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1.
Int J Mol Sci ; 24(21)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37958557

RESUMO

In this study, we report a novel splice variant in the TRA2B gene identified in a patient presenting with seizures and neurodevelopmental delay. This paper represents the second investigation of pathogenic variants in the TRA2B gene in humans, reaffirming the conclusions of the initial study and underscoring the importance of this research. Comprehensive genetic testing, including whole genome sequencing, Sanger sequencing, and mRNA analysis, was performed on the proband and her parents. The proband harbored a de novo c.170+1G>A variant in the RS1 domain of Tra2ß, which was confirmed to be pathogenic through mRNA analysis, resulting in exon 2 deletion and a frameshift (p.Glu13Valfs*2). The clinical presentation of the patient was consistent with phenotypes described in one of the previous studies. These findings contribute to the dissemination and reinforcement of prior discoveries in the context of TRA2B-related syndrome and highlight the need for further investigation into the functional consequences and underlying pathogenic mechanisms associated with TRA2B mutations.


Assuntos
Mutação da Fase de Leitura , Malformações do Sistema Nervoso , Humanos , Feminino , Mutação , Éxons/genética , RNA Mensageiro/genética , Malformações do Sistema Nervoso/genética , Convulsões/genética , Fatores de Processamento de Serina-Arginina/genética , Proteínas do Tecido Nervoso/genética
2.
Int J Mol Sci ; 24(18)2023 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-37762628

RESUMO

Type 2 diabetes mellitus is a chronic metabolic disease with no cure. Adipose tissue is a major site of systemic insulin resistance. Sortilin is a central component of the glucose transporter -Glut4 storage vesicles (GSV) which translocate to the plasma membrane to uptake glucose from circulation. Here, using human adipocytes we demonstrate the presence of the alternatively spliced, truncated sortilin variant (Sort_T) whose expression is significantly increased in diabetic adipose tissue. Artificial-intelligence-based modeling, molecular dynamics, intrinsically disordered region analysis, and co-immunoprecipitation demonstrated association of Sort_T with Glut4 and decreased glucose uptake in adipocytes. The results show that glucagon-like peptide-1 (GLP1) hormone decreases Sort_T. We deciphered the molecular mechanism underlying GLP1 regulation of alternative splicing of human sortilin. Using splicing minigenes and RNA-immunoprecipitation assays, the results show that GLP1 regulates Sort_T alternative splicing via the splice factor, TRA2B. We demonstrate that targeted antisense oligonucleotide morpholinos reduces Sort_T levels and improves glucose uptake in diabetic adipocytes. Thus, we demonstrate that GLP1 regulates alternative splicing of sortilin in human diabetic adipocytes.


Assuntos
Processamento Alternativo , Diabetes Mellitus Tipo 2 , Humanos , Adipócitos , Peptídeo 1 Semelhante ao Glucagon/genética , Glucose
3.
Oncol Res ; 31(5): 769-785, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37547760

RESUMO

Late-stage ovarian cancer (OC) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is unclear. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. In this study, we explored the molecular functions of a canonical RBP, Transformer 2ß homolog (TRA2B), in cancer cells. TRA2B knockdown in HeLa cells and subsequent whole-transcriptome RNA sequencing (RNA-seq) analysis revealed the TRA2B-regulated alternative splicing (AS) profile. We disrupted TRA2B expression in epithelial OC cells and performed a series of experiments to confirm the resulting effects on OC cell proliferation, apoptosis and invasion. TRA2B-regulated AS was tightly associated with the mitotic cell cycle, apoptosis and several cancer pathways. Moreover, the expression of hundreds of genes was regulated by TRA2B, and these genes were enriched in the functions of cell proliferation, cell adhesion and angiogenesis, which are related to the malignant phenotype of OC. By integrating the alternatively spliced and differentially expressed genes, we found that AS events and gene expression were regulated independently. We then explored and validated the oncogenic functions of TRA2B by knocking down its expression in OC cells. The high TRA2B expression was associated with poor prognosis in patients with OC. In ovarian tissue, TRA2B expression showed a gradual increasing trend with increasing malignancy. We demonstrated the important roles of TRA2B in ovarian neoplasia and aggressive OC behaviors and identified the underlying molecular mechanisms, facilitating the targeted treatment of OC.


