Добірка наукової літератури з теми "FOXP2, alternative splicing, PTBP1"

We performed transcriptome analysis in the hippocampus 24 h after lipopolysaccharide (LPS) administration. We observed glial-specific genes, comprised of two-thirds of all differentially expressed genes (DEGs). We found microglial DEGs that were the most numerous in LPS group. On the contrary, differential alternative splicing (DAS) analysis revealed the most numerous DAS events in astrocytes. Besides, we observed distinct major isoform switching in the Ptbp1 gene, with skipping of exon 8 in LPS group. Ptbp1 usually considered a pluripotency sustaining agent in brain embryonic development, according to the previous studies. Analyzing the splicing tune-up upon LPS exposure, we came to a supposition that the short Ptbp1 isoform de-represses immune-specific response by Ptbp1 adjusted splicing architecture. Additionally, the Ptbp3 (NOD1) immune-specific splicing factor has apparently been de-repressed by the Ptbp1 short isoform in glial cells. Notably, both the Ptbp1 and Ptbp3 genes express primarily in microglial/endothelial brain cells. We also report immune-related genes, altering their major isoforms upon LPS exposure. The results revealed immune modulating role of alternative splicing in brain.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

Introduction : Ibrutinib, an oral, selective inhibitor of Bruton's tyrosine kinase (BTK), dramatically improved Progression-free survival (PFS) and Overall survival (OS) compared with immunochemotherapy in CLL both in first line and relapsed/refractory patients. However, some patients did progress on ibrutinb with dismal outcome. The underlying mechanism remains to be investigated beyond evolving of BTK and/or PLCg2 mutation, the dysfunction of apoptotic protein and mitochondrial apoptotic dependencies may be involves in ibrutinib resistance. PTBP1 (Polypyrimidine tract binding protein 1), a splicing factor, was found to be necessary for B cell selection in germinal centers. Knocking out PTBP1 in B cell resulted in impaired BCR-mediated B-cell activation and antibody production. Here, we investigate the regulation of PTBP1 on alternative splicing of apoptotic protein and its implications in CLL and ibrutinib resistance. Methods: Eighty-one CLL patients and 5 healthy controls were enrolled in this study from January 2010 to May 2018. The PTBP1 mRNA expression was measured by real-time polymerase chain reaction (RT-PCR) and Western-blot. We analyzed the PTBP1 expression with established CLL prognostic factors such as p53 and IGHV mutation status, and treatment to first treatment (TTFT). Resistant MEC-1 cell line was established by intermittently incubating with ibrutinib at a low concentration for short intervals and then gradually increased to 2-fold of IC50 value. Cells were allowed to recover every time after washing off the drug. RT-PCR was performed for both long and short isoform of MCL-1 by using specific primer in both parent and resistant cell lines and series ibrutinb-treated (both sensitive and resistant) patients' primary cells. Resistant MEC-1 cell line was cultured in RPMI 1640 without ibrutinib for 48hrs before transfection, siRNA targeting with PTBP1 mRNA and non-targeting siRNA were transfected into cells by using lipofectamine 3000. The transfection efficiency were verified by Western blot after 24 h and ibrutinib was added into resistant cell line. Apoptosis was then analyzed using flow cytometry (FCM) after 24 hrs. Receiver operating characteristic curve (ROC) and area under the ROC curve (AUC) were established to verify the best cut-off value in differentiating the high or low expression of PTBP1 mRNA. Time-to-first-treatment (TTFT) interval was defined as interval from diagnosis to first treatment. All statistical analyses were performed using the SPSS software program. Results: The expression of PTBP1 in CLL primary patients was significantly increased than 5 healthy donors (p < 0.01)(A). Patients with IGHV-mutated had higher level of PTBP1 as compared with patients with IGHV-unmutated (p < 0.05). Furthermore, Higher level of PTBP1 was associated with shorter TTFT in whole cohort, also in IGHV-mutated and unmutated subgroup (p < 0.05)(B). We further demonstrated that PTBP1 was aberrant expressed in ibrutinib resistant MEC-1 cell line or ibrutinib resistant primary patients' samples, as compared with parent cell line or patients' baseline samples. We also found the dysregulation of alternative splicing of MCL-1 in ibrutinib resistant models, presented with increased anti-apoptotic MCL-1L and decreased pro-apoptotic MCL-1S(C). Moreover, knocking down of PTBP1 sensitized CLL to ibrutinib via switching alternative splicing of MCL-1 to its pro-apoptotic isform MCL-1S(D). Conclusions: The splicing factor PTBP1 is involved in the pathogenesis of CLL. Its aberrant expression may lead to the dysregulation of alternative splicing of MCL-1, resulted in increased MCL-1L/s ratio. PTBP1 can be as a promising target for the treatment of CLL patients progressed on ibrutinib. Figure Disclosures No relevant conflicts of interest to declare.

