmic CELF1 activity to normal and pathogenic skeletal muscle biology, we generated transgenic mice for doxycycline-inducible and skeletal muscle-specific expression of active CELF1 mutants engineered to be localized predominantly to either the nucleus or the cytoplasm. Adult mice expressing nuclear, but not cytoplasmic, CELF1 are characterized by strong histopathological defects, muscle loss within 10 days and changes in AS. In contrast, mice expressing cytoplasmic CELF1 display changes in protein levels of targets known to be regulated at the level of translation by CELF1, with minimal changes in AS. These changes are in the absence of overt histopathological changes or muscle loss. RNA-sequencing revealed extensive gene expression and AS changes in mice overexpressing nuclear and naturally localized CELF1 protein, with affected genes involved in cytoskeleton dynamics, membrane dynamics, RNA processing and zinc ion binding. These results support a stronger role for nuclear CELF1 functions as compared to cytoplasmic CELF1 functions in skeletal muscle wasting.
Assuntos
Proteínas CELF1/genética , Atrofia Muscular/genética , Distrofia Miotônica/genética , Estabilidade de RNA/genética , Processamento Alternativo/genética , Animais , Nucléolo Celular/genética , Citoplasma/genética , Humanos , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Distrofia Miotônica/patologia , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genéticaRESUMO
Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUGexp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSALR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUGexp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.
Assuntos
Processamento Alternativo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Distrofia Miotônica/genética , Animais , Proteínas CELF1/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feto , Humanos , Camundongos , Camundongos Transgênicos , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNA-binding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Distrofia Miotônica/tratamento farmacológico , Proteínas de Ligação a RNA/genética , Animais , Anti-Inflamatórios não Esteroides/classificação , Proteínas CELF1/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/genética , Fenilbutazona/farmacologiaRESUMO
INTRODUCTION: Microstructural abnormalities in white matter (WM) are often reported in Alzheimer's disease (AD). However, it is unclear which brain regions have the strongest WM changes in presymptomatic AD and what biological processes underlie WM abnormality during disease progression. METHODS: We developed a systems biology framework to integrate matched diffusion tensor imaging (DTI), genetic and transcriptomic data to investigate regional vulnerability to AD and identify genetic risk factors and gene subnetworks underlying WM abnormality in AD. RESULTS: We quantified regional WM abnormality and identified most vulnerable brain regions. A SNP rs2203712 in CELF1 was most significantly associated with several DTI-derived features in the hippocampus, the top ranked brain region. An immune response gene subnetwork in the blood was most correlated with DTI features across all the brain regions. DISCUSSION: Incorporation of image analysis with gene network analysis enhances our understanding of disease progression and facilitates identification of novel therapeutic strategies for AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Proteínas CELF1/genética , Biologia de Sistemas/métodos , Idoso , Doença de Alzheimer/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Imagem de Tensor de Difusão/métodos , Endofenótipos , Feminino , Redes Reguladoras de Genes/genética , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Polimorfismo de Nucleotídeo Único , TranscriptomaRESUMO
The insulin receptor gene (INSR) undergoes alternative splicing to give rise to two functionally related, but also distinct, isoforms IR-A and IR-B, which dictate proliferative and metabolic regulations, respectively. Previous studies identified the RNA-binding protein CUGBP1 as a key regulator of INSR splicing. In this study, we show that the differential splicing of INSR occurs more frequently in breast cancer than in non-tumor breast tissues. In breast cancer cell lines, the IR-A:IR-B ratio varies in different molecular subtypes, knockdown or overexpression of CUGBP1 gene in breast cancer cells altered IR-A:IR-B ratio through modulation of IR-A expression, thereby reversed or enhanced the insulin-induced oncogenic behavior of breast cancer cells, respectively. Our data revealed the predominant mitogenic role of IR-A isoform in breast cancer and depicted a novel interplay between INSR and CUGBP1, implicating CUGBP1 and IR-A isoform as the potential therapeutic targets and biomarkers for breast cancer.
Assuntos
Antígenos CD/genética , Neoplasias da Mama/patologia , Proteínas CELF1/metabolismo , Splicing de RNA , Receptor ErbB-2/metabolismo , Receptor de Insulina/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas CELF1/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Isoformas de ProteínasRESUMO
Myotonic dystrophy involves two types of chronically debilitating rare neuromuscular diseases: type 1 (DM1) and type 2 (DM2). Both share similarities in molecular cause, clinical signs, and symptoms with DM2 patients usually displaying milder phenotypes. It is well documented that key clinical symptoms in DM are associated with a strong mis-regulation of RNA metabolism observed in patient's cells. This mis-regulation is triggered by two leading DM-linked events: the sequestration of Muscleblind-like proteins (MBNL) and the mis-regulation of the CUGBP RNA-Binding Protein Elav-Like Family Member 1 (CELF1) that cause significant alterations to their important functions in RNA processing. It has been suggested that DM1 may be treatable through endogenous modulation of the expression of MBNL and CELF1 proteins. In this study, we analyzed the recent identification of the involvement of microRNA (miRNA) molecules in DM and focus on the modulation of these miRNAs to therapeutically restore normal MBNL or CELF1 function. We also discuss additional prospective miRNA targets, the use of miRNAs as disease biomarkers, and additional promising miRNA-based and miRNA-targeting drug development strategies. This review provides a unifying overview of the dispersed data on miRNA available in the context of DM.