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Epithelial to mesenchymal transition (EMT) is believed to be a principal factor contributing to cancer metastasis. The post-transcriptional and post-translational mechanisms underlying EMT are comparatively underexplored. We previously demonstrated that the CELF1 RNA binding protein is necessary and sufficient to drive the EMT of breast epithelial cells, and that the relative protein expression of CELF1 in this context was dictated at the post-translational level. Here, we elucidate the mechanism of this regulation. Mass spectrometric analysis of CELF1 isolated from mesenchymal MCF-10A cells identified multiple sites of serine and threonine phosphorylation on the protein, correlating with the increased stability of this protein in this cellular state. Analysis of phosphomimetic and serine/threonine-to-alanine phosphomutant variants of CELF1 revealed that these phosphorylation sites indeed dictate CELF1 stability, ubiquitination state, and function in vitro. Via co-immunoprecipitation and in vitro kinase assays, we identified the protein kinase C alpha and epsilon isozymes as the kinases responsible for CELF1 phosphorylation in a breast cell line. Genetic epistasis experiments confirmed that these PKCs function upstream of CELF1 in this EMT program, and CELF1 phosphorylation impacts tumor metastasis in a xenograft model. This work is the first to formally establish the mechanisms underlying post-translational control of CELF1 expression and function during EMT of breast epithelial cells. Given the broad dysregulation of CELF1 expression in human breast cancer, our results may ultimately provide knowledge that may be leveraged for novel therapeutic interventions in this context., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA-binding proteins (RBPs) are attractive targets in human pathologies. Despite a number of efforts to target RBPs with small molecules, it is still difficult to develop RBP inhibitors, asking for a deeper understanding of how to chemically perturb RNA-binding activity. In this study, we found that the thiopurine drugs (6-mercaptopurine and 6-thioguanine) effectively disrupt CELF1-RNA interaction. The disrupting activity relies on the formation of disulfide bonds between the thiopurine drugs and CELF1. Mutating the cysteine residue proximal to the RNA recognition motifs (RRMs), or adding reducing agents, abolishes the disrupting activity. Furthermore, the 1,2,4-triazole-3-thione, a thiopurine analogue, was identified with 20-fold higher disrupting activity. Based on this analogue, we found that compound 9 disrupts CELF1-RNA interaction in living cells and ameliorates CELF1-mediated myogenesis deficiency. In summary, we identified a thiol-mediated binding mechanism for thiopurine drugs and their derivatives to perturb protein-RNA interaction, which provides novel insight for developing RBP inhibitors. Additionally, this work may benefit the pharmacological and toxicity research of thiopurine drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the 3' untranslated region of the DM1 protein kinase gene. Characteristic degenerative muscle symptoms include myotonia, atrophy, and weakness. We previously proposed an Musashi homolog 2 (MSI2)>miR-7>autophagy axis whereby MSI2 overexpression repressed miR-7 biogenesis that subsequently de-repressed muscle catabolism through excessive autophagy. Because the DM1 HSA LR mouse model expressing expanded CUG repeats shows weak muscle-wasting phenotypes, we hypothesized that MSI2 overexpression was sufficient to promote muscle dysfunction in vivo., Methods: By means of recombinant AAV murine MSI2 was overexpressed in neonates HSA LR mice skeletal muscle to induce DM1-like phenotypes., Results: Sustained overexpression of the murine MSI2 protein in HSA LR neonates induced autophagic flux and expression of critical autophagy proteins, increased central nuclei and reduced myofibers area, and weakened muscle strength. Importantly, these changes were independent of MBNL1, MBNL2, and Celf1 protein levels, which remained unchanged upon Msi2 overexpression., Conclusions: Globally, molecular, histological, and functional data from these experiments in the HSA LR mouse model confirms the pathological role of MSI2 expression levels as an atrophy-associated component that impacts the characteristic muscle dysfunction symptoms in DM1 patients., Competing Interests: Declaration of competing interest M.S-A., R.A., and A.B. are inventors in patent PCT/EP2022/054129., (© 2023 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Hepatic fibrosis, a common pathological feature of chronic liver injuries, is a serious public health problem and lacks effective therapy. Glycyrrhizic acid (GA) is a bioactive ingredient in the root of traditional Chinese medicine licorice, and exhibits remarkable anti-viral, anti-inflammatory and hepatoprotective actions., Purpose: Here we aimed to investigated whether GA provided a therapeutic efficacy in hepatic fibrosis and uncover its molecular mechanisms., Study Design and Methods: We investigated the anti-fibrosis effects of GA using CCl 4 -induced mouse mode of liver fibrosis as well as TGF-β1-activated human LX-2 cells and primary hepatic stellate cells (HSCs). CUGBP1-mediated IFN-γ/STAT1/Smad7 signaling was examined with immunofluorescence staining and western blot analysis. We designed and studied the binding of GA to CUGBP1 using in silico docking, and validated by microscale thermophoresis (MST) assay., Results: GA obviously attenuated CCl 4 -induced liver histological damage, and reduced serum ALT and AST levels. Meanwhile, GA decreased liver fibrogenesis markers such as α-SMA, collagen α1, HA, COL-III, and LN in the hepatic tissues. Mechanistically, GA remarkably elevated the levels of IFN-γ, p-STAT1, Smad7, and decreased CUGBP1 in vivo and in vitro. Over-expression of CUGBP1 completely abolished the anti-fibrotic effect of GA and regulation on IFN-γ/STAT1/Smad7 pathway in LX-2 cells and primary HSCs, confirming CUGBP1 played a pivotal role in the protection by GA from liver fibrosis. Further molecular docking and MST assay indicated that GA had a good binding affinity with the CUGBP1 protein. The dissociation constant (Kd) of GA and CUGBP1 was 0.293 μM., Conclusion: Our study demonstrated for the first time that GA attenuated liver fibrosis and hepatic stellate cell activation by promoting CUGBP1-mediated IFN-γ/STAT1/Smad7 signalling pathways. GA may be a potential candidate compound for preventing or reliving liver fibrosis., Competing Interests: Declaration of Competing Interests The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier GmbH.)

