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1.
Cell Stem Cell ; 31(5): 754-771.e6, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38701759

RESUMO

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.


Assuntos
Processamento Alternativo , Diferenciação Celular , Cromatina , Ribonucleoproteínas Nucleares Heterogêneas , Neurônios , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Fatores de Transcrição , Processamento Alternativo/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Transcrição Gênica , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Éxons/genética , Humanos , Autorrenovação Celular/genética
2.
Front Immunol ; 15: 1375168, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38690287

RESUMO

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma. The HTLV-1 Tax constitutively activates nuclear factor-κB (NF-κB) to promote the survival and transformation of HTLV-1-infected T cells. Despite extensive study of Tax, how Tax interacts with host factors to regulate NF-κB activation and HTLV-1-driven cell proliferation is not entirely clear. Here, we showed that overexpression of Poly (rC)-binding protein 1 (PCBP1) promoted Tax-mediated IκB kinase (IKK)-NF-κB signaling activation, whereas knockdown of PCBP1 attenuated Tax-dependent IKK-NF-κB activation. However, Tax activation of HTLV-1 long terminal repeat was unaffected by PCBP1. Furthermore, depletion of PCBP1 led to apoptosis and reduced proliferation of HTLV-1-transformed cells. Mechanistically, PCBP1 interacted and co-localized with Tax in the cytoplasm, and PCBP1 KH3 domain was indispensable for the interaction between PCBP1 and Tax. Moreover, PCBP1 facilitated the assembly of Tax/IKK complex. Collectively, our results demonstrated that PCBP1 may exert an essential effect in Tax/IKK complex combination and subsequent NF-κB activation, which provides a novel insight into the pathogenetic mechanisms of HTLV-1.


Assuntos
Proteínas de Ligação a DNA , Produtos do Gene tax , Ribonucleoproteínas Nucleares Heterogêneas , Vírus Linfotrópico T Tipo 1 Humano , NF-kappa B , Proteínas de Ligação a RNA , Humanos , Produtos do Gene tax/metabolismo , NF-kappa B/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Transdução de Sinais , Células HEK293 , Ligação Proteica , Proliferação de Células , Infecções por HTLV-I/metabolismo , Infecções por HTLV-I/virologia , Apoptose , Quinase I-kappa B/metabolismo , Interações Hospedeiro-Patógeno
3.
Folia Histochem Cytobiol ; 62(1): 25-36, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38563050

RESUMO

INTRODUCTION: Endometriosis (EMs), manifested by pain and infertility, is a chronic inflammatory disease. The precise pathophysiology of this disease remains uncertain. Insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) and polypyrimidine tract-binding protein 1 (PTBP1) have both been found to regulate proliferation, apoptosis, and invasion. This study aimed to investigate the effects of IGF2BP1/PTBP1 in treating EMs. MATERIALS AND METHODS: qRT-PCR and western blotting were employed to quantify IGF2BP1 and PTBP1 expression in six patients with EMs (mean age 33.83 years). The correlation analysis, STRING database prediction, and RNA immunoprecipitation were utilized to identify the relationship between IGF2BP1 and PTBP1. Ectopic endometrial volume, weight, HE staining, and IGF2BP1 silencing were utilized to estimate the effects of IGF2BP1 in EMs model rats. qRT-PCR, CCK-8, 5-ethynyl-2'-deoxyuridine (EDU) labeling, Transwell assay, and flow cytometry were utilized to assess the effects of IGF2BP1/PTBP1 on the proliferation, migration, invasion, and apoptosis of ectopic endometrial stromal cells (eESCs). Furthermore, western blotting was employed to evaluate expressions of PCNA, VEGF, and E-cadherin in EMs rats and eESCs. RESULTS: The mRNA and protein levels of IGF2BP1 and PTBP1 in the ectopic and eutopic endometrium of EMs patients were significantly increased. RNA immunoprecipitation revealed a close interaction of IGF2BP1 with PTBP1. Additionally, the endometrial volume, weight, and histopathologic scores in rats were significantly reduced after IGF2BP1 silencing. IGF2BP1 silencing also decreased the expression of PCNA and VEGF, and increased E-cadherin expression in endometrial tissues of EMs rats. Moreover, IGF2BP1 silencing inhibited proliferation, migration, and invasion and promoted apoptosis through PTBP1 in eESCs. CONCLUSIONS: IGF2BP1 exhibits potential beneficial properties in the management of EMs by interacting with PTBP1, thereby highlighting IGF2BP1 as a promising therapeutic target for EMs.