Assuntos
Processamento Alternativo , Neoplasias Ovarianas , Feminino , Humanos , Processamento Alternativo/genética , Células HeLa , Neoplasias Ovarianas/genética , Ciclo Celular/genética , Genes Reguladores , Proliferação de Células/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética
4.
Genet Med ; 25(4): 100003, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36549593

RESUMO

PURPOSE: Transformer2 proteins (Tra2α and Tra2ß) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder. METHODS: A total of 12 individuals from 11 unrelated families who harbored predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA sequencing and western blot analyses of Tra2ß-1 and Tra2ß-3 isoforms from patient-derived cells were performed. Tra2ß1-GFP, Tra2ß3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells. RESULTS: All variants clustered in the 5' part of TRA2B, upstream of an alternative translation start site responsible for the expression of the noncanonical Tra2ß-3 isoform. All affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2ß-1 isoform, whereas they increased the expression of the Tra2ß-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2ß-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2ß-1. CONCLUSION: Predicted loss-of-function variants clustered in the 5' portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2ß protein.


Assuntos
Transtorno do Espectro Autista , Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Humanos , Processamento Alternativo , Proteínas de Ligação a RNA/genética , Células HEK293 , Isoformas de Proteínas/genética , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
5.
Environ Toxicol Pharmacol ; 94: 103921, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35764259

RESUMO

Environmentally relevant (100 nM) inorganic arsenic (iAs) exposure displaces zinc from zinc fingers of upstream splice regulator ZRANB2 disrupting the splicing of its target TRA2B. Excess zinc displaced iAs from ZRANB2 zinc fingers in cell free system. Thus, the hypothesis that zinc supplementation could prevent iAs-mediated disruption of ZRANB2 splice function in human keratinocytes was tested. The data show that zinc supplementation prevented iAs-induced dysregulation of TRA2B splicing by ZRANB2 as well as the induction of ZRANB2 protein expression. These results provide additional support for the hypothesis that zinc supplementation could prevent iAs-mediated disease in iAs-exposed populations.


Assuntos
Arsênio , Arsênio/metabolismo , Arsênio/toxicidade , Suplementos Nutricionais , Humanos , Proteínas de Ligação a RNA/metabolismo , Zinco/metabolismo , Zinco/farmacologia
6.
Mol Ther Nucleic Acids ; 27: 304-318, 2022 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-35024243

RESUMO

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.

7.
J Bone Oncol ; 29: 100372, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34258182

RESUMO

Mesenchymal stem cells (MSCs) are multipotent stem cells with significant potential for regenerative medicine. The tumorigenesis of osteosarcoma is an intricate system and MSCs act as an indispensable part of this, interacting with the tumor microenvironment (TME) during the process. MSCs link to cells by acting on each component in the TME via autocrine or paracrine extracellular vesicles for cellular communication. Because of their unique characteristics, MSCs can be modified and processed into good biological carriers, loaded with drugs, and transfected with anticancer genes for the targeted treatment of osteosarcoma. Previous high-quality reviews have described the biological characteristics of MSCs; this review will discuss the effects of MSCs on the components of the TME and cellular communication and the prospects for clinical applications of MSCs.

8.
RNA ; 23(3): 378-394, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27940503

RESUMO

Nonsense-mediated RNA decay (NMD) selectively degrades mutated and aberrantly processed transcripts that contain premature termination codons (PTC). Cellular NMD activity is typically assessed using exogenous PTC-containing reporters. We overcame some inherently problematic aspects of assaying endogenous targets and developed a broadly applicable strategy to reliably and easily monitor changes in cellular NMD activity. Our new method was genetically validated for distinguishing NMD regulation from transcriptional control and alternative splicing regulation, and unexpectedly disclosed a different sensitivity of NMD targets to NMD inhibition. Applying this robust method for screening, we identified NMD-inhibiting stressors but also found that NMD inactivation was not universal to cellular stresses. The high sensitivity and broad dynamic range of our method revealed a strong correlation between NMD inhibition, endoplasmic reticulum (ER) stress, and polysome disassembly upon thapsigargin treatment in a temporal and dose-dependent manner. We found little evidence of calcium signaling mediating thapsigargin-induced NMD inhibition. Instead, we discovered that of the three unfolded protein response (UPR) pathways activated by thapsigargin, mainly protein kinase RNA-like endoplasmic reticulum kinase (PERK) was required for NMD inhibition. Finally, we showed that ER stress compounded TDP-43 depletion in the up-regulation of NMD isoforms that had been implicated in the pathogenic mechanisms of amyotrophic lateral sclerosis and frontotemporal dementia, and that the additive effect of ER stress was completely blocked by PERK deficiency.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Tapsigargina/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Códon sem Sentido , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteína 4 Homóloga a Disks-Large , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ensaios de Triagem em Larga Escala , Indóis/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Ligação a RNA , Transdução de Sinais , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
9.
J Biol Chem ; 289(46): 31662-31672, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25261467