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 ( Celf1 cKO ) 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 Celf1 cKO mouse lenses at postnatal day (P) 0. Celf1 cKO 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 Celf1 cKO 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 Celf1 cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1 cKO 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.

CUGBP Elav-like family member 1 (CELF1), an RNA-binding protein (RBP), plays important roles in the pathogenesis of diseases such as myotonic dystrophy, liver fibrosis and cancers. However, targeting CELF1 is still a challenge, as RBPs are considered largely undruggable. Here, we discovered that compound 27 disrupted CELF1-RNA binding via structure-based virtual screening and biochemical assays. Compound 27 binds directly to CELF1 and competes with RNA for binding to CELF1. Compound 27 promotes IFN-γ secretion and suppresses TGF-β1-induced hepatic stellate cell (HSC) activation by inhibiting CELF1-mediated IFN-γ mRNA decay. In vivo, compound 27 attenuates CCl4-induced murine liver fibrosis. Furthermore, the structure-activity relationship analysis was performed and compound 841, a derivative of compound 27, was identified as a selective CELF1 inhibitor. In conclusion, targeting CELF1 RNA-binding activity with small molecules was achieved, which provides a novel strategy for treating liver fibrosis and other CELF1-mediated diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2022. The Author(s).)

Development of the ocular lens - a transparent tissue capable of sustaining frequent shape changes for optimal focusing power - pushes the boundaries of what cells can achieve using the molecular toolkit encoded by their genomes. The mammalian lens contains broadly two types of cells, the anteriorly located monolayer of epithelial cells which, at the equatorial region of the lens, initiate differentiation into fiber cells that contribute to the bulk of the tissue. This differentiation program involves massive upregulation of select fiber cell-expressed RNAs and their subsequent translation into high amounts of proteins, such as crystallins. But intriguingly, fiber cells achieve this while also simultaneously undergoing significant morphological changes such as elongation - involving about 1000-fold length-wise increase - and migration, which requires modulation of cytoskeletal and cell adhesion factors. Adding further to the challenges, these molecular and cellular events have to be coordinated as fiber cells progress toward loss of their nuclei and organelles, which irreversibly compromises their potential for harnessing genetically hardwired information. A long-standing question is how processes downstream of signaling and transcription, which may also participate in feedback regulation, contribute toward orchestrating these cellular differentiation events in the lens. It is now becoming clear from findings over the past decade that post-transcriptional gene expression regulatory mechanisms are critical in controlling cellular proteomes and coordinating key processes in lens development and fiber cell differentiation. Indeed, RNA-binding proteins (RBPs) such as Caprin2, Celf1, Rbm24 and Tdrd7 have now been described in mediating post-transcriptional control over key factors (e.g. Actn2, Cdkn1a (p21 Cip1 ), Cdkn1b (p27 Kip1 ), various crystallins, Dnase2b, Hspb1, Pax6, Prox1, Sox2) that are variously involved in cell cycle, transcription, cytoskeleton maintenance and differentiation in the lens. Furthermore, deficiencies of these RBPs have been shown to result in various eye and lens defects and/or cataract. Because fiber cell differentiation in the lens occurs throughout life, the underlying regulatory mechanisms operational in development are expected to also be recruited for the maintenance of transparency in aged lenses. Indeed, in support of this, TDRD7 and CAPRIN2 loci have been linked to age-related cataract in humans. Here, I will review the role of key RBPs in the lens and their importance in understanding the pathology of lens defects. I will discuss advances in RBP-based gene expression control, in general, and the important challenges that need to be addressed in the lens to define the mechanisms that determine the epithelial and fiber cell proteome. Finally, I will also discuss in detail several key future directions including the application of bioinformatics approaches such as iSyTE to study RBP-based post-transcriptional gene expression control in the aging lens and in the context of age-related cataract., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cardiac hypertrophy is considered as a common pathophysiological process in various cardiovascular diseases. CUG triplet repeat-binding protein 1 (CELF1) is an RNA-binding protein that has been shown to be an important post-transcription regulator and involved in several types of cancer, whereas its role in cardiac remodeling remains unclear. Herein, we found that the expression of CELF1 was significantly increased in pressure overload-induced hypertrophic hearts and angiotensin II (Ang II)-induced neonatal cardiomyocytes. Based on transverse aortic constriction-induced cardiac hypertrophy model, CELF1 deficiency markedly ameliorated cardiac hypertrophy, cardiac fibrosis, oxidative stress, and apoptosis. Accordingly, CELF1 deficiency alleviated the production of reactive oxygen species (ROS) and apoptosis of neonatal cardiomyocytes via inhibition of Raf1, TAK1, ERK1/2, and p38 phosphorylation. Mechanistically, depletion or overexpression of CELF1 negatively regulated the protein expression of phosphatidylethanolamine-binding protein 1 (PEBP1), while the mRNA expression of PEBP1 remained unchanged. RNA immunoprecipitation revealed that CELF1 directly interacted with PEBP1 mRNA. Biotin pull-down analysis and dual-luciferase assay showed that CELF1 directly bound to the fragment 1 within 3'UTR of PEBP1. Moreover, knockdown of PEBP1 partially enhanced the production of ROS and apoptosis of neonatal cardiomyocytes inhibited by CELF1 deficiency. In conclusion, CELF1 binds to the 3'UTR of PEBP1 and acts as an endogenous activator of MAPK signaling pathway. Inhibition of CELF1 attenuates pathological cardiac hypertrophy, oxidative stress, and apoptosis, thus could be a potential therapeutic strategy of pathological cardiac hypertrophy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cardiac hypertrophy is imposed much pressure on heart and threatening our live. Previous study suggested that dysregulation of Celf1 is largely connecting to neonatal cardiac dysfunction. Hence, we aimed to explore the precise function and probable regulatory mechanism upstream of Celf1in cardiac hypertrophy. Here, Ang-II treatment was implemented to stimulate hypertrophic phenotypes inH9C2 and MCM cells. Immunofluorescence assay was conducted to measure the surface area of cardiomyocytes. And qRT-PCR assay was conducted to investigate gene expression. Moreover, western blot assay was conducted to probe the protein levels. Results uncovered that Celf1 expression was increased dependent on elevated Ang-II concentration, and that inhibited Celf1 could relieve the Ang-II-caused cardiac hypertrophy. Significantly, Celf1was found to be targeted by miR-129-5p but then released via the sponging role of circ-Jarid2. Furthermore, circ-Jarid2 was found to promote cardiac hypertrophy, whereas miR-129-5p played suppressing parts in hypertrophic cardiomyocytes. Moreover, we verified circ-Jarid2 contributed to cardiac hypertrophy via miR-129-5p/Celf1 axis both in vitro and in vivo. In conclusion, circ-Jarid2/miR-129-5p/Celf1 axis aggravates cardiac hypertrophy, which provides new ideas for developing treatment strategies for patients with cardiac hypertrophy., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