Assuntos
Endometriose , Adulto , Animais , Feminino , Humanos , Ratos , Caderinas/metabolismo , Proliferação de Células , Endometriose/patologia , Endométrio/patologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Elife ; 132024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38597390

RESUMO

Alternative RNA splicing is an essential and dynamic process in neuronal differentiation and synapse maturation, and dysregulation of this process has been associated with neurodegenerative diseases. Recent studies have revealed the importance of RNA-binding proteins in the regulation of neuronal splicing programs. However, the molecular mechanisms involved in the control of these splicing regulators are still unclear. Here, we show that KIS, a kinase upregulated in the developmental brain, imposes a genome-wide alteration in exon usage during neuronal differentiation in mice. KIS contains a protein-recognition domain common to spliceosomal components and phosphorylates PTBP2, counteracting the role of this splicing factor in exon exclusion. At the molecular level, phosphorylation of unstructured domains within PTBP2 causes its dissociation from two co-regulators, Matrin3 and hnRNPM, and hinders the RNA-binding capability of the complex. Furthermore, KIS and PTBP2 display strong and opposing functional interactions in synaptic spine emergence and maturation. Taken together, our data uncover a post-translational control of splicing regulators that link transcriptional and alternative exon usage programs in neuronal development.


Assuntos
Processamento Alternativo , Éxons , Neurônios , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas Serina-Treonina Quinases , Animais , Humanos , Camundongos , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosforilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
5.
Cancer Commun (Lond) ; 44(4): 469-490, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38512764

RESUMO

BACKGROUND: Chemoresistance is a major cause of treatment failure in gastric cancer (GC). Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is an N6-methyladenosine (m6A)-binding protein involved in a variety of cancers. However, whether m6A modification and hnRNPA2B1 play a role in GC chemoresistance is largely unknown. In this study, we aimed to investigate the role of hnRNPA2B1 and the downstream mechanism in GC chemoresistance. METHODS: The expression of hnRNPA2B1 among public datasets were analyzed and validated by quantitative PCR (qPCR), Western blotting, immunofluorescence, and immunohistochemical staining. The biological functions of hnRNPA2B1 in GC chemoresistance were investigated both in vitro and in vivo. RNA sequencing, methylated RNA immunoprecipitation, RNA immunoprecipitation, and RNA stability assay were performed to assess the association between hnRNPA2B1 and the binding RNA. The role of hnRNPA2B1 in maintenance of GC stemness was evaluated by bioinformatic analysis, qPCR, Western blotting, immunofluorescence, and sphere formation assays. The expression patterns of hnRNPA2B1 and downstream regulators in GC specimens from patients who received adjuvant chemotherapy were analyzed by RNAscope and multiplex immunohistochemistry. RESULTS: Elevated expression of hnRNPA2B1 was found in GC cells and tissues, especially in multidrug-resistant (MDR) GC cell lines. The expression of hnRNPA2B1 was associated with poor outcomes of GC patients, especially in those who received 5-fluorouracil treatment. Silencing hnRNPA2B1 effectively sensitized GC cells to chemotherapy by inhibiting cell proliferation and inducing apoptosis both in vitro and in vivo. Mechanically, hnRNPA2B1 interacted with and stabilized long noncoding RNA NEAT1 in an m6A-dependent manner. Furthermore, hnRNPA2B1 and NEAT1 worked together to enhance the stemness properties of GC cells via Wnt/ß-catenin signaling pathway. In clinical specimens from GC patients subjected to chemotherapy, the expression levels of hnRNPA2B1, NEAT1, CD133, and CD44 were markedly elevated in non-responders compared with responders. CONCLUSION: Our findings indicated that hnRNPA2B1 interacts with and stabilizes lncRNA NEAT1, which contribute to the maintenance of stemness property via Wnt/ß-catenin pathway and exacerbate chemoresistance in GC.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ribonucleoproteínas Nucleares Heterogêneas , RNA Longo não Codificante , Neoplasias Gástricas , Humanos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , RNA Longo não Codificante/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
6.
Biol Cell ; 116(5): e2300128, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38538536

RESUMO

BACKGROUND INFORMATION: The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. RESULTS: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. CONCLUSIONS AND SIGNIFICANCE: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.