The output of alternative splicing depends on the cooperative or antagonistic activities of several RNA-binding proteins (RBPs), like Ptbp1 and Esrp1 inXenopus. Fine-tuning of the RBP abundance is therefore of prime importance to achieve tissue- or cell-specific splicing patterns. Here, we addressed the mechanisms leading to the high expression of theptbp1gene, which encodes Ptbp1, inXenopusepidermis. Two splice isoforms ofptbp1mRNA differ by the presence of an alternative exon 11, and only the isoform including exon 11 can be translated to a full-length protein.In vivominigene assays revealed that the nonproductive isoform was predominantly produced. Knockdown experiments demonstrated that Esrp1, which is specific to the epidermis, strongly stimulated the expression ofptbp1by favoring the productive isoform. Consequently, knocking downesrp1phenocopiedptbp1inactivation. Conversely, Ptbp1 repressed the expression of its own gene by favoring the nonproductive isoform. Hence, a complex posttranscriptional mechanism controls Ptbp1 abundance inXenopusepidermis: skipping of exon 11 is the default splicing pattern, but Esrp1 stimulatesptbp1expression by favoring the inclusion of exon 11 up to a level that is limited by Ptbp1 itself. These results decipher a posttranscriptional mechanism that achieves various abundances of the ubiquitous RBP Ptbp1 in different tissues.

Alternative splicing plays a pivotal role in modulating the function of eukaryotic proteins. In the inner ear, many genes undergo alternative splicing, and errors in this process lead to hearing loss. Cadherin 23 (CDH23) forms part of the so-called tip links, which are indispensable for mechanoelectrical transduction (MET) in the hair cells. Cdh23 gene contains 69 exons, and exon 68 is subjected to alternative splicing. Exon 68 of the Cdh23 gene is spliced into its mRNA only in a few cell types including hair cells. The mechanism responsible for the alternative splicing of Cdh23 exon 68 remains elusive. In the present work, we performed a cell-based screening to look for splicing factors that regulate the splicing of Cdh23 exon 68. RBM24 and RBM38 were identified to enhance the inclusion of Cdh23 exon 68. The splicing of Cdh23 exon 68 is affected in Rbm24 knockdown or knockout cells. Moreover, we also found that PTBP1 inhibits the inclusion of Cdh23 exon 68. Taken together, we show here that alternative splicing of Cdh23 exon 68 is regulated by RBM24, RBM38, and PTBP1.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Polypyrimidine tract binding proteins, PTBP1 and PTBP2, are paralogous RNA binding proteins that play a critical role in the process of neuronal differentiation and maturation; changes in the concentration of PTBP proteins during neuronal development direct splicing changes in many transcripts that code for proteins critical for neuronal differentiation. How the two related proteins regulate different sets of neuronal exons is unclear. The distinct splicing activities of PTBP1 and PTBP2 can be recapitulated in an in vitro splicing system with the differentially regulated N1 exon of the c-src pre-mRNA. Here, we conducted experiments under these in vitro splicing conditions to identify PTBP1 and PTBP2 interacting partner proteins. Our results highlight that both PTBPs interact with proteins that participate in chromatin remodeling and transcription regulation. Our data reveal that PTBP1 interacts with many proteins involved in mRNA processing including splicing regulation while PTBP2 does not. Our results also highlight enzymes that can serve as potential “writers” and “erasers” in adding chemical modifications to the PTB proteins. Overall, our study highlights important differences in protein-protein interactions between the PTBP proteins under splicing conditions and supports a role for post-translational modifications in dictating their distinct splicing activities.