RESUMO

Obesity is characterized by adipocyte hyperplasia and hypertrophy. We previously showed that PKCδ expression is dysregulated in obesity (Carter, G., Apostolatos, A., Patel, R., Mathur, A., Cooper, D., Murr, M., and Patel, N. A. (2013) ISRN Obes. 2013, 161345). Using 3T3L1 preadipocytes, we studied adipogenesis in vitro and showed that expression of PKCδ splice variants, PKCδI and PKCδII, have different expression patterns during adipogenesis (Patel, R., Apostolatos, A., Carter, G., Ajmo, J., Gali, M., Cooper, D. R., You, M., Bisht, K. S., and Patel, N. A. (2013) J. Biol. Chem. 288, 26834-26846). Here, we evaluated the role of PKCδI splice variant during adipogenesis. Our results indicate that PKCδI expression level is high in preadipocytes and decreasing PKCδI accelerated terminal differentiation. Our results indicate that PKCδI is required for mitotic clonal expansion of preadipocytes. We next evaluated the splice factor regulating the expression of PKCδI during 3T3L1 adipogenesis. Our results show TRA2B increased PKCδI expression. To investigate the molecular mechanism, we cloned a heterologous splicing PKCδ minigene and showed that inclusion of PKCδ exon 9 is increased by TRA2B. Using mutagenesis and a RNA-immunoprecipitation assay, we evaluated the binding of Tra2ß on PKCδI exon 9 and show that its association is required for PKCδI splicing. These results provide a better understanding of the role of PKCδI in adipogenesis. Determination of this molecular mechanism of alternative splicing presents a novel therapeutic target in the management of obesity and its co-morbidities.


Assuntos
Ciclo Celular , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteína Quinase C-delta/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipogenia , Processamento Alternativo , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Camundongos , Mutação , Fatores de Processamento de Serina-Arginina
10.
RNA Biol ; 11(4): 351-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24658338

RESUMO

BRCA1 exon 11 is one of the biggest human exons, spanning 3426 bases. This gene is potentially involved in DNA repair as well as cell growth and cell cycle control. Exon 11 is regulated at the splicing level producing three main different combinations of BRCA1 mature transcripts; one including the whole of exon 11 (full isoform), one skipping the entire exon (D11 isoform), and one including only 117 base pairs of exon 11 (D11q isoform). Using minigene and deletion analyses, we have previously described important splicing regulatory sequences located at the beginning of this exon (5' end). We have now found additional important sequences located at its 3' end. In particular, we describe the presence of a strong splicing enhancer adjacent to the downstream 5' splice site, which minimizes competition from an upstream 5' splice site and so ensures long exon inclusion. Analyses of the proteins binding these RNA sequences have revealed that Tra2beta and hnRNP L are involved in the regulation of BRCA1 exon 11 by influencing the recognition of donor sites. Interestingly, BRCA1 exon 11 carrying deletion of the regulatory sequences bound by these factors also showed unexpected responses to up- or downregulation of these regulatory proteins, suggesting that they can also bind elsewhere in this large exon and elicit different effects on its recognition.   The identification of sequences and proteins relevant for the regulation of BRCA1 exon 11 now provides better knowledge on how this exon is recognized and may represent an important step toward understanding how large exons are regulated.


Assuntos
Éxons , Regulação da Expressão Gênica , Genes BRCA1 , Splicing de RNA , Processamento Alternativo , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Humanos , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Isoformas de RNA , Sítios de Splice de RNA , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , Fatores de Processamento de Serina-Arginina
11.
J Comp Neurol ; 522(2): 372-92, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23818142

RESUMO

Alternative splicing of pre-mRNAs can rapidly regulate the expression of large groups of proteins. The RNA binding protein TRA2B (SFRS10) plays well-established roles in developmentally regulated alternative splicing during Drosophila sexual differentiation. TRA2B is also essential for mammalian embryogenesis and is implicated in numerous human diseases. Precise regulation of alternative splicing is critical to the development and function of the central nervous system; however, the requirements for specific splicing factors in neurogenesis are poorly understood. This study focuses on the role of TRA2B in mammalian brain development. We show that, during murine cortical neurogenesis, TRA2B is expressed in both neural progenitors and cortical projection neurons. Using cortex-specific Tra2b mutant mice, we show that TRA2B depletion results in apoptosis of the neural progenitor cells as well as disorganization of the cortical plate. Thus, TRA2B is essential for proper development of the cerebral cortex.


Assuntos
Córtex Cerebral/embriologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Córtex Cerebral/metabolismo , Imunoprecipitação , Hibridização In Situ , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Fatores de Processamento de Serina-Arginina
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