Aims: Hepatocellular carcinoma (HCC) is a malignant cancer that threatened human life seriously. Long non-coding RNA (lncRNA) BACE1-AS has been reported as a key regulator in tumorigenesis. Yet the specific correlation between BACE1-AS and HCC still needs further investigation. The primary purpose of our study is to reveal the exact correlation between BACE1-AS and HCC., Main Methods: Bioinformatics via TCGA database revealed BACE1-AS closely related with HCC. qRT-PCR confirmed the abnormal BACE1-AS level in HCC tissues and cells. Databases prediction suggested that miR-377-3p might be a modulatory target of BACE1-AS and luciferase assay confirmed this hypothesis. Further study discovered that CELF1 also partook in the regulatory axis of BACE1-AS/miR-377-3p. Wound healing assays and transwell assays were utilized to investigate the impact of BACE1-AS, miR-377-3p and CELF1 in vitro. In vivo metastasis was examined by pulmonary metastasis model., Key Findings: This study found that BACE1-AS was overexpressed in HCC tissues and cell lines. Knockdown of BACE1-AS could restrain HCC progression in vitro, and inhibit pulmonary metastasis in vivo. MiR-377-3p was negatively modulated by BACE1-AS in HCC tumor tissues and cells. MiR-377-3p up-regulation inhibited HCC cells migration and invasion via inactivating EMT process. Moreover, CELF1 was identified as a downstream regulator of miR-377-3p and served as an oncogene in HCC cells., Significance: Our findings supported that lncRNA BACE1-AS was up-regulated in HCC, promoting invasion and metastasis of hepatocellular carcinoma cells by modulating miR-377-3p/CELF1 axis via contributing to EMT pathway. BACE1-AS could be a potential biomarker in HCC for future treatment., (Copyright © 2021. Published by Elsevier Inc.)

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-β) 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 Federation of American Societies for Experimental Biology.)

Nasopharyngeal carcinoma is a type of otolaryngological malignancy with high incidence. Long non-coding RNAs (lncRNAs) are closely related to nasopharyngeal carcinoma. LncRNA AFAP1-AS1 (AFAP1-AS1) has been found to play important roles in nasopharyngeal carcinoma progression and poor prognosis. However, the mechanism underlying AFAP1-AS1 in regulating nasopharyngeal carcinoma is still unclear. In current study, AFAP1-AS1 was found to be up-regulated in nasopharyngeal carcinoma tissues and cells. AFAP1-AS1 overexpression and knockdown were conducted in nasopharyngeal carcinoma cells. The results proved that AFAP1-AS1 promoted the survival and migration of nasopharyngeal carcinoma cells. Additionally, specificity protein 1 (SP1) was enhanced in nasopharyngeal carcinoma tissues and cells, and induced AFAP1-AS1 expression. The interaction between AFAP1-AS1 and miR-497-5p was confirmed. AFAP1-AS1 was demonstrated to regulate CELF1, a target gene of miR-497-5p. Further functional analysis revealed that AFAP1-AS1 knockdown attenuated SP1-induced nasopharyngeal carcinoma progression. These results indicate that SP1-induced AFAP1-AS1 facilitates nasopharyngeal carcinoma progression by regulating miR-497-5p/CELF1 pathway, which provides a new target for nasopharyngeal carcinoma treatment.