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.

Objective. Polypyrimidine tract-binding protein 1 (PTBP1) is an RNA-binding protein, which plays a role in pre-mRNA splicing and in the regulation of alternative splicing events. However, little was known about the correlation between PTBP1 and glioma and its prognostic significance in glioma patients. Our aim was to investigate the expression, functional role, and prognostic value of PTBP1 in glioma. Methods. We explored the expression of PTBP1 protein using immunohistochemistry in 150 adult malignant glioma tissues and 20 normal brain tissues and evaluated its association with clinicopathological parameters by chi-square test. Kaplan-Meier method was used to evaluate the prognostic effect of PTBP1 in glioma. Univariate/multivariate Cox analyses were used to identify independent prognostic factors. Transcriptional regulation network was constructed based on differentially expressed genes (DEGs) of PTBP1 from TCGA/CGGA database. GO and KEGG enrichment analyses were used to explore the function and pathways of DEGs. Results. Out of the 150 malignant glioma tissues (60 LGG and 90 GBMs) and 20 normal brain tissues in our cohort, PTBP1 protein was high expressed in glioma tissues (79/150, 52.7%), but no expression was detected in normal brain tissues (0/20, 0%). The expression of PTBP1 was significantly higher in GBMs ( P < 0.001 ). More than half of GBMs (62/90, 68.9%) were PTBP1 high expression. Chi-square test showed that the expression of PTBP1 was correlated with patient age, WHO grade, Ki-67 index, and IDH status. High expression of PTBP1 was significantly associated with poor prognosis in glioma, and it was an independent risk factor in glioma patients. Furthermore, we shed light on the underlying mechanism of PTBP1 by constructing a miR-218-TCF3-PTBP1 transcriptional network in glioma. Conclusion. PTBP1 was high expressed in glioma, and it significantly correlated with poor prognosis, suggesting a potential therapeutic target for glioma, particularly for GBM.

Neurotrophins and their receptors are key molecules in the development of the
nervous system. Neurotrophin-3 binds preferentially to its high-affinity receptor
NTRK3, which exists in two major isoforms in humans, the full-length kinaseactive
form (150 kDa) and a truncated non-catalytic form (50 kDa). The two
variants show different 3'UTR regions, indicating that they might be differentially
regulated at the post-transcriptional level. In this work we explore how
microRNAs take part in the regulation of full-length and truncated NTRK3,
demonstrating that the two isoforms are targeted by different sets of microRNAs.
We analyze the physiological consequences of the overexpression of some of the
regulating microRNAs in human neuroblastoma cells. Finally, we provide
preliminary evidence for a possible involvement of miR-124 - a microRNA with no
putative target site in either NTRK3 isoform - in the control of the alternative
spicing of NTRK3 through the downregulation of the splicing repressor PTBP1.
Las neurotrofinas y sus receptores constituyen una familia de factores cruciales
para el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su función
principalmente a través de una unión de gran afinidad al receptor NTRK3, del cual
se conocen dos isoformas principales, una larga de 150KDa con actividad de tipo
tirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformas
no comparten la misma región 3'UTR, lo que sugiere la existencia de una
regulación postranscripcional diferente. En el presente trabajo se ha explorado
como los microRNAs intervienen en la regulación de NTRK3, demostrando que las
dos isoformas son reguladas por diferentes miRNAs. Se han analizado las
consecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizando
células de neuroblastoma. Finalmente, se ha estudiado la posible implicación del
microRNA miR-124 en el control del splicing alternativo de NTRK3 a través de la
regulación de represor de splicing PTBP1.