Assuntos
Dano ao DNA , Fosfatase 3 de Especificidade Dupla , RNA Mensageiro , Humanos , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fosfatase 3 de Especificidade Dupla/metabolismo , Fosfatase 3 de Especificidade Dupla/genética , Biossíntese de Proteínas , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Células HeLa
7.
Neurobiol Dis ; 193: 106454, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38408684

RESUMO

Axonal mitochondria defects are early events in the pathogenesis of motoneuron disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. The RNA-binding protein hnRNP R interacts with different motoneuron disease-related proteins such as SMN and TDP-43 and has important roles in axons of motoneurons, including axonal mRNA transport. However, whether hnRNP R also modulates axonal mitochondria is currently unknown. Here, we show that axonal mitochondria exhibit altered function and motility in hnRNP R-deficient motoneurons. Motoneurons lacking hnRNP R show decreased anterograde and increased retrograde transport of mitochondria in axons. Furthermore, hnRNP R-deficiency leads to mitochondrial hyperpolarization, caused by decreased complex I and reversed complex V activity within the respiratory chain. Taken together, our data indicate a role for hnRNP R in regulating transport and maintaining functionality of axonal mitochondria in motoneurons.


Assuntos
Axônios , Neurônios Motores , Potenciais da Membrana , Neurônios Motores/metabolismo , Axônios/patologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Mitocôndrias/metabolismo
8.
Biochim Biophys Acta Mol Basis Dis ; 1870(3): 167050, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38331110

RESUMO

HNRNPA2B1 and HNRNPR stabilize ASCL1 mRNA in neuroblastoma, but whether their regulatory effects depend on m6A modification and whether their function involves ASCL1 remain unknown. This study investigated the m6A-dependent binding of HNRNPA2B1 and HNRNPR to ASCL1 and subsequent regulation, as well as the expression, clinical significance, and function of HNRNPA2B1 and HNRNPR in neuroblastoma. We revealed that METTL14 mediated ASCL1 m6A modification to stabilize ASCL1. HNRNPA2B1 and HNRNPR significantly enriched ASCL1 mRNA by binding to the 5' and 3' untranslated regions, respectively, and METTL14 knockdown reduced this enrichment. Mutations in m6A sites in the untranslated regions of ASCL1 mRNA considerably decreased probe capacity to engage HNRNPA2B1 and HNRNPR. HNRNPR interacts with IGF2BP1, and knocking down either impaired binding to ASCL1 mRNA. HNRNPA2B1 and HNRNPR knockdown suppressed neuroblastoma cell growth and invasion, while ASCL1 overexpression restored these effects. The high HNRNPA2B1 and HNRNPR expression in neuroblastoma correlated with ASCL1 expression. Thus, HNRNPA2B1 and HNRNPR bind and stabilize ASCL1 mRNA in an m6A-dependent manner to promote neuroblastoma progression. This study not only discovered a new mechanism underlying the high ASCL1 expression in neuroblastoma but also identified the HNRNPA2B1/HNRNPR/ASCL1 axis as a promising target for inhibiting neuroblastoma progression.


Assuntos
Adenina/análogos & derivados , Neuroblastoma , Humanos , Neuroblastoma/genética , Regiões 3' não Traduzidas , RNA Mensageiro/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ribonucleoproteínas Nucleares Heterogêneas
9.
J Neuropathol Exp Neurol ; 83(4): 276-288, 2024 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-38324733