Background & Aims: The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms., Methods: Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities., Results: DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction., Conclusions: To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis., (Published by Elsevier Inc.)

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.

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear 1 . The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus 2 and binding diverse proteins 3-5 to achieve X-chromosome inactivation (XCI) 6,7 . The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off 8 . Here we show that the Xist RNA-binding proteins PTBP1 9 , MATR3 10 , TDP-43 11 and CELF1 12 assemble on the multivalent E-repeat element of Xist 7 and, via self-aggregation and heterotypic protein-protein interactions, form a condensate 1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase 8 , indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Colorectal cancer (CRC) is often diagnosed at later stages after it has metastasized to other organs. The development of chemoresistance also contributes to a poor prognosis. Therefore, an increased understanding of the metastatic properties of CRC and chemoresistance could improve patient survival. CUGBP elav-like family member 1 (CELF1) is an RNA-binding protein, which is overexpressed in many human malignant tumors. However, the influence of CELF1 in CRC is unclear. V-ets erythroblastosis virus E26 oncogene homologue 2 (ETS2) is an evolutionarily conserved proto-oncogene known to be overexpressed in a variety of human cancers including CRC. In thespresent tudy, we investigated the association between CELF1 and ETS2 in CRC tumorigenesis and oxaliplatin (L-OHP) resistance. We found a positive correlation between the elevated expression of CELF1 and ETS2 in human CRC tissues. Overexpression of CELF1 increased CRC cell proliferation, migration, and invasion in vitro and in a xenograft tumor growth model in vivo, and induced resistance to L-OHP. In contrast, CELF1 knockdown improved the response of CRC cells to L-OHP. Overexpression of ETS2 increased the malignant behavior of CRC cells (growth, migration, and invasion) and L-OHP resistance in vitro. Moreover, L-OHP resistance induced by CELF1 overexpression was reversed by ETS2 knockdown. The results of luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that CELF1 up-regulates ETS2 by binding to its 3'-UTR. Taken together, our findings have identified that CELF1 regulates ETS2 in a mechanism that results in CRC tumorigenesis and L-OHP resistance, and CELF1 may be a promising target for overcoming chemoresistance in CRC., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

Myotonic dystrophy type 1 (DM1) is a complex genetic disease affecting many tissues. DM1 is caused by an expansion of CTG repeats in the 3'-UTR of the DMPK gene. The mechanistic studies of DM1 suggested that DMPK mRNA, containing expanded CUG repeats, is a major therapeutic target in DM1. Therefore, the removal of the toxic RNA became a primary focus of the therapeutic development in DM1 during the last decade. However, a cure for this devastating disease has not been found. Whereas the degradation of toxic RNA remains a preferential approach for the reduction of DM1 pathology, other approaches targeting early toxic events downstream of the mutant RNA could be also considered. In this review, we discuss the beneficial role of the restoring of the RNA-binding protein, CUGBP1/CELF1, in the correction of DM1 pathology. It has been recently found that the normalization of CUGBP1 activity with the inhibitors of GSK3 has a positive effect on the reduction of skeletal muscle and CNS pathologies in DM1 mouse models. Surprisingly, the inhibitor of GSK3, tideglusib also reduced the toxic CUG-containing RNA. Thus, the development of the therapeutics, based on the correction of the GSK3β-CUGBP1 pathway, is a promising option for this complex disease.

Myotonic dystrophy type 1 (DM1) is a multisystem neuromuscular disease without cure. One of the possible therapeutic approaches for DM1 is correction of the RNA-binding proteins CUGBP1 and MBNL1, misregulated in DM1. CUGBP1 activity is controlled by glycogen synthase kinase 3β (GSK3β), which is elevated in skeletal muscle of patients with DM1, and inhibitors of GSK3 were suggested as therapeutic molecules to correct CUGBP1 activity in DM1. Here, we describe that correction of GSK3β with a small-molecule inhibitor of GSK3, tideglusib (TG), not only normalizes the GSK3β-CUGBP1 pathway but also reduces the mutant DMPK mRNA in myoblasts from patients with adult DM1 and congenital DM1 (CDM1). Correction of GSK3β in a mouse model of DM1 ( HSA LR mice) with TG also reduces the levels of CUG-containing RNA, normalizing a number of CUGBP1- and MBNL1-regulated mRNA targets. We also found that the GSK3β-CUGBP1 pathway is abnormal in skeletal muscle and brain of DMSXL mice, expressing more than 1,000 CUG repeats, and that the correction of this pathway with TG increases postnatal survival and improves growth and neuromotor activity of DMSXL mice. These findings show that the inhibitors of GSK3, such as TG, may correct pathology in DM1 and CDM1 via several pathways., (Copyright © 2019 American Society for Microbiology.)

CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE 2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1β and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE 2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

Alternative splicing (AS) is dysregulated in Type 1 diabetic (T1D) hearts but mechanisms responsible are unclear. Here, we provide evidence that the RNA binding protein (RBP) PTBP1 is modulated in adult T1D hearts contributing to AS changes. We show that a spliced variant of PTBP1 that is highly expressed in normal newborn mouse hearts is aberrantly expressed in adult T1D mouse hearts. Comparing known PTBP1-target datasets to our T1D mouse transcriptome datasets, we discovered a group of genes with PTBP1 binding sites in their pre-mRNAs that are differentially spliced in T1D mouse hearts. We demonstrated that inducible expression of diabetes-induced PTBP1 spliced variant has less repressive splicing function. Notably, PTBP1 regulates AS of some of its targets antagonistically to RBFOX2. In sum, our results indicate that diabetic conditions disrupt developmental regulation of PTBP1 leading to differential AS of PTBP1 target genes. Identification of PTBP1 and PTBP1-regulated RNA networks can provide RNA-based therapies for the treatment of diabetes cardiac complications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dysregulated alternative splicing (AS) that contributes to diabetes pathogenesis has been identified, but little is known about the RNA binding proteins (RBPs) involved. We have previously found that the RBP CELF1 is upregulated in the diabetic heart; however, it is unclear if CELF1 contributes to diabetes-induced AS changes. Utilizing genome wide approaches, we identified extensive changes in AS patterns in Type 1 diabetic (T1D) mouse hearts. We discovered that many aberrantly spliced genes in T1D hearts have CELF1 binding sites. CELF1-regulated AS affects key genes within signaling pathways relevant to diabetes pathogenesis. Disruption of CELF1 binding sites impairs AS regulation by CELF1. In sum, our results indicate that CELF1 target RNAs are aberrantly spliced in the T1D heart leading to abnormal gene expression. These discoveries pave the way for targeting RBPs and their RNA networks as novel therapies for cardiac complications of diabetes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Primate-specific NBL2 macrosatellite is hypomethylated in several types of tumors, yet the consequences of this DNA hypomethylation remain unknown. We show that NBL2 conserved repeats are close to the centromeres of most acrocentric chromosomes. NBL2 associates with the perinucleolar region and undergoes severe demethylation in a subset of colorectal cancer (CRC). Upon DNA hypomethylation and histone acetylation, NBL2 repeats are transcribed in tumor cell lines and primary CRCs. NBL2 monomers exhibit promoter activity, and are contained within novel, non-polyA antisense lncRNAs, which we designated TNBL (Tumor-associated NBL2 transcript). TNBL is stable throughout the mitotic cycle, and in interphase nuclei preferentially forms a perinucleolar aggregate in the proximity of a subset of NBL2 loci. TNBL aggregates interact with the SAM68 perinucleolar body in a mirror-image cancer specific perinucleolar structure. TNBL binds with high affinity to several proteins involved in nuclear functions and RNA metabolism, such as CELF1 and NPM1. Our data unveil novel DNA and RNA structural features of a non-coding macrosatellite frequently altered in cancer.

Peroxisome proliferator-activated receptor-γ (PPARγ), is a transcription factor that governs pathways, such as lipid metabolism and immune response, that have been implicated in the etiology of LOAD. Previously, we established HepG2-derived cell-lines with stable knockdown of PPARγ gene, and showed an increase in mRNA levels of genes mapped in the APOE linkage disequilibrium (LD) region on chromosome 19q13.32, with the greatest effect observed for APOE-mRNA. Here, we extended the analysis using our PPARγ knockdown model system and investigated the broader effect on expression changes of genes implicated in LOAD via genome wide association studies (GWAS). We applied the nCounter gene expression assay (NanoString) using a panel of twenty-four LOAD-associated genes inferred by proximity to the top significantly associated SNPs. Two independent PPARγ knockdown cell-lines showed changes in mRNA levels of a total of seven genes compared to a control HepG2 cell-line; six of which, ABCA7, APOE, CASS4, CELF1, PTK2B, and ZCWPW1, were upregulated and one, DSG2, was downregulated upon PPARγ knockdown. Our results propose that PPARγ may act as a master regulator of the transcription of several genes involved in LOAD pathogenesis. Our study provided the premise for further analyses including a larger set of genes positioned within a wider range of linkage disequilibrium (LD) regions tagged by all LOAD significantly associated SNPs.

Enterocyte apoptosis induced by lipid emulsions is a key cause of intestinal atrophy under total parenteral nutrition (TPN) support, and our previous work demonstrated that olive oil lipid emulsion (OOLE) could induce enterocyte apoptosis via CUGBP, Elav-like family member 1 (CELF1)/ apoptosis-inducing factor (AIF) pathway. As TPN-associated complications are partially related to choline deficiency, we aimed to address whether choline supplementation could attenuate OOLE-induced enterocyte apoptosis. Herein we present evidence that supplementary choline exhibits protective effect against OOLE-induced enterocyte apoptosis both in vivo and in vitro. In a rat model of TPN, substantial reduction in apoptotic rate along with decreased expression of CELF1 was observed when supplementary choline was added to OOLE. In cultured Caco-2 cells, supplementary choline attenuated OOLE-induced apoptosis and mitochondria dysfunction by suppressing CELF1/AIF pathway. Compared to OOLE alone, the expression of CELF1 and AIF was significantly decreased by supplementary choline, whereas the expression of Bcl-2 was evidently increased. No obvious alterations were observed in Bax expression and caspase-3 activation. Mechanistically, supplementary choline repressed the expression of CELF1 by increasing the recruitment of CELF1 mRNA to processing bodies, thus resulting in suppression of its protein translation. Taken together, our data suggest that supplementary choline exhibits effective protection against OOLE-induced enterocyte apoptosis, and thus, it has the potential to be used for the prevention and treatment of TPN-induced intestinal atrophy., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Melanomas are well-known for their altered mRNA expression profiles. Yet, the specific contribution of mRNA binding proteins (mRBPs) to melanoma development remains unclear. Here we identify a cluster of melanoma-enriched genes under the control of CUGBP Elav-like family member 1 (CELF1). CELF1 was discovered with a distinct prognostic value in melanoma after mining the genomic landscape of the 692 known mRBPs across different cancer types. Genome-wide transcriptomic, proteomic, and RNA-immunoprecipitation studies, together with loss-of-function analyses in cell lines, and histopathological evaluation in clinical biopsies, revealed an intricate repertoire of CELF1-RNA interactors with minimal overlap with other malignancies. This systems approach uncovered the oncogene DEK as an unexpected target and downstream effector of CELF1. Importantly, CELF1 and DEK were found to represent early-induced melanoma genes and adverse indicators of overall patient survival. These results underscore novel roles of CELF1 in melanoma, illustrating tumor type-restricted functions of RBPs in cancer.

