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1.
Nat Commun ; 12(1): 4961, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400640

RESUMO

Esophageal cancer (EC) is a type of aggressive cancer without clinically relevant molecular subtypes, hindering the development of effective strategies for treatment. To define molecular subtypes of EC, we perform mass spectrometry-based proteomic and phosphoproteomics profiling of EC tumors and adjacent non-tumor tissues, revealing a catalog of proteins and phosphosites that are dysregulated in ECs. The EC cohort is stratified into two molecular subtypes-S1 and S2-based on proteomic analysis, with the S2 subtype characterized by the upregulation of spliceosomal and ribosomal proteins, and being more aggressive. Moreover, we identify a subtype signature composed of ELOA and SCAF4, and construct a subtype diagnostic and prognostic model. Potential drugs are predicted for treating patients of S2 subtype, and three candidate drugs are validated to inhibit EC. Taken together, our proteomic analysis define molecular subtypes of EC, thus providing a potential therapeutic outlook for improving disease outcomes in patients with EC.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Espectrometria de Massas/métodos , Proteômica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Estudos de Coortes , Elonguina/genética , Elonguina/metabolismo , Humanos , Prognóstico , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
2.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445242

RESUMO

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced ß-catenin protein, but not mRNA levels. The reduction of ß-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.


Assuntos
Proliferação de Células , Fibroblastos/metabolismo , Pulmão/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Linhagem Celular , Humanos , RNA Longo não Codificante/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , beta Catenina/genética
3.
Elife ; 102021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34338635

RESUMO

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus, and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.


Assuntos
Cílios/metabolismo , Citoplasma/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Animais , Núcleo Celular/metabolismo , Masculino , Camundongos , Fatores de Processamento de Serina-Arginina/metabolismo
4.
Nat Commun ; 12(1): 4507, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301951

RESUMO

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.


Assuntos
Processamento Alternativo/genética , Disautonomia Familiar/genética , Mutação , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/efeitos dos fármacos , Animais , Células Cultivadas , Modelos Animais de Doenças , Disautonomia Familiar/tratamento farmacológico , Disautonomia Familiar/metabolismo , Elementos Facilitadores Genéticos/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Camundongos Transgênicos , Estrutura Molecular , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Sítios de Splice de RNA/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Elongação da Transcrição/metabolismo
5.
Nucleic Acids Res ; 49(12): 7103-7121, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34161584

RESUMO

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a 3D structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable 3D structural scaffolds to the splicing code across the mammalian transcriptome.


Assuntos
Precursores de RNA/química , Splicing de RNA , RNA Mensageiro/química , Células HeLa , Humanos , Íntrons , Mutação , Conformação de Ácido Nucleico , Domínios Proteicos , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Fatores de Processamento de Serina-Arginina/química , Fatores de Processamento de Serina-Arginina/metabolismo , Fator de Processamento U2AF/metabolismo
6.
Nucleic Acids Res ; 49(11): 6420-6436, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096602

RESUMO

The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function.


Assuntos
Núcleo Celular/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Movimento Celular , Citoplasma/genética , RNA Helicases DEAD-box/metabolismo , DNA Complementar/metabolismo , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Motivos de Nucleotídeos , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo
7.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34184034

RESUMO

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) is crucial for mammalian craniofacial development, although the mechanisms by which the activity of downstream intracellular effectors is regulated to mediate gene expression changes have not been defined. We find that the RNA-binding protein Srsf3 is phosphorylated at Akt consensus sites downstream of PI3K-mediated PDGFRα signaling in mouse palatal mesenchyme cells, leading to its nuclear translocation. We further demonstrate that ablation of Srsf3 in the mouse neural crest lineage leads to facial clefting due to defective cranial neural crest cell proliferation and survival. Finally, we show that Srsf3 regulates the alternative RNA splicing of transcripts encoding protein kinases in the mouse facial process mesenchyme to regulate PDGFRα-dependent intracellular signaling. Collectively, our findings reveal that alternative RNA splicing is an important mechanism of gene expression regulation downstream of PI3K/Akt-mediated PDGFRα signaling in the facial mesenchyme and identify Srsf3 as a critical regulator of craniofacial development.


Assuntos
Processamento Alternativo , Mesoderma/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ligantes , Masculino , Camundongos , Camundongos Knockout , Crista Neural/embriologia , Crista Neural/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética
8.
Pain ; 162(7): 2097-2109, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33938719

RESUMO

ABSTRACT: Activation of transient receptor potential ankyrin 1 (TRPA1) channels by both environmental irritants and endogenous inflammatory mediators leads to excitation of the nerve endings, resulting in acute sensation of pain, itch, or chronic neurogenic inflammation. As such, TRPA1 channels are actively pursued as therapeutic targets for various pathological nociception and pain disorders. We uncovered that exon 27 of human TRPA1 (hTRPA1) could be alternatively spliced into hTRPA1_27A and hTRPA1_27B splice variants. The resulting channel variants displayed reduced expression, weakened affinity to interact with WT, and suffered from complete loss of function because of disruption of the C-terminal coiled-coil domain. Using a human minigene construct, we revealed that binding of splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to the exonic splicing enhancer was critical for the inclusion of intact exon 27. Knockdown of SRSF1, mutation within exonic splicing enhancer, or masking SRSF1 binding with antisense oligonucleotides promoted alternative splicing within exon 27. Finally, antisense oligonucleotides-induced alternative splicing produced transcript and protein variants that could be functionally determined as diminished endogenous TRPA1 activity in human Schwann cell-line SNF96.2 and hiPSCs-derived sensory neurons. The outcome of the work could potentially offer a novel therapeutic strategy for treating pain by targeting alternative splicing of hTRPA1.


Assuntos
Anquirinas , Oligonucleotídeos Antissenso , Processamento Alternativo/genética , Anquirinas/genética , Humanos , Mutação/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
9.
Nucleic Acids Res ; 49(10): 5760-5778, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037780

RESUMO

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.


Assuntos
Processamento Alternativo/genética , Carcinogênese/metabolismo , Desenvolvimento Embrionário/genética , Hematopoese/genética , Melanoma/metabolismo , Timócitos/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Apoptose/genética , Carcinogênese/genética , Proliferação de Células/genética , Genômica , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Linfopenia/genética , Linfopenia/metabolismo , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Timo/embriologia , Timo/metabolismo
10.
J Immunol ; 206(10): 2353-2365, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33941656

RESUMO

IL-17A plays an essential role in the pathogenesis of many autoimmune diseases, including psoriasis and multiple sclerosis. Act1 is a critical adaptor in the IL-17A signaling pathway. In this study, we report that an anti-sense long noncoding RNA, TRAF3IP2-AS1, regulates Act1 expression and IL-17A signaling by recruiting SRSF10, which downregulates the expression of IRF1, a transcriptional factor of Act1. Interestingly, we found that a psoriasis-susceptible variant of TRAF3IP2-AS1 A4165G (rs13210247) is a gain-of-function mutant. Furthermore, we identified a mouse gene E130307A14-Rik that is homologous to TRAF3IP2-AS1 and has a similar ability to regulate Act1 expression and IL-17A signaling. Importantly, treatment with lentiviruses expressing E130307A14-Rik or SRSF10 yielded therapeutic effects in mouse models of psoriasis and experimental autoimmune encephalomyelitis. These findings suggest that TRAF3IP2-AS1 and/or SRSF10 may represent attractive therapeutic targets in the treatment of IL-17-related autoimmune diseases, such as psoriasis and multiple sclerosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Interleucina-17/metabolismo , Psoríase/metabolismo , RNA Longo não Codificante/metabolismo , RNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais/genética , Animais , Proteínas de Ciclo Celular/genética , Técnicas de Inativação de Genes , Células HaCaT , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA/genética , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Fatores de Processamento de Serina-Arginina/genética , Transfecção
11.
Mol Med Rep ; 24(1)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33982782

RESUMO

Atherosclerosis is a primary cause of multiple types of cardiovascular disease, including myocardial infarction. In addition, injury of human umbilical vein endothelial cells (HUVECs) can lead to the development of atherosclerosis. Circular (circ)RNAs participate in atherosclerosis. It has previously been shown that circRNA cSMARCA5 (hsa_circ_0001445) expression is downregulated in atherosclerosis. However, the effects of hsa_circ_0001445 on the proliferation of HUVECs remain unclear. In order to mimic atherosclerosis in vitro, HUVECs were treated with oxidized low­density lipoprotein (oxLDL). The expression levels of specific genes and proteins were detected in HUVECs by reverse transcription­quantitative PCR and western blot analysis, respectively. Cell proliferation was assessed by Cell Counting Kit­8 and 5­Ethynyl­2'­deoxyuridine staining. Cell apoptosis and 5,5',6,6'­Tetrachloro­1,1',3,3'­tetraethyl­imidacarbocyanine staining were examined by flow cytometry. In addition, the association between hsa_circ_0001445 and serine/arginine­rich splicing factor 1 (SRSF1) was investigated by RNA pull­down assay. hsa_circ_0001445 expression was downregulated in oxLDL­treated HUVECs. Moreover, oxLDL­induced inhibition of HUVEC proliferation was significantly reversed by overexpression of hsa_circ_0001445. oxLDL notably inhibited tube formation and mitochondrial membrane potential in HUVECs, while these effects were markedly reversed by hsa_circ_0001445 overexpression. Furthermore, overexpression of hsa_circ_0001445 reversed oxLDL­induced activation of ß­catenin by binding to SRSF1. Collectively, these data demonstrated that overexpression of hsa_circ_0001445 reversed oxLDL­induced inhibition of HUVEC proliferation via activation of the SRSF1/ß­catenin axis. These findings may provide novel targets for the treatment of atherosclerosis.


Assuntos
Proliferação de Células/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Transição Epitelial-Mesenquimal/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Lipoproteínas LDL/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Neovascularização Patológica/genética , beta Catenina/metabolismo
12.
J Hematol Oncol ; 14(1): 60, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33849617

RESUMO

BACKGROUND: Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear. METHODS: RNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5250-Ser (SRSF5250-Ser) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14exon10 (METTL14exon10) and Cyclin L2exon6.3 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14exon 10 and Cyclin L2exon6.3 skipping were surveyed. RESULTS: CLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5250-Ser, which inhibited METTL14exon10 skipping while promoted Cyclin L2exon6.3 skipping. In addition, aberrant METTL14exon 10 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2exon6.3 promoted proliferation of PDAC cells. CONCLUSIONS: The CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.


Assuntos
Ciclinas/metabolismo , Éxons , Metiltransferases/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Ciclinas/genética , Feminino , Células HEK293 , Xenoenxertos , Humanos , Masculino , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Transcrição/genética
13.
Biochim Biophys Acta Mol Cell Res ; 1868(7): 119045, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33872670

RESUMO

PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.


Assuntos
Proteínas Mitocondriais/metabolismo , Mitofagia/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Antígenos Nucleares/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mitofagia/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/fisiologia , Ubiquitina-Proteína Ligases/metabolismo
14.
Cell Mol Life Sci ; 78(11): 4893-4905, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33835194

RESUMO

Emerging evidence shows that m6A, one of the most abundant RNA modifications in mammals, is involved in the entire process of spermatogenesis, including mitosis, meiosis, and spermiogenesis. "Writers" catalyze m6A formation on stage-specific transcripts during male germline development, while "erasers" remove m6A modification to maintain a balance between methylation and demethylation. The different functions of RNA-m6A transcripts depend on their recognition by "readers". m6A modification mediates RNA metabolism, including mRNA splicing, translation, and degradation, as well as the maturity and biosynthesis of non-coding RNAs. Sperm RNA profiles are easily affected by environmental exposure and can even be inherited for several generations, similar to epigenetic inheritance. Here, we review and summarize the critical role of m6A in different developmental stages of male germ cells, to understand of the mechanisms and epigenetic regulation of m6A modifications. In addition, we also outline and discuss the important role of non-coding RNAs in spermatogenesis and RNA modifications in epigenetic inheritance.


Assuntos
Epigênese Genética , RNA/metabolismo , Espermatozoides/metabolismo , Enzimas AlkB/metabolismo , Animais , Humanos , Masculino , Metiltransferases/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Espermatogênese
15.
Cell Immunol ; 362: 104299, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33607522

RESUMO

Acute lung injury (ALI) is a severe disease with a high rate of morbidity and mortality, characterized by excessive and uncontrolled inflammatory response in lung. Recent studies demonstrated that serine arginine-rich splicing factor 1 (SRSF1) is involved in inflammation. However, whether SRSF1 modulates ALI remains to be determined. In this study, we established an ALI mouse model that induced by lipopolysaccharide (LPS), with or without the treatment of SRSF1 antibody. Our result showed that SRSF1 expression was elevated in LPS-induced ALI. Importantly, treatment with SRSF1 antibody notably ameliorated ALI in mice, as determined by reduction in lung W/D ratios, histopathological changes, lung inflammation and TSLP expression. Besides, exposure of human alveolar epithelial A549 cells to LPS enhanced the expression of both SRSF1 and TSLP, while knockdown or overexpression of SRSF1 significantly lowered or upregulated the expression of TSLP induced by LPS. Interestingly, the expression of SRSF1 and TSLP showed a positive correlation in normal human lung tissues. Mechanistically, we found that SRSF1 directly bound with the mRNA of TSLP and may exert its function by stabilizing the mRNA of TSLP in LPS-induced ALI. Therefore, our results indicated that SRSF1 may be an important contributor in lung inflammation of LPS-induced ALI and SRSF1 signaling blocking may serve as a potential treatment of ALI.


Assuntos
Lesão Pulmonar Aguda/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Modelos Animais de Doenças , Humanos , Inflamação/genética , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Transdução de Sinais/efeitos dos fármacos
16.
Am J Physiol Lung Cell Mol Physiol ; 320(5): L726-L738, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33565360

RESUMO

Pulmonary arterial hypertension (PAH) refers to a set of heterogeneous vascular diseases defined by elevation of pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR), leading to right ventricular (RV) remodeling and often death. Early increases in pulmonary artery stiffness in PAH drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling. Dysregulation of microRNAs can drive PAEC dysfunction. However, the role of vascular stiffness in regulating pathogenic microRNAs in PAH is incompletely understood. Here, we demonstrated that extracellular matrix (ECM) stiffening downregulated miR-7 levels in PAECs. The RNA-binding protein quaking (QKI) has been implicated in the biogenesis of miR-7. Correspondingly, we found that ECM stiffness upregulated QKI, and QKI knockdown led to increased miR-7. Downstream of the QKI-miR-7 axis, the serine and arginine-rich splicing factor 1 (SRSF1) was identified as a direct target of miR-7. Correspondingly, SRSF1 was reciprocally upregulated in PAECs exposed to stiff ECM and was negatively correlated with miR-7. Decreased miR-7 and increased QKI and SRSF1 were observed in lungs from patients with PAH and PAH rats exposed to SU5416/hypoxia. Lastly, miR-7 upregulation inhibited human PAEC migration, whereas forced SRSF1 expression reversed this phenotype, proving that miR-7 depended upon SRSF1 to control migration. In aggregate, these results define the QKI-miR-7-SRSF1 axis as a mechanosensitive mechanism linking pulmonary arterial vascular stiffness to pathogenic endothelial function. These findings emphasize implications relevant to PAH and suggest the potential benefit of developing therapies that target this miRNA-dependent axis in PAH.


Assuntos
Endotélio Vascular/patologia , Matriz Extracelular/patologia , MicroRNAs/genética , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/patologia , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Fatores de Processamento de Serina-Arginina/genética , Transdução de Sinais , Remodelação Vascular
17.
Int J Biochem Cell Biol ; 134: 105948, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33609745

RESUMO

Ferroptosis, a newly iron-dependent form of cell death, is often accompanied by the damage of membrane lipid peroxide. Recently, the ferroptosis inducer erastin has been reported to exhibit potential anti-cancer activities. The aim of this study was to investigate the effects of SRSF9 on the sensitivity of colorectal cancer (CRC) to erastin and explore the underlying molecular mechanism. Short hairpin RNAs (shRNAs) or SRSF9 overexpression vector (SRSF9-OE) was transfected into erastin-induced human CRC cells to inhibit or overexpress SRSF9. Results showed that SRSF9 inhibition promoted the cell death induced by erastin, conversely, SRSF9 overexpression augmented the resistance to erastin-induced death in human CRC cells. SRSF9 decreased lipid peroxide damage which was a key event during erastin-induced ferroptosis in human CRC cells. Furthermore, we found that SRSF9 inhibition increased erastin-induced ferroptosis by downregulating GPX4 level. In an In vivo study, SRSF9 shRNA or SRSF9-OE stably transfected human CRC cells were subcutaneously injected into the right flank of nude mice. SRSF9 overexpression partly abolished the tumor growth inhibition and ferroptosis induced by erastin. Our data indicated SRSF9's regulation of GPX4 as an essential mechanism driving CRC tumorigenesis and resistance of erastin-induced ferroptosis. This molecular mechanism may provide a novel method for improving the sensitivity of CRC to erastin.


Assuntos
Neoplasias Colorretais/metabolismo , Ferroptose , Ferro/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Piperazinas/farmacologia , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Animais , Células CACO-2 , Morte Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Nus , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Phytomedicine ; 84: 153491, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33601237

RESUMO

BACKGROUND: Bitter tastants can activate bitter taste receptors (TAS2Rs) and thus initiate relaxation of airway smooth muscle cells (ASMCs), which have great potential in the development of novel bronchodilator drugs for asthma therapy. However, the canonical bitter substance, denatonium is known to induce apoptosis of airway epithelial cells (AECs), indicating that other bitter tastants may also impair the epithelial integrity to prevent hazardous particulate matters such as coronaviruses. Therefore, any bitter tastants intended for treating airway disease should be carefully evaluated for potential toxicity to AECs. HYPOTHESIS/PURPOSE: Considering the vast diversity of bitter tastants in nature and different types of TAS2Rs expressed in airway cells, we hypothesized that there must be some natural bitter tastants to be not only potent in inducing relaxation of ASMCs but also unharmful to AECs. STUDY DESIGN AND METHODS: Here we evaluated a group of bitter flavonoids that are derived from fruits and commonly used in traditional herbal medicine, including apigenin, hesperetin, kaempferol, naringenin, quercetin, and naringin, for their effects on the proliferation of human airway epithelial-like (16HBE14o-, BEAS-2B, and A549) cells cultured in vitro. Cell proliferation and associated signaling pathways were assessed by cell counting, ATP assay, cell cycling assay, quantitative RT-PCR, Fluo-4 labeling, and fluorescence resonance energy transfer, respectively. RESULTS: The results show that five of the six tested bitter tastants inhibited, but only naringin promoted the proliferation of the 16HBE14o-, BEAS-2B, and A549 cells at the dose of a few hundred micromoles. Furthermore, the naringin-promoted proliferation of the 16HBE14o- cells was associated with enhanced cell cycle progression, mRNA expression of cyclin E, and evoked calcium signaling/ERK signaling, which were all attenuated by inhibition of the TAS2R signaling pathways with specific blockers. CONCLUSION: These findings indicate that although the majority of the bitter flavonoids may inhibit the proliferation of AECs, naringin emerged as one to promote the proliferation of AECs via cell cycle progression and TAS2R-activated intracellular signaling. It suggests that naringin and not a few other bitter tastants can be proven with nontoxicity to the airway epithelial structure and function, which provides further confidence in the development of safe and effective TAS2R-based bronchodilators for asthma therapy.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Flavanonas/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Animais , Asma/tratamento farmacológico , Broncodilatadores/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Receptores Acoplados a Proteínas G/metabolismo
19.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562358

RESUMO

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the "Find Individual Motif Occurrences" (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.


Assuntos
Adenosina Trifosfatases/genética , Movimento Celular , Proteínas Cromossômicas não Histona/genética , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Neovascularização Patológica/patologia , RNA Circular/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Motivos de Nucleotídeos , Prognóstico , RNA Circular/genética , Fatores de Processamento de Serina-Arginina/genética , Células Tumorais Cultivadas
20.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33483422

RESUMO

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.


Assuntos
Fator 4 Ativador da Transcrição/genética , Edição de Genes/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mitocôndrias/genética , Proteínas Serina-Treonina Quinases/genética , 3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminoácidos/deficiência , Aminoácidos/farmacologia , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação da Expressão Gênica , Genoma Humano , Glucose/deficiência , Glucose/farmacologia , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligomicinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
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