RESUMO

Cerebral infarction (CI) is one of the leading causes of disability and death. LncRNAs are key factors in CI progression. Herein, we studied the function of long noncoding RNA KCNQ1OT1 in CI patient plasma samples and in CI models. Quantitative real-time PCR and Western blotting tested gene and protein expressions. The interactions of KCNQ1OT1/PTBP1 and miR-16-5p were analyzed using dual-luciferase reporter and RNA immunoprecipitation assays; MTT assays measured cell viability. Cell migration and angiogenesis were tested by wound healing and tube formation assays. Pathological changes were analyzed by triphenyltetrazolium chloride and routine staining. We found that KCNQ1OT1 and PTBP1 were overexpressed and miR-16-5p was downregulated in CI patient plasma and in oxygen-glucose deprived (OGD) induced mouse brain microvascular endothelial (bEnd.3) cells. KCNQ1OT1 knockdown suppressed pro-inflammatory cytokine production and stimulated angiogenic responses in OGD-bEnd.3 cells. KCNQ1OT1 upregulated PTBP1 by sponging miR-16-5p. PTBP1 overexpression or miR-16-5p inhibition attenuated the effects of KCNQ1OT1 knockdown. PTBP1 silencing protected against OGD-bEnd.3 cell injury by enhancing SIRT1. KCNQ1OT1 silencing or miR-16-5p overexpression also alleviated ischemic injury in a mice middle cerebral artery occlusion model. Thus, KCNQ1OT1 silencing alleviates CI by regulating the miR-16-5p/PTBP1/SIRT1 pathway, providing a theoretical basis for novel therapeutic strategies targeting CI.


Assuntos
MicroRNAs , RNA Longo não Codificante , Animais , Humanos , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células Endoteliais/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Apoptose/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Oxigênio , Ribonucleoproteínas Nucleares Heterogêneas , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética
10.
J Biol Chem ; 300(3): 105733, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38336291

RESUMO

RNA Binding Proteins regulate, in part, alternative pre-mRNA splicing and, in turn, gene expression patterns. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins sharing 74% amino acid sequence identity. Both proteins contain four structured RNA-recognition motifs (RRMs) connected by linker regions and an N-terminal region. Despite their similarities, the paralogs have distinct tissue-specific expression patterns and can regulate discrete sets of target exons. How two highly structurally similar proteins can exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1 signifying a role for these regions in dictating the paralog's distinct splicing activities. To this end, we conducted bioinformatics analysis to determine the evolutionary conservation of RRMs versus linker regions in PTBP1 and PTBP2 across species. To determine the role of PTBP2 unstructured regions in splicing activity, we created hybrid PTBP1-PTBP2 constructs that had counterpart PTBP1 regions swapped to an otherwise PTBP2 protein and assayed on differentially regulated exons. We also conducted molecular dynamics studies to investigate how negative charges introduced by phosphorylation in PTBP2 unstructured regions can alter their physical properties. Collectively, results from our studies reveal an important role for PTBP2 unstructured regions and suggest a role for phosphorylation in the differential splicing activities of the paralogs on certain regulated exons.


Assuntos
Processamento Alternativo , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Vertebrados , Animais , Humanos , Camundongos , Ratos , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Fosforilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Especificidade da Espécie , Vertebrados/genética , Galinhas/genética
11.
Nan Fang Yi Ke Da Xue Xue Bao ; 44(1): 9-16, 2024 Jan 20.
Artigo em Chinês | MEDLINE | ID: mdl-38293971

RESUMO

OBJECTIVE: To investigate the role of RNA-binding motif protein X-linked (RBMX) in regulating the proliferation, migration, invasion and glycolysis in human bladder cancer cells. METHODS: A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown, respectively, and successful cell modeling was verified using RT-qPCR and Western blotting. Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay, and cell migration and invasion abilities were determined using Transwell experiment. The expressions of glycolysis-related proteins M1 pyruvate kinase (PKM1) and M2 pyruvate kinase (PKM2) were detected using Western blotting. The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits. RESULTS: RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown. RBMX overexpression significantly inhibited the proliferation, clone formation, migration and invasion of bladder cancer cells, while RBMX knockdown produced the opposite effects. Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2, while RBMX knockdown produced the opposite effects. Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression (P < 0.05) but increased significantly following RBMX knockdown (P < 0.05). CONCLUSION: RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression, suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.


Assuntos
Piruvato Quinase , Neoplasias da Bexiga Urinária , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Glucose/farmacologia , Glicólise , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Neoplasias da Bexiga Urinária/genética
12.
Biochem Soc Trans ; 52(1): 111-122, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38174726

RESUMO

Cells encounter a variety of stresses throughout their lifetimes. Oxidative stress can occur via a myriad of factors, including exposure to chemical toxins or UV light. Importantly, these stressors induce chemical changes (e.g. chemical modifications) to biomolecules, such as RNA. Commonly, guanine is oxidized to form 8-oxo-7,8-hydroxyguanine (8-oxoG) and this modification can disrupt a plethora of cellular processes including messenger RNA translation and stability. Polynucleotide phosphorylase (PNPase), heterogeneous nuclear ribonucleoprotein D (HNRPD/Auf1), poly(C)-binding protein (PCBP1/HNRNP E1), and Y-box binding protein 1 (YB-1) have been identified as four RNA-binding proteins that preferentially bind 8-oxoG-modified RNA over unmodified RNA. All four proteins are native to humans and PNPase is additionally found in bacteria. Additionally, under oxidative stress, cell survival declines in mutants that lack PNPase, Auf1, or PCBP1, suggesting they are critical to the oxidative stress response. This mini-review captures the current understanding of the PNPase, HNRPD/Auf1, PCBP1, and YB-1 proteins and the mechanism that has been outlined so far by which they recognize and interact with 8-oxoG-modified RNAs.


Assuntos
Proteínas de Ligação a RNA , RNA , Humanos , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica
13.
Mol Cancer ; 23(1): 4, 2024 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-38184608

RESUMO

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown. METHODS: High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis. RESULTS: In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC. CONCLUSIONS: Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , MicroRNAs , Humanos , Carcinoma de Células Renais/genética , Processamento Alternativo , RNA Circular/genética , MicroRNAs/genética , Neoplasias Renais/genética , Ribonucleoproteínas Nucleares Heterogêneas , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Citocromo P-450 CYP1B1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética
14.
Virology ; 592: 109986, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38290414

RESUMO

The large amount of viral RNA produced during infections has the potential to interact with and effectively sequester cellular RNA binding proteins, thereby influencing aspects of post-transcriptional gene regulation in the infected cell. Here we demonstrate that the abundant 5' leader RNA region of SARS-CoV-2 viral RNAs can interact with the cellular polypyrimidine tract binding protein (PTBP1). Interestingly, the effect of a knockdown of PTBP1 protein on cellular gene expression is also mimicked during SARS-CoV-2 infection, suggesting that this protein may be functionally sequestered by viral RNAs. Consistent with this model, the alternative splicing of mRNAs that is normally controlled by PTBP1 is dysregulated during SARS-CoV-2 infection. Collectively, these data suggest that the SARS-CoV-2 leader RNA sequesters the cellular PTBP1 protein during infection, resulting in significant impacts on the RNA biology of the host cell. These alterations in post-transcriptional gene regulation may play a role in SARS-CoV-2 mediated molecular pathogenesis.


Assuntos
COVID-19 , Ribonucleoproteínas Nucleares Heterogêneas , Proteína de Ligação a Regiões Ricas em Polipirimidinas , SARS-CoV-2 , Humanos , Processamento Alternativo , COVID-19/metabolismo , COVID-19/virologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , SARS-CoV-2/fisiologia
15.
BMC Vet Res ; 20(1): 25, 2024 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-38218813

RESUMO

BACKGROUND: Poly C Binding Protein 1 (PCBP1) belongs to the heterogeneous nuclear ribonucleoprotein family. It is a multifunctional protein that participates in several functional circuits and plays a variety of roles in cellular processes. Although PCBP1 has been identified in several mammals, its function in porcine was unclear. RESULTS: In this study, we cloned the gene of porcine PCBP1 and analyzed its evolutionary relationships among different species. We found porcine PCBP1 protein sequence was similar to that of other animals. The subcellular localization of PCBP1 in porcine kidney cells 15 (PK-15) cells was analyzed by immunofluorescence assay (IFA) and revealed that PCBP1 was mainly localized to the nucleus. Reverse transcription-quantitative PCR (RT-qPCR) was used to compare PCBP1 mRNA levels in different tissues of 30-day-old pigs. Results indicated that PCBP1 was expressed in various tissues and was most abundant in the liver. Finally, the effects of PCBP1 on cell cycle and apoptosis were investigated following its overexpression or knockdown in PK-15 cells. The findings demonstrated that PCBP1 knockdown arrested cell cycle in G0/G1 phase, and enhanced cell apoptosis. CONCLUSIONS: Porcine PCBP1 is a highly conserved protein, plays an important role in determining cell fate, and its functions need further study.


Assuntos
Proteínas de Transporte , Proteínas de Ligação a RNA , Suínos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Apoptose/fisiologia , Ligação Proteica , Mamíferos
16.
Artigo em Inglês | MEDLINE | ID: mdl-38281704

RESUMO

Alternative splicing (AS) plays an important role in various physiological processes in eukaryotes, such as the stress response. However, patterns of AS events remain largely unexplored during salinity acclimation in fishes. In this study, we conducted AS analysis using RNA-seq datasets to explore splicing patterns in the gill tissues of rainbow trout exposed to altered salinity environments, ranging from 0 ‰ (T0) to 30 ‰ (T30). The results revealed 1441, 351, 483, 1051 and 1049 differentially alternatively spliced (DAS) events in 5 pairwise comparisons, including T6 vs. T0, T12 vs. T0, T18 vs. T0, T24 vs. T0, and T30 vs. T0, respectively. These DAS events were derived from 1290, 328, 444, 963 and 948 genes. Enrichment analysis indicated that these DAS genes were related to RNA splicing and processing. Among these, 14 DAS genes were identified as members of the large heterogeneous nuclear RNP (hnRNP) gene family. Alternative 3' splice site (A3SS), exon skipping (SE) and intron retention (RI) events resulted in the fragmentation or even loss of the functional RNA recognition motif (RRM) domains in hnrnpa0, hnrnp1a, hnrnp1b and hnrnpc genes. The incomplete RRM domains would hinder the interactions between hnRNP genes and pre-mRNAs. It would in turn influence the splicing patterns and mRNA stability of downstream target genes in response to salinity changes. The study provides insights into salinity acclimation in gill tissues of rainbow trout and serves as a significant reference on the osmoregulation mechanisms at post-transcription regulation levels in fish.


Assuntos
Oncorhynchus mykiss , Animais , Oncorhynchus mykiss/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Salinidade , Processamento Alternativo , Brânquias/metabolismo
17.
Aging (Albany NY) ; 16(1): 226-245, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38214653

RESUMO

Cancer accounts for the highest rates of morbidity and mortality worldwide. RNA binding motif protein X-linked (RBMX) is a nuclear RNA-binding protein, associated with certain types of cancer by participating in the integration of sister chromatids and a combination of ribonucleoprotein complexes. However, the specific role of RBMX in cancer immunity remains unknown. This study presents the aberrant expression levels, single-cell distributions, effective prognostic roles, immune cell infiltration associations, and immunotherapy responses of RBMX as a biomarker in various types of cancer. Moreover, it validates the aberrant expression of RBMX in clinical cancer samples. Furthermore, we also evaluated the relationships between RBMX expression and myeloid-derived suppressor cells in clinical samples by immunofluorescent staining. The results showed that knockdown of RBMX can impair the proliferation, migration, and invasion of liver cancer cells. Finally, we indicated that RBMX may play an immunoregulatory role in cancer progression, affecting the therapeutic effects of immune checkpoint inhibitors in patients with cancer.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas , Neoplasias , Humanos , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Prognóstico , Imunoterapia , Neoplasias/genética , Neoplasias/terapia
18.
Yi Chuan ; 46(1): 46-62, 2024 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-38230456

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer accounting for 90% of cases. It is a highly invasive and deadly cancer with a gradual onset. Polypyrimidine tract-binding protein 1 (PTBP1) is an important RNA-binding protein involved in RNA metabolism and has been linked to oncogenic splicing events. While the oncogenic role of PTBP1 in HCC cells has been established, the exact mechanism of action remains unclear. This study aimed to investigate the functional connection between PTBP1 and dysregulated splicing events in HCC. Through immunoprecipitation-mass spectrometry analyses, we discovered that the proteins bound to PTBP1 were significantly enriched in the complex responsible for the alternative splicing of FGFR2 (fibroblast growth factor receptor 2). Further RNA immunoprecipitation and quantitative PCR assays confirmed that PTBP1 down-regulated the FGFR2-IIIb isoform levels and up-regulated the FGFR2-IIIc isoform levels in HCC cells, leading to a switch from FGFR2-IIIb to FGFR2-IIIc isoforms. Subsequent functional evaluations using CCK-8, transwell, and plate clone formation assays in HCC cell lines HepG2 and Huh7 demonstrated that FGFR2-IIIb exhibited tumor-suppressive effects, while FGFR2-IIIc displayed tumor-promoting effects. In conclusion, this study provides insights into the PTBP1-mediated alternative splicing mechanism in HCC progression, offering a new theoretical basis for the prevention and treatment of this malignancy. Mechanistically, the isoform switch from FGFR2-IIIb to FGFR2-IIIc promoted epithelial-mesenchymal transformation (EMT) of HCC cells and activated the FGFR cascades ERK and AKT pathways.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Isoformas de Proteínas/genética , Processamento Alternativo , RNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo
19.
Commun Biol ; 7(1): 130, 2024 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-38273088

RESUMO

Colorectal cancer is a grievous health concern, we have proved long non-coding RNA LINC00689 is considered as a potential diagnosis biomarker for colorectal cancer, and it is necessary to further investigate its upstream and downstream mechanisms. Here, we show that KLF15, a transcription factor, exhibits the reduced expression in colorectal cancer. KLF15 suppresses the proliferative and metastatic capacities of colorectal cancer cells both in vitro and in vivo by transcriptionally activating LINC00689. Subsequently, LINC00689 recruits PTBP1 protein to enhance the stability of LATS2 mRNA in the cytoplasm. This stabilization causes the suppression of the YAP1/ß-catenin pathway and its target downstream genes. Our findings highlight a regulatory network involving KLF15, LINC00689, PTBP1, LATS2, and the YAP1/ß-catenin pathway in colorectal cancer, shedding light on potential therapeutic targets for colorectal cancer therapy.


Assuntos
Neoplasias Colorretais , beta Catenina , Humanos , beta Catenina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo
20.
Biochim Biophys Acta Mol Basis Dis ; 1870(3): 167011, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38176460

RESUMO

Tamoxifen (TAM) is the primary drug for treating estrogen receptor alpha-positive (ER+) breast cancer (BC). However, resistance to TAM can develop in some patients, limiting its therapeutic efficacy. The ubiquitin-specific protease (USP) family has been associated with the development, progression, and drug resistance of various cancers. To explore the role of USPs in TAM resistance in BC, we used qRT-PCR to compare USP expression between TAM-sensitive (MCF-7 and T47D) and TAM-resistant cells (MCF-7R and T47DR). We then modulated USP46 expression and examined its impact on cell proliferation, drug resistance (via CCK-8 and EdU experiments), glycolysis levels (using a glycolysis detection assay), protein interactions (confirmed by co-IP), and protein changes (analyzed through Western blotting). Our findings revealed that USP46 was significantly overexpressed in TAM-resistant BC cells, leading to the inhibition of the ubiquitin degradation of polypyrimidine tract-binding protein 1 (PTBP1). Overexpression of PTBP1 increased the PKM2/PKM1 ratio, promoted glycolysis, and intensified TAM resistance in BC cells. Knockdown of USP46 induced downregulation of PTBP1 protein by promoting its K48-linked ubiquitination, resulting in a decreased PKM2/PKM1 ratio, reduced glycolysis, and heightened TAM sensitivity in BC cells. In conclusion, this study highlights the critical role of the USP46/PTBP1/PKM2 axis in TAM resistance in BC. Targeted therapy against USP46 may represent a promising strategy to improve the prognosis of TAM-resistant patients.


Assuntos
Neoplasias da Mama , Tamoxifeno , Humanos , Feminino , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células MCF-7 , Resistencia a Medicamentos Antineoplásicos/genética , Glicólise , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo
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