Il "gene del linguaggio" Forkhead Box P2 (FOXP2) codifica per un fattore di trascrizione e diverse mutazioni nella regione codificante sono state correlate con il disturbo della parola e del linguaggio di tipo 1 (SPCH1). Esperimenti di linkage disequilibrium e genome wide association hanno associato FOXP2 anche a disturbi dello sviluppo neuronale, tra i quali i disturbi dello spettro autistico (ASDs). Tuttavia non sono note mutazioni nella regione codificante di FOXP2 in pazienti ASD, portando all'ipotesi di possibili difetti a livello post-trascrizionale. A tal proposito, mentre sono disponibili informazioni sulla regolazione da parte di miRNA, poco è noto sulla regolazione dello splicing alternativo di FOXP2 e sulle ribonucleoproteine coinvolte. La proteina FOXP2 è codificata dal trascritto full-length (FOXP2-FL), formato da 17 esoni canonici più due piccoli esoni alternativi, 3b e 4a, in grado di mantenere la cornice di lettura. L'analisi bioinformatica della sequenza genomica di FOXP2 ha rivelato la presenza di molteplici ipotetici siti di legame per la ribonucleoproteina Polypyrimidine Binding Tract Protein 1 (PTBP1) nelle regioni introniche attorno agli esoni 11 e 3b. PTBP1 è una ribonucleoproteina ubiquitaria in grado di regolare l’esclusione esonica. Inoltre, PTBP1 ed il suo paralogo PTBP2, che spesso riconosce gli stessi siti di legame, svolgono un ruolo chiave durante il differenziamento neuronale. Mediante immunoprecipitazione di PTBP1 e degli mRNA ad esso legati (RIP) e sovraespressione di PTBP1 in cellule HEK293 abbiamo dimostrato che PTBP1 è in grado di legare i trascritti di FOXP2 e promuovere l’esclusione dell'esone 11, generando il trascritto FOXP2-△11. L'aumentato livello del trascritto FOXP2-△11 è stato accompagnato da una significativa diminuzione del trascritto FOXP2-FL e della proteina da esso codificata. Abbiamo ulteriormente confermato il ruolo di PTBP1 nel promuovere l’esclusione dell'esone 11 di FOXP2 tramite un saggio con minigene. Similmente, abbiamo dimostrato che anche la proteina PTBP2 marcata con Myc è in grado di promuovere la generazione del trascritto FOXP2-△11. Successivamente, abbiamo analizzato l'abilità di PTBP1 di regolare l'esone 3b. Malgrado la presenza di numerosi siti putativi di legame per PTBP1 attorno all'esone 3b, PTBP1 non è stato in grado di promuoverne l’esclusione, suggerendo la specificità di PTBP1 nella regolazione dell'esone 11. L'esclusione dell'esone 11 introduce un codone di terminazione prematuro (PTC) all'interno dell'esone 12 nel trascritto di FOXP2 che, se tradotto, codificherebbe per una proteina tronca mancante del dominio FOX per il legame al DNA, tuttavia un simile peptide non è stato rilevato nel nostro sistema cellulare in vitro. L'analisi della sequenza di FOXP2-△11 ci ha fatto ipotizzare una sua degradazione mediante Nonsense Mediated Decay (NMD), che elimina gli mRNA contenenti dei PTC. Usando inibitori specifici abbiamo dimostrato che FOXP2-△11 è un target di NMD. Infine, il silenziamento genico di PTBP1 non ha modificato l'espressione proteica di FOXP2, ma poiché il silenziamento di PTBP1 promuove l'espressione di PTBP2, abbiamo co-silenziato PTBP1 e PTBP2, ed osservato un aumento della proteina FOXP2 di 2.5 volte. L'analisi dei trascritti ha rilevato un incremento di FOXP2-FL di circa il 26%, ma nessuna variazione nell'espressione di FOXP2-△11, suggerendo una regolazione operata da PTBP1 e PTBP2 indipendente dallo splicing alternativo. Poichè PTBP1 è in grado anche di controllare la trascrizione dei trascritti, abbiamo analizzato bioinformaticamente le regioni 5' dei trascritti e trovato un cluster di siti putativi di legame di PTBP1 in uno dei 5' alternativi di FOXP2, tuttavia ulteriori studi sono necessari. In conclusione, proponiamo un modello di controllo dei livelli di espressione di FOXP2 durante lo sviluppo neuronale mediante uno splicing alternativo regolato da PTBP1, che genera il trascritto non codificante FOXP2-△11 poi degradato attraverso NMD.