Aim: Myocardial infarction (MI) is one of the leading causes of death in elderly people. Expanding the knowledge of the molecular mechanisms underlying MI is of profound importance to developing a cure for MI. The CUGBP- and ETR-3-like factor (CELF) proteins, a family of RNA-binding proteins, play key roles in RNA metabolism. To determine the functions and molecular mechanisms of CELF proteins in MI, an animal model of acute myocardial infarction (AMI) was used in our study., Results: We found that the CUG triplet repeat RNA-binding protein 1 (CUGBP1)/CELF1 expression levels were decreased in AMI-injured hearts, and further studies showed that two highly conserved adenylate-uridylate-rich (AU-rich) elements in the 3'UTR of CUGBP1 were responsible for the decreased CUGBP1 expression. Upon AMI, human antigen R (HuR) was relocated to the cytoplasm from the nucleus and interacted with these AU-rich elements to affect the expression of CUGBP1. Reintroduction of CUGBP1 via gene delivery by recombinant adenovirus improved cardiac function in AMI mice. Our studies also indicated that CUGBP1 protected cardiomyocytes from ischemia-induced injury through the promotion of angiogenesis and inhibition of apoptosis by regulating the vascular endothelial growth factor-A gene. Innovation and Conclusion: Our studies indicate a role for CUGBP1 in cardiac disease and reveal a novel MI post-transcriptional gene regulatory mechanism. The reconstitution of CUGBP1 could be developed as a potential therapeutic option for the management of MI. Antioxid. Redox Signal. 27, 1013-1026.

Insulin-like growth factor type 2 (IGF2) receptor (IGF2R) recognizes mannose 6-phosphate-containing molecules and IGF2 and plays an important role in many pathophysiological processes, including gut mucosal adaptation. However, the mechanisms that control cellular IGF2R abundance are poorly known. MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) critically regulate gene expression programs in mammalian cells by modulating the stability and translation of target mRNAs. Here we report that miRNA 195 (miR-195) and RBP CUG-binding protein 1 (CUGBP1) jointly regulate IGF2R expression at the posttranscriptional level in intestinal epithelial cells. Both miR-195 and CUGBP1 interacted with the 3' untranslated region (3'-UTR) of Igf2r mRNA, and the association of CUGBP1 with Igf2r mRNA enhanced miR-195 binding to Igf2r mRNA. Ectopically expressed CUGBP1 and miR-195 repressed IGF2R translation cooperatively without altering the stability of Igf2r mRNA. Importantly, the miR-195- and CUGBP1-repressed levels of cellular IGF2R led to a disruption in the structure of the trans -Golgi network. These findings indicate that IGF2R expression is controlled posttranscriptionally by two factors that associate with Igf2r mRNA and suggest that miR-195 and CUGBP1 dampen IGF signaling by inhibiting IGF2R translation., (Copyright © 2017 American Society for Microbiology.)

The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enhancing brown fat activity and promoting white fat browning are attractive therapeutic strategies for treating obesity and associated metabolic disorders. To provide a comprehensive picture of the gene regulatory network in these processes, we conducted a series of transcriptome studies by RNA sequencing (RNA-seq) and quantified the mRNA and long noncoding RNA (lncRNA) changes during white fat browning (chronic cold exposure, beta-adrenergic agonist treatment, and intense exercise) and brown fat activation or inactivation (acute cold exposure or thermoneutrality, respectively). mRNA-lncRNA coexpression networks revealed dynamically regulated lncRNAs to be largely embedded in nutrient and energy metabolism pathways. We identified a brown adipose tissue-enriched lncRNA, lncBATE10, that was governed by the cAMP-cAMP response element-binding protein (Creb) axis and required for a full brown fat differentiation and white fat browning program. Mechanistically, lncBATE10 can decoy Celf1 from Pgc1α, thereby protecting Pgc1α mRNA from repression by Celf1. Together, these studies provide a comprehensive data framework to interrogate the transcriptomic changes accompanying energy homeostasis transition in adipose tissue.

Despite intensive investigations, mechanisms of liver cancer are not known. Here, we identified an important step of liver cancer, which is the neutralization of tumor suppressor activities of an RNA binding protein, CUGBP1. The translational activity of CUGBP1 is activated by dephosphorylation at Ser302. We generated CUGBP1-S302A knock-in mice and found that the reduction of translational activity of CUGBP1 causes development of a fatty liver phenotype in young S302A mice. Examination of liver cancer in diethylnitrosamine (DEN)-treated CUGBP1-S302A mice showed these mice develop much more severe liver cancer that is associated with elimination of the mutant CUGBP1. Searching for mechanisms of this elimination, we found that the oncoprotein gankyrin (Gank) preferentially binds to and triggers degradation of dephosphorylated CUGBP1 (de-ph-S302-CUGBP1) or S302A mutant CUGBP1. To test the role of Gank in degradation of CUGBP1, we generated mice with liver-specific deletion of Gank. In these mice, the tumor suppressor isoform of CUGBP1 is protected from Gank-mediated degradation. Consistent with reduction of CUGBP1 in animal models, CUGBP1 is reduced in patients with pediatric liver cancer. Thus, this work presents evidence that de-ph-S302-CUGBP1 is a tumor suppressor protein and that the Gank-UPS-mediated reduction of CUGBP1 is a key event in the development of liver cancer., (Copyright © 2017 American Society for Microbiology.)

Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6., Competing Interests: The authors declare no competing financial interests.

Background and Aims: Parenterally-administered lipid emulsion (LE) is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1) has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro., Methods: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting., Results: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE)-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF) was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation., Conclusion: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3'-untranslated region (3'-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3'-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.

Excessive activation of hepatic stellate cells (HSCs) is a key step in liver fibrogenesis. Here we report that CUG-binding protein 1 (CUGBP1) expression is elevated in HSCs and positively correlates with liver fibrosis severity in human liver biopsies. Transforming growth factor-beta (TGF-β) selectively increases CUGBP1 expression in cultured HSCs in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Knockdown of CUGBP1 inhibits alpha smooth muscle actin (α-SMA) expression and promotes interferon gamma (IFN-γ) production in HSCs in vitro. We further show that CUGBP1 specifically binds to the 3' untranslated region (UTR) of human IFN-γ mRNA and promotes its decay. In mice, knockdown of CUGBP1 alleviates, whereas its overexpression exacerbates, bile duct ligation (BDL)-induced hepatic fibrosis. Therefore, CUGBP1-mediated IFN-γ mRNA decay is a key event for profibrotic TGF-β-dependent activation of HSCs, and inhibiting CUGBP1 to promote IFN-γ signalling in activated HSCs could be a novel strategy to treat liver fibrosis.

The RNA binding protein Celf1 regulates alternative splicing in the nucleus and mRNA stability and translation in the cytoplasm. Celf1 is strongly down-regulated during mouse postnatal heart development. Its re-induction in adults induced severe heart failure and reversion to fetal splicing and gene expression patterns. However, the impact of Celf1 depletion on cardiac transcriptional and posttranscriptional dynamics in neonates has not been addressed. We found that homozygous Celf1 knock-out neonates exhibited cardiac dysfunction not observed in older homozygous animals, although homozygous mice are smaller than wild type littermates throughout development. RNA-sequencing of mRNA from homozygous neonatal hearts identified a network of cell cycle genes significantly up-regulated and down-regulation of ion transport and circadian genes. Cell cycle genes are enriched for Celf1 binding sites supporting a regulatory role in mRNA stability of these transcripts. We also identified a cardiac splicing network coordinated by Celf1 depletion. Target events contain multiple Celf1 binding sites and enrichment in GU-rich motifs. Identification of direct Celf1 targets will advance our knowledge in the mechanisms behind developmental networks regulated by Celf1 and diseases where Celf1 is mis-regulated.

RNA-binding protein activities are highly regulated through protein levels, intracellular localization, and post-translation modifications. During development, mRNA processing of specific gene sets is regulated through manipulation of functional RNA-binding protein activities. The impact of altered RNA-binding protein activities also affects human diseases in which there are either a gain-of-function or loss-of-function causes pathogenesis. We will discuss RNA-binding proteins and their normal developmental RNA metabolism and contrast how their function is disrupted in disease. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims/hypothesis: CUG-binding protein 1 (CUGBP1) is a multifunctional RNA-binding protein that regulates RNA processing at several stages including translation, deadenylation and alternative splicing, as well as RNA stability. Recent studies indicate that CUGBP1 may play a role in metabolic disorders. Our objective was to examine its role in endocrine pancreas function through gain- and loss-of-function experiments and to further decipher the underlying molecular mechanisms., Methods: A mouse model in which type 2 diabetes was induced by a high-fat diet (HFD; 60% energy from fat) and mice on a standard chow diet (10% energy from fat) were compared. Pancreas-specific CUGBP1 overexpression and knockdown mice were generated. Different lengths of the phosphodiesterase subtype 3B (PDE3B) 3' untranslated region (UTR) were cloned for luciferase reporter analysis. Purified CUGBP1 protein was used for gel shift experiments., Results: CUGBP1 is present in rodent islets and in beta cell lines; it is overexpressed in the islets of diabetic mice. Compared with control mice, the plasma insulin level after a glucose load was significantly lower and glucose clearance was greatly delayed in mice with pancreas-specific CUGBP1 overexpression; the opposite results were obtained upon pancreas-specific CUGBP1 knockdown. Glucose- and glucagon-like peptide1 (GLP-1)-stimulated insulin secretion was significantly attenuated in mouse islets upon CUGBP1 overexpression. This was associated with a strong decrease in intracellular cAMP levels, pointing to a potential role for cAMP PDEs. CUGBP1 overexpression had no effect on the mRNA levels of PDE1A, 1C, 2A, 3A, 4A, 4B, 4D, 7A and 8B subtypes, but resulted in increased PDE3B expression. CUGBP1 was found to directly bind to a specific ATTTGTT sequence residing in the 3' UTR of PDE3B and stabilised PDE3B mRNA. In the presence of the PDE3 inhibitor cilostamide, glucose- and GLP-1-stimulated insulin secretion was no longer reduced by CUGBP1 overexpression. Similar to CUGBP1, PDE3B was overexpressed in the islets of diabetic mice., Conclusions/interpretation: We conclude that CUGBP1 is a critical regulator of insulin secretion via activating PDE3B. Repressing this protein might provide a potential strategy for treating type 2 diabetes.

Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate., Competing Interests: The authors declare no conflict of interest.

Background: Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, and lncRNA taurine-upregulated gene 1 (TUG1) has been proven to be associated with several human cancers. However, the mechanisms of TUG1-involved regulation remain largely unknown., Methods: We examined the expressions of TUG1 in a cohort of 89 patients with non-small cell lung cancer (NSCLC) to determine the association between TUG1 expression and clinical parameters. We used circular chromosome conformation capture (4C) coupled with next-generation sequencing to explore the genome regions that interact with TUG1 and the TUG1-mediated regulation., Results: TUG1 was significantly downregulated, and the TUG1 downregulation correlated with sex (p = 0.006), smoking status (p = 0.016), and tumor differentiation grade (p = 0.001). Knockdown of TUG1 significantly promoted the proliferation of NSCLC cells. According to the bioinformatic analysis result of TUG1 4C sequencing data, 83 candidate genes and their interaction regions were identified. Among these candidate genes, CUGBP and Elav-like family member 1 (CELF1) are potential targets of TUG1 in-trans regulation. To confirm the interaction between TUG1 and CELF1, relative expressions of CELF1 were examined in TUG1 knockdown H520 cells; results showed that CELF1 was significantly upregulated in TUG1 knockdown H520 cells. RNA immunoprecipitation was then performed to examine whether TUG1 RNA was bound to PRC2, a TUG1-involved regulation mechanism reported in previous studies. The results demonstrated that TUG1 RNA was bound to enhancer of zeste protein 2/embryonic ectoderm development (EZH2/EED), which is essential for PRC2. Finally, our designed ChIP assay revealed that the EZH2/EED was bound to the promotor region of CELF1 within 992 bp upstream of the transcript start site., Conclusion: TUG1 is downregulated in NSCLC. Using TUG1 4C sequencing and bioinformatic analysis, we found CELF1 to be a potential target of TUG1 RNA in in-trans regulation. Moreover, subsequent experiments showed that TUG1 RNA could bind to PRC2 in the promotor region of CELF1 and negatively regulate CELF1 expressions in H520 cells. Our results may facilitate developing new treatment modalities targeting TUG1/PRC2/CELF1 interactions in patients with NSCLC.

Background: CUG-BP, Elav-like family member 1 (CELF1) is a multifunctional RNA binding protein found in a variety of adult and embryonic tissues. In the heart, CELF1 is found exclusively in the myocardium. However, the roles of CELF1 during cardiac development have not been completely elucidated., Results: Myofibrillar organization is disrupted and proliferation is reduced following knockdown of CELF1 in cultured chicken primary embryonic cardiomyocytes. In vivo knockdown of Celf1 in developing Xenopus laevis embryos resulted in myofibrillar disorganization and a trend toward reduced proliferation in heart muscle, indicating conserved roles for CELF1 orthologs in embryonic cardiomyocytes. Loss of Celf1 also resulted in morphogenetic abnormalities in the developing heart and gut. Using optical coherence tomography, we showed that cardiac contraction was impaired following depletion of Celf1, while heart rhythm remained unperturbed. In contrast to cardiac muscle, loss of Celf1 did not disrupt myofibril organization in skeletal muscle cells, although it did lead to fragmentation of skeletal muscle bundles., Conclusions: CELF1 is required for normal myofibril organization, proliferation, morphogenesis, and contractile performance in the developing myocardium. Developmental Dynamics 245:854-873, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Objective: Human macrophages can shift phenotype across the inflammatory M1 and reparative M2 spectrum in response to environmental challenges, but the mechanisms promoting inflammatory and cardiometabolic disease-associated M1 phenotypes remain incompletely understood. Alternative splicing (AS) is emerging as an important regulator of cellular function, yet its role in macrophage activation is largely unknown. We investigated the extent to which AS occurs in M1 activation within the cardiometabolic disease context and validated a functional genomic cell model for studying human macrophage-related AS events., Approach and Results: From deep RNA-sequencing of resting, M1, and M2 primary human monocyte-derived macrophages, we found 3860 differentially expressed genes in M1 activation and detected 233 M1-induced AS events; the majority of AS events were cell- and M1-specific with enrichment for pathways relevant to macrophage inflammation. Using genetic variant data for 10 cardiometabolic traits, we identified 28 trait-associated variants within the genomic loci of 21 alternatively spliced genes and 15 variants within 7 differentially expressed regulatory splicing factors in M1 activation. Knockdown of 1 such splicing factor, CELF1, in primary human macrophages led to increased inflammatory response to M1 stimulation, demonstrating CELF1's potential modulation of the M1 phenotype. Finally, we demonstrated that an induced pluripotent stem cell-derived macrophage system recapitulates M1-associated AS events and provides a high-fidelity macrophage AS model., Conclusions: AS plays a role in defining macrophage phenotype in a cell- and stimulus-specific fashion. Alternatively spliced genes and splicing factors with trait-associated variants may reveal novel pathways and targets in cardiometabolic diseases., (© 2016 American Heart Association, Inc.)

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions., (Copyright © 2016 the American Physiological Society.)

The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention.