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1.
Brain Res Bull ; 163: 1-13, 2020 10.
Article in English | MEDLINE | ID: mdl-32562719

ABSTRACT

BACKGROUND: Glioma is a primary intracranial malignancy with poor prognosis, of which the pathogenesis remains to be elucidated. Therein, the aim of this study is to discuss the impacts of lncRNA plasmacytoma variant translocation 1 (PVT1)/microRNA-128-1-5p (miR-128-1-5p)/polypyrimidine tract-binding protein 1 (PTBP1) axis on the biological characteristics of glioma cells. METHODS: Glioma tissue samples (72 cases) and normal brain tissue samples (35 cases) were harvested. The expression of PVT1, miR-128-1-5p and PTBP1 in glioma tissues and cells was detected. Glioma cells were transfected with sh-PVT1, miR-128-1-5p mimics or miR-128-1-5p inhibitors to verify the impacts of PVT1 and miR-128-1-5p on DNA damage, cell colony formation, invasion, proliferation, migration and apoptosis of glioma U87 and U251 cells. The growth of transplanted tumor was tested by tumor xenograft in nude mice. The combination of PVT1 and miR-128-1-5p and the targeting relationship between miR-128-1-5p and PTBP1 were verified. RESULTS: PVT1 and PTBP1 expression was enhanced and miR-128-1-5p expression was degraded in glioma tissues and cells. Overexpressed miR-128-1-5p and lowly-expressed PVT1 promoted DNA damage, suppressed colony formation, invasion, proliferation and migration as well as boosted apoptosis of U251 and U87 cells. Up-regulating miR-128-1-5p and down-regulating PVT1 reduced transplanted tumor volume and weight of glioma in mice. Low expression miR-128-1-5p reversed the effect of low expression PVT1 on the biological characteristics of glioma cells. PVT1 specifically bound to miR-128-1-5p and PTBP1 was the target gene of miR-128-1-5p. CONCLUSION: This study suggests that down-regulated PVT1 or up-regulated miR-128-1-5p boosts apoptosis and attenuates proliferation of glioma cells by inhibiting PTBP1 expression. This study is essential for finding new therapeutic targets for glioma.


Subject(s)
Apoptosis/physiology , Brain Neoplasms/metabolism , Glioma/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , MicroRNAs/biosynthesis , Polypyrimidine Tract-Binding Protein/biosynthesis , RNA, Long Noncoding/biosynthesis , Animals , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/physiology , Cells, Cultured , Female , Gene Expression Regulation, Neoplastic/physiology , Glioma/genetics , Glioma/pathology , Heterogeneous-Nuclear Ribonucleoproteins/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Polypyrimidine Tract-Binding Protein/antagonists & inhibitors , Polypyrimidine Tract-Binding Protein/genetics , RNA, Long Noncoding/genetics , Up-Regulation/physiology , Xenograft Model Antitumor Assays/methods
2.
J Cell Physiol ; 235(2): 1711-1722, 2020 02.
Article in English | MEDLINE | ID: mdl-31304601

ABSTRACT

Multifarious biological functions of long noncoding RNAs (lncRNAs) have been reported in various cancers including bladder cancer (BCa). This study aims to determine the biological role of a certain lncRNA in BCa. Consistent with the data of The Cancer Genome Atlas database, it was validated that lncRNA HLA complex group 22 (HCG22) was weakly expressed in BCa samples and lowly expressed HCG22 was closely correlated with low overall survival of the BCa patient. To verify the role of HCG22 in BCa progression, functional experiments were carried out in two representative BCa cells (J82 and T24) and the negative effects of HCG22 expression on the cell proliferation, migration, and epithelial-mesenchymal transition were identified. Mechanistically, polypyrimidine tract-binding protein 1 (PTBP1), which was highly expressed in BCa tissues and cell lines, was negatively regulated by HCG22 and the PTBP1-mediated Warburg effect was also obstructed by HCG22. Furthermore, HCG22 modulated the expression of PTBP1 through destabilizing human antigen R (HuR). And functional rescue assays confirmed that HCG22 functioned in bladder cancer through downregulating PTBP1. In conclusion, the present study revealed that HCG22 inhibited BCa progression via the HuR/PTBP1 axis, opening new prospects for potent therapeutic regimens for BCa patients.


Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation/genetics , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , Polypyrimidine Tract-Binding Protein/biosynthesis , RNA, Long Noncoding/genetics , Urinary Bladder Neoplasms/pathology , Adult , Aged , Cell Line, Tumor , Cell Proliferation/genetics , ELAV-Like Protein 1/biosynthesis , ELAV-Like Protein 1/genetics , Epithelial-Mesenchymal Transition/genetics , Female , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Male , Middle Aged , Neoplasm Invasiveness/genetics , Polypyrimidine Tract-Binding Protein/genetics , Urinary Bladder Neoplasms/genetics
3.
Mol Cells ; 41(10): 909-916, 2018 Oct 31.
Article in English | MEDLINE | ID: mdl-30165730

ABSTRACT

In pancreatic ß cells, glucose stimulates the biosynthesis of insulin at transcriptional and post-transcriptional levels. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), also named hnRNP I, acts as a critical mediator of insulin biosynthesis through binding to the pyrimidine-rich region in the 3'-untranslated region (UTR) of insulin mRNA. However, the underlying mechanism that regulates its expression in ß cells is unclear. Here, we report that glucose induces the expression of PTBP1 via the insulin receptor (IR) signaling pathway in ß cells. PTBP1 is present in ß cells of both mouse and monkey, where its levels are increased by glucose and insulin, but not by insulin-like growth factor 1. PTBP1 levels in immortalized ß cells established from wild-type (ßIRWT) mice are higher than levels in ß cells established from IR-null (ßIRKO) mice, and ectopic re-expression of IR-WT in ßIRKO cells restored PTBP1 levels. However, PTBP1 levels were not altered in ßIRKO cells transfected with IR-3YA, in which the Tyr1158/1162/1163 residues are substituted with Ala. Consistently, treatment with glucose or insulin elevated PTBP1 levels in ßIRWT cells, but not in ßIRKO cells. In addition, silencing Akt significantly lowered PTBP1 levels. Thus, our results identify insulin as a pivotal mediator of glucose-induced PTBP1 expression in pancreatic ß cells.


Subject(s)
Glucose/metabolism , Insulin-Secreting Cells/metabolism , Polypyrimidine Tract-Binding Protein/biosynthesis , Receptor, Insulin/metabolism , Animals , Cell Line, Tumor , Gene Knockout Techniques , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Macaca mulatta , Male , Mice , Mice, Inbred C57BL , Polypyrimidine Tract-Binding Protein/genetics , Signal Transduction , Transfection
4.
Biochemistry ; 57(26): 3873-3882, 2018 07 03.
Article in English | MEDLINE | ID: mdl-29851470

ABSTRACT

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue-specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post-translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N-terminal, linker 1, and linker 2 regions. We find that the two proteins have many overlapping acetate modifications in the RNA recognition motifs (RRMs) with a few distinct sites in PTBP1 RRM2 and RRM3. Our data also reveal that lysine residues in the nuclear localization sequence of PTBP2 are acetylated. Collectively, our results highlight important differences in post-translational modifications between the paralogs and suggest a role for them in the differential splicing activity of PTBP1 and PTBP2.


Subject(s)
Escherichia coli/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , Nerve Tissue Proteins/biosynthesis , Polypyrimidine Tract-Binding Protein/biosynthesis , Protein Processing, Post-Translational , Acetylation , Amino Acid Motifs , Escherichia coli/genetics , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Nerve Tissue Proteins/genetics , Phosphorylation , Polypyrimidine Tract-Binding Protein/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
5.
Cell Death Dis ; 9(5): 569, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29752441

ABSTRACT

Polypyrimidine tract-binding protein 3 (PTBP3) is an essential RNA-binding protein with roles in RNA splicing, 3' end processing and translation. Although increasing evidence implicates PTBP3 in several cancers, its role in gastric cancer metastasis remains poorly explored. In this study, we found that PTBP3 was upregulated in the gastric cancer tissues of patients with lymph node metastasis. Patients with high PTBP3 expression levels had significantly shorter survival than those with low PTBP3 expression. Overexpression/knockdown of PTBP3 expression had no effect on proliferation, whereas it regulated migration and invasion in vitro. In addition, when a mouse xenotransplant model of MKN45 was established, knockdown of PTBP3 in MKN45 cells caused the formation of tumours that were smaller in size than their counterparts, with suppression of tumour lymphangiogenesis and metastasis to regional lymph nodes. Furthermore, we identified caveolin 1 (CAV1) as a downstream target of PTBP3. RNA immunoprecipitation (RIP) assays and dual-luciferase reporter gene assays indicated that PTBP3 interacted with the CU-rich region of the CAV1 gene to downregulate CAV1α expression. Knockdown of CAV1α abrogated the reduction of FAK and Src induced by PTBP3 knockdown. In summary, our findings provide experimental evidence that PTBP3 may function as a metastatic gene in gastric cancer by regulating CAV1 through alternative splicing.


Subject(s)
Alternative Splicing , Caveolin 1/biosynthesis , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Polypyrimidine Tract-Binding Protein/biosynthesis , Stomach Neoplasms/metabolism , Animals , Caveolin 1/genetics , Cell Line, Tumor , Female , Humans , Mice , Mice, SCID , Neoplasm Metastasis , Neoplasm Proteins/genetics , Polypyrimidine Tract-Binding Protein/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology
6.
Neurotoxicol Teratol ; 59: 1-15, 2017.
Article in English | MEDLINE | ID: mdl-27751817

ABSTRACT

Exposure to arsenic, a common environmental toxin found in drinking water, leads to a host of neurological pathologies. We have previously demonstrated that developmental exposure to a low level of arsenic (50ppb) alters epigenetic processes that underlie deficits in adult hippocampal neurogenesis leading to aberrant behavior. It is unclear if arsenic impacts the programming and regulation of embryonic neurogenesis during development when exposure occurs. The master negative regulator of neural-lineage, REST/NRSF, controls the precise timing of fate specification and differentiation of neural stem cells (NSCs). Early in development (embryonic day 14), we observed increased expression of Rest, its co-repressor, CoREST, and the inhibitory RNA binding/splicing protein, Ptbp1, and altered expression of mRNA spliced isoforms of Pbx1 that are directly regulated by these factors in the male brain in response to prenatal 50ppb arsenic exposure. These increases were concurrent with decreased expression of microRNA-9 (miR-9), miR-9*, and miR-124, all of which are REST/NRSF targets and inversely regulate Rest expression to allow for maturation of NSCs. Exposure to arsenic decreased the formation of neuroblasts in vitro from NSCs derived from male pup brains. The female response to arsenic was limited to increased expression of CoREST and Ptbp2, an RNA binding protein that allows for appropriate splicing of genes involved in the progression of neurogenesis. These changes were accompanied by increased neuroblast formation in vitro from NSCs derived from female pups. Unexposed male mice express transcriptomic factors to induce differentiation earlier in development compared to unexposed females. Thus, arsenic exposure likely delays differentiation of NSCs in males while potentially inducing precocious differentiation in females early in development. These effects are mitigated by embryonic day 18 of development. Arsenic-induced dysregulation of the regulatory loop formed by REST/NRSF, its target microRNAs, miR-9 and miR-124, and RNA splicing proteins, PTBP1 and 2, leads to aberrant programming of NSC function that is perhaps perpetuated into adulthood inducing deficits in differentiation we have previously observed.


Subject(s)
Arsenic/toxicity , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , Nerve Tissue Proteins/biosynthesis , Neural Stem Cells/drug effects , Polypyrimidine Tract-Binding Protein/biosynthesis , Prenatal Exposure Delayed Effects/metabolism , Repressor Proteins/biosynthesis , Sex Characteristics , Animals , Cells, Cultured , Co-Repressor Proteins , Female , Male , Mice , MicroRNAs/biosynthesis , Neurogenesis/drug effects , Pre-B-Cell Leukemia Transcription Factor 1/biosynthesis , Pregnancy
7.
Hum Mol Genet ; 25(16): 3454-3466, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27378699

ABSTRACT

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene-encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component, however, has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of the BA4 (Brodmann area 4) motor cortex from seven human HD brains and seven controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using two expanded panels with a total of 108 BA4 tissues from patients and controls, we identified four splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD.


Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/genetics , Huntington Disease/genetics , Motor Cortex/metabolism , Polypyrimidine Tract-Binding Protein/genetics , Transcriptome/genetics , Adult , Aged , Alternative Splicing/genetics , Animals , Autopsy , Female , Gene Expression Regulation , Genetic Predisposition to Disease , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , High-Throughput Nucleotide Sequencing , Humans , Huntingtin Protein/genetics , Huntington Disease/physiopathology , Male , Mice , Middle Aged , Motor Cortex/pathology , Polypyrimidine Tract-Binding Protein/biosynthesis
8.
Sci Rep ; 6: 20665, 2016 Feb 09.
Article in English | MEDLINE | ID: mdl-26857472

ABSTRACT

RNA-binding motif protein 4a (RBM4a) reportedly reprograms splicing profiles of the insulin receptor (IR) and myocyte enhancer factor 2C (MEF2C) genes, facilitating the differentiation of brown adipocytes. Using an RNA-sequencing analysis, we first compared the gene expressing profiles between wild-type and RBM4a(-/-) brown adipocytes. The ablation of RBM4a led to increases in the PTBP1, PTBP2 (nPTB), and Nova1 proteins, whereas elevated RBM4a reduced the expression of PTBP1 and PTBP2 proteins in brown adipocytes through an alternative splicing-coupled nonsense-mediated decay mechanism. Subsequently, RBM4a indirectly shortened the half-life of the Nova1 transcript which was comparatively stable in the presence of PTBP2. RBM4a diminished the influence of PTBP2 in adipogenic development by reprogramming the splicing profiles of the FGFR2 and PKM genes. These results constitute a mechanistic understanding of the RBM4a-modulated splicing cascade during the brown adipogenesis.


Subject(s)
Adipocytes, Brown/metabolism , Adipogenesis/physiology , Cell Differentiation/physiology , RNA Splicing/physiology , RNA-Binding Proteins/metabolism , Adipocytes, Brown/cytology , Animals , Heterogeneous-Nuclear Ribonucleoproteins/biosynthesis , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Mice , Mice, Knockout , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neuro-Oncological Ventral Antigen , Polypyrimidine Tract-Binding Protein/biosynthesis , Polypyrimidine Tract-Binding Protein/genetics , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics
9.
BMC Infect Dis ; 16: 29, 2016 Jan 27.
Article in English | MEDLINE | ID: mdl-26818704

ABSTRACT

BACKGROUND: Dengue virus (DENV) is the most common vector-borne viral infection worldwide with approximately 390 million cases and 25,000 reported deaths each year. MicroRNAs (miRNAs) are small non-coding RNA molecules responsible for the regulation of gene expression by repressing mRNA translation or inducing mRNA degradation. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in DENV replication is poorly understood. METHODS: Here, we explored the relationship between DENV and cellular microRNAs using bioinformatics tools. We overexpressed miRNA-133a in Vero cells to test its role in DENV replication and analyzed its expression using RT-qPCR. Furthermore, the expression of polypyrimidine tract binding protein (PTB), a protein involved in DENV replication, was analyzed by western blot. In addition, we profiled miRNA-133a expression in Vero cells challenged with DENV-2, using Taqman miRNA. RESULTS: Bioinformatic analysis revealed that the 3' untranslated region (3'UTR) of the DENV genome of all four DENV serotypes is targeted by several cellular miRNAs, including miRNA-133a. We found that overexpression of synthetic miRNA-133a suppressed DENV replication. Additionally, we observed that PTB transcription , a miRNA-133a target, is down-regulated during DENV infection. Based in our results we propose that 3'UTR of DENV down-regulates endogenous expression of miRNA-133a in Vero cells during the first hours of infection. CONCLUSIONS: miRNA-133a regulates DENV replication possibly through the modulation of a host factor such as PTB. Further investigations are needed to verify whether miRNA-133a has an anti-DENV effect in vivo.


Subject(s)
Dengue Virus/physiology , MicroRNAs/biosynthesis , RNA, Viral/biosynthesis , Animals , Cell Line , Chlorocebus aethiops , Dengue/virology , Dengue Virus/genetics , Humans , Polypyrimidine Tract-Binding Protein/biosynthesis , Vero Cells , Virus Replication
10.
Nucleic Acids Res ; 43(11): 5586-600, 2015 Jun 23.
Article in English | MEDLINE | ID: mdl-25940628

ABSTRACT

Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3.


Subject(s)
Alternative Splicing , Peptide Chain Initiation, Translational , Polypyrimidine Tract-Binding Protein/genetics , Animals , Cells, Cultured , Codon, Initiator , Humans , K562 Cells , Mice, Inbred C57BL , Polypyrimidine Tract-Binding Protein/biosynthesis , Polypyrimidine Tract-Binding Protein/metabolism , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/metabolism
11.
PLoS Genet ; 10(11): e1004771, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25375251

ABSTRACT

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.