The language gene Forkhead Box P2 (FOXP2) encodes a transcription factor and mutations in gene coding region have been associated with human speech and language disorder type 1 (SPCH1). Linkage disequilibrium and genome wide association studies (GWAS) linked FOXP2 also to neurodevelopmental diseases, among others autism spectrum disorders (ASDs), but since no mutations in the coding region were found, post-transcriptional defects were hypothesized. While some data on miRNA regulation are available, little is known on alternative splicing regulation of FOXP2 and the ribonucleoproteins (RBPs) involved. FOXP2 protein is encoded by full-length (FOXP2-FL) transcript, composed of 17 canonical exons and two alternative small in frame alternatively spliced exons, 3b and 4a. Bioinformatic analysis of FOXP2 genomic sequence revealed several putative binding sites in the intronic regions around FOXP2 exon 11 and exon 3b for the Polypyrimidine Tract Binding Protein 1 (PTBP1). PTBP1 is a ubiquitous ribonucleoprotein often regulating exon skipping. Moreover, PTBP1 and its paralog PTBP2, that recognizes the same binding sequences in most cases, play key roles during neuronal differentiation. By RNA ImmunoPrecipitation (RIP) and PTBP1 overexpression in HEK293 cells we demonstrated that PTBP1 binds FOXP2 transcripts and promotes the exclusion of exon 11 generating a FOXP2-△11 transcript. Interestingly, up-regulation of FOXP2-△11 transcript was accompanied by down-regulation of FOXP2-FL transcript and subsequent FOXP2 protein decrement of about 50%. These findings were further confirmed by in vitro minigene assays performed on FOXP2 gene region from exon 9 to exon 13 subcloned in a suitable expression vector. Interestingly, Myc-tagged PTBP2 was also able to up-regulate FOXP2-△11. The minigene approach allowed us also to restrict the intronic regions required for PTBP1 binding sufficient to promote the exclusion of exon 11. Similarly, we analyzed the ability of PTBP1 to regulate the alternatively spliced 3b exon. Despite the presence of numerous predicted binding sites for PTBP1 around exon 3b, PTBP1 appeared unable to promote its skipping, suggesting specificity towards exon 11. The exclusion of exon 11 creates a Premature Termination Codon (PTC) within exon 12, which will generate a truncated FOXP2 protein lacking the DNA binding domain, if translated. Nevertheless, we could not find FOXP2-△11-derived peptides in our cell system. Feature analysis of FOXP2-△11 nucleotide sequence led us to hypothesize this transcript could be degraded by Nonsense-Mediated Decay (NMD), a conserved degradation pathway for aberrant or PTC-bearing mRNAs. By using selective inhibitors, we showed that FOXP2-△11 transcript is indeed a target of NMD. Finally, siRNA-mediated PTBP1 silencing did not show a significative variation of FOXP2 protein or transcript in HEK293 cells, but since the silencing of PTBP1 will promote PTBP2 expression, we co-silenced PTBP1 and PTBP2 and observed a FOXP2 protein overexpression of about 2.5 fold. Transcripts analysis of the same samples revealed a FOXP2-FL increment of about 26%, but no variation of FOXP2-△11 transcript expression, thus pointing to an alternative splicing-indipendent regulation performed by PTBP1 and PTBP2. Since PTBP1 may also control transcript translation, we performed a bioinformatic analysis of FOXP2 5' UTR regions and found a cluster of putative binding sites for PTBP1 in one of the alternatively spliced 5’ UTR of FOXP2 gene, whose function is yet to be elucidated. In conclusion, we propose a model for the control of FOXP2 expression mediated by PTBP1 via upregulation of the noncoding FOPX2-△11 transcript by alternative splicing and degraded by NMD.