Subject(s)
Alternative Splicing/genetics , Hermanski-Pudlak Syndrome/genetics , Polypyrimidine Tract-Binding Protein/genetics , RNA, Messenger/genetics , Animals , Exons , Gene Expression Regulation , HeLa Cells , Hermanski-Pudlak Syndrome/pathology , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Polypyrimidine Tract-Binding Protein/biosynthesis , RNA Splice Sites/genetics
12.
J Mol Cell Cardiol ; 53(4): 578-87, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22850285

ABSTRACT

The Myocyte Enhancer Factor-2 (MEF2) family of transcription factors regulates gene expression during cardiomyocyte differentiation and adaptation of the myocardium to stress. MEF2 activity is enhanced by increasing its transcription and by MAPK-dependent phosphorylation, and is reduced by binding to class-II Histone Deacetylases and by miR-1-mediated degradation of its transcript. Here we show that MEF2 protein abundance is regulated at the translational level, determining myocyte size, during hypertrophy. In order to reduce MEF2 protein expression, its silencing through RNA interference required serum deprivation and, even in this condition, MEF2 protein abundance recovered to basal levels in presence of phenylephrine. Hypertrophic agonist stimulation of neonatal ventricular cardiomyocytes increased Mef2 expression by enhancing its translation, without changing its transcription or blocking degradation of the protein. MEF2 abundance was increased by Calcineurin overexpression in vivo and was reduced by Calcineurin inhibition in vitro, without affecting Mef2 mRNA levels. Calcineurin activity influenced expression of Polypyrimidine Tract Protein (PTB), contributing to MEF2 translation. Thus, our results show a previously unrecognized but relevant level of MEF2 activity regulation through the control of its translation that involves Calcineurin and PTB.


Subject(s)
Calcineurin/metabolism , MADS Domain Proteins/biosynthesis , MADS Domain Proteins/genetics , Myocytes, Cardiac/metabolism , Myogenic Regulatory Factors/biosynthesis , Myogenic Regulatory Factors/genetics , Polypyrimidine Tract-Binding Protein/biosynthesis , Animals , Cells, Cultured , HEK293 Cells , Humans , MEF2 Transcription Factors , Male , Mice , Mice, Transgenic , Pyrimidines/pharmacology , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , Signal Transduction
13.
Biol Reprod ; 75(3): 352-9, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16641145

ABSTRACT

In Sertoli cells of testis, androgen receptor-regulated gene transcription plays an indispensable role in maintaining spermatogenesis. Androgen receptor activity is modulated by a number of coregulators which are associated with the androgen receptor. Non-POU-domain-containing, octamer binding protein (NONO), a member of the DBHS-containing proteins, complexes with androgen receptor and functions as a coactivator for the receptor. Paraspeckle protein 1 alpha isoform (PSPC1, previously known as PSP1) and Splicing factor, proline- and glutamine-rich (SFPQ, previously known as PSF), other members of the DBHS-containing proteins, are also found in androgen receptor complexes, suggesting that these DBHS-containing proteins may cooperatively regulate androgen receptor-mediated gene transcription. We demonstrated that PSPC1, NONO, and SFPQ are coexpressed in Sertoli cell line TTE3 and interact reciprocally. The effect of the DBHS-containing proteins on the transcriptional activity was assessed using the construct containing androgen-responsive elements followed by a luciferase gene. The results showed that all the DBHS-containing proteins activate androgen receptor-mediated transcription, and PSPC1 is the most effective coactivator among them. Furthermore, we confirmed the presence of PSPC1, NONO, and SFPQ proteins in Sertoli cells of adult mouse testis sections. These observations suggest that PSPC1, NONO, and SFPQ form complexes with each other in Sertoli cells and may regulate androgen receptor-mediated transcriptional activity.


Subject(s)
DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Nuclear Proteins/biosynthesis , Nuclear Proteins/physiology , Polypyrimidine Tract-Binding Protein/biosynthesis , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/physiology , Receptors, Androgen/physiology , Sertoli Cells/metabolism , Transcription, Genetic/physiology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Blotting, Western , Cell Line , DNA-Binding Proteins/genetics , Genes, Reporter , Immunohistochemistry , Immunoprecipitation , Luciferases/genetics , Male , Mice , Nuclear Proteins/genetics , Plasmids/genetics , Polypyrimidine Tract-Binding Protein/genetics , Polypyrimidine Tract-Binding Protein/physiology , RNA-Binding Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Testis/cytology , Testis/metabolism , Transcriptional Activation
14.
Mol Cell Biol ; 25(21): 9595-607, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16227608

ABSTRACT

The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the alpha-tropomyosin pre-mRNA 3' end processing to the somite-specific pattern that results from the utilization of an upstream 3'-terminal exon designed exon 9A9'. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9' and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3' end processing of the alpha-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9', PTB regulates the cleavage/polyadenylation of this 3'-terminal exon.


Subject(s)
Polypyrimidine Tract-Binding Protein/physiology , RNA 3' End Processing , Tropomyosin/genetics , Actinin/genetics , Animals , Animals, Genetically Modified , Body Patterning , Cell Differentiation , Embryo, Nonmammalian , Endoderm/metabolism , Exons , Polyadenylation , Polypyrimidine Tract-Binding Protein/biosynthesis , Polypyrimidine Tract-Binding Protein/genetics , Protein Isoforms/genetics , RNA Precursors/metabolism , RNA Splicing , Skin/metabolism , Somites/cytology , Somites/metabolism , Xenopus laevis
15.
Neuro Oncol ; 6(1): 9-14, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14769134

ABSTRACT

Polypyrimidine tract-binding protein (PTB) is a nuclear factor that binds to the polypyrimidine tract of pre-mRNA introns, where it is associated with negative regulation of RNA splicing and with exon silencing. We have previously demonstrated that PTB expression is increased during glial cell transformation and that this increase correlates brain and in glial and neuronal tumors. Paraffin sections were stained by using a primary monoclonal antibody against PTB. Tissues that were analyzed included normal with changes in the RNA splicing of the fibroblast growth factor receptor 1. In this paper we examine the specific cellular distribution of PTB expression in normal brain (n = 2) and tumors of various types (low-grade astrocytoma, n = 2; anaplastic astrocytoma, n = 2; glioblastoma, n = 4; medulloblastoma, n = 4; central neurocytoma, n = 2; dysplastic gangliocytoma, n = 1; ganglioglioma, n = 1; paraganglioma, n = 1). In glial cell populations the majority of astrocytes and oligodendrocytes were negative, but occasional positively staining cells were observed. Strongly positive PTB staining was observed in ependymocytes, choroid plexus epithelium, microglia, arachnoid membrane, and adenohypophysis, and weak staining was found in the neurohypophysis. In all cases vascular endothelium and smooth muscle stained strongly. In tumor samples, intense positive nuclear staining was observed in transformed cells of low-grade astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, medulloblastoma, paraganglioma, and the glial population of both ganglioglioma and dysplastic gangliocytoma (the neuronal cells of both were negative). In medulloblastoma, neoplastic neuronal cells were positive, as were other cell lineages. In normal brain, all neuron populations and pineocytes were negative for PTB. We conclude that although glial cells show derepression of PTB expression, a similar mechanism is absent in both nonneoplastic neurons and in most neuronally derived tumor cells. Strong upregulation of PTB expression in tumor cells of glial or primitive neuroectodermal origin suggests involvement of this protein in cellular transformation. Whether PTB affects splicing of RNAs critical to cellular transformation or proliferation is an important question for future research.


Subject(s)
Brain Neoplasms/metabolism , Brain/metabolism , Gene Expression Regulation , Polypyrimidine Tract-Binding Protein/biosynthesis , RNA Splicing , Brain/pathology , Brain Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Humans , Polypyrimidine Tract-Binding Protein/genetics
16.
Cancer Res ; 63(19): 6154-7, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-14559795

ABSTRACT

Exclusion of the alpha-exon by alternative RNA splicing of the fibroblast growth factor receptor 1 (FGFR1) primary transcript leads to the production of FGFR1beta. Glial cell transformation is associated with a progressive increase in FGFR1beta expression that coincides with a dramatic increase in the expression of the splicing factor polypyrimidine tract-binding protein (PTB). Cell-specific overexpression of PTB increased alpha-exon skipping, and a reduction in PTB increased alpha-exon inclusion. Targeted disruption of PTB interaction with FGFR1 precursor RNA in vivo by an antisense oligonucleotide also increased alpha-exon inclusion. These results suggest that PTB plays a direct role in alpha-exon splicing.


Subject(s)
Alternative Splicing/physiology , Polypyrimidine Tract-Binding Protein/physiology , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Fibroblast Growth Factor/genetics , Alternative Splicing/drug effects , Alternative Splicing/genetics , Animals , Cell Line, Tumor , Down-Regulation , Exons/drug effects , Exons/genetics , Humans , Mice , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Polypyrimidine Tract-Binding Protein/biosynthesis , Polypyrimidine Tract-Binding Protein/genetics , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA Precursors/drug effects , RNA Precursors/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction
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