Journal articles on the topic 'FOXP2, alternative splicing, PTBP1'
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Babenko, Vladimir N., Galina T. Shishkina, Dmitriy A. Lanshakov, Ekaterina V. Sukhareva, and Nikolay N. Dygalo. "LPS Administration Impacts Glial Immune Programs by Alternative Splicing." Biomolecules 12, no. 2 (February 8, 2022): 277. http://dx.doi.org/10.3390/biom12020277.
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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.
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Hinkle, Emma R., Hannah J. Wiedner, Eduardo V. Torres, Micaela Jackson, Adam J. Black, R. Eric Blue, Sarah E. Harris, et al. "Alternative splicing regulation of membrane trafficking genes during myogenesis." RNA 28, no. 4 (January 26, 2022): 523–40. http://dx.doi.org/10.1261/rna.078993.121.
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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.
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Zhu, Huayuan, Xiaotong Li, Xinqi Zheng, Juejin Wang, Hanning Tang, Wei Xu, and Jianyong Li. "PTBP1 Regulates Alternative Splicing of Apoptotic Protein: Implications in CLL and Ibrutinib Resistance." Blood 134, Supplement_1 (November 13, 2019): 1290. http://dx.doi.org/10.1182/blood-2019-126945.
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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.
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Méreau, Agnès, Vincent Anquetil, Hubert Lerivray, Justine Viet, Claire Schirmer, Yann Audic, Vincent Legagneux, Serge Hardy, and Luc Paillard. "A Posttranscriptional Mechanism That Controls Ptbp1 Abundance in the Xenopus Epidermis." Molecular and Cellular Biology 35, no. 4 (December 15, 2014): 758–68. http://dx.doi.org/10.1128/mcb.01040-14.
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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.
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Li, Nana, Haibo Du, Rui Ren, Yanfei Wang, and Zhigang Xu. "Alternative Splicing of Cdh23 Exon 68 Is Regulated by RBM24, RBM38, and PTBP1." Neural Plasticity 2020 (July 25, 2020): 1–11. http://dx.doi.org/10.1155/2020/8898811.
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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.
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Pina, Jeffrey M., Luis A. Hernandez, and Niroshika M. Keppetipola. "Polypyrimidine tract binding proteins PTBP1 and PTBP2 interact with distinct proteins under splicing conditions." PLOS ONE 17, no. 2 (February 3, 2022): e0263287. http://dx.doi.org/10.1371/journal.pone.0263287.
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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.
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Fochi, Stefania, Pamela Lorenzi, Marilisa Galasso, Chiara Stefani, Elisabetta Trabetti, Donato Zipeto, and Maria Grazia Romanelli. "The Emerging Role of the RBM20 and PTBP1 Ribonucleoproteins in Heart Development and Cardiovascular Diseases." Genes 11, no. 4 (April 8, 2020): 402. http://dx.doi.org/10.3390/genes11040402.
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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.
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Zhu, Wei, Bo-lun Zhou, Li-juan Rong, Li Ye, Hong-juan Xu, Yao Zhou, Xue-jun Yan, et al. "Roles of PTBP1 in alternative splicing, glycolysis, and oncogensis." Journal of Zhejiang University-SCIENCE B 21, no. 2 (February 2020): 122–36. http://dx.doi.org/10.1631/jzus.b1900422.
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Liu, Pan, Guo-Chao He, Yu-Zhen Tan, Ge-Xin Liu, An-Min Liu, Xiao-Peng Zhu, Yang Zhou, and Wan-Ming Hu. "PTBP1 is a Novel Poor Prognostic Factor for Glioma." BioMed Research International 2022 (March 8, 2022): 1–11. http://dx.doi.org/10.1155/2022/7590997.
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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.
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Sasabe, Toshikazu, Eugene Futai, and Shoichi Ishiura. "PTBP1 regulates the alternative splicing of dopamine receptor D2 (DRD2)." Neuroscience Research 65 (January 2009): S90. http://dx.doi.org/10.1016/j.neures.2009.09.369.
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Bai, Hua, and Bing Chen. "Abnormal PTBP1 Expression Sustains the Disease Progression of Multiple Myeloma." Disease Markers 2020 (June 19, 2020): 1–10. http://dx.doi.org/10.1155/2020/4013658.
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Multiple myeloma (MM) is a hematopoietic malignancy characterized by heterogeneity, which corresponds to alternative splicing (AS) profiles and disadjust gene expression. Bioinformatics analysis of AS factors possibly related to MM progression identified the polypyrimidine tract binding protein (PTBP1) as candidate. The purpose of this study was to confirm the incidence and prognostic value of PTBP1 in MM patients. Several cohorts of 2971 patients presenting newly diagnosed and relapsed MM were enrolled. Correlations between PTBP1 expression and clinicopathological characteristics, proliferative activity, and response to therapy of myeloma cells were analyzed. Moreover, the effect of PTBP1 on the AS pattern of specific aerobic glycolysis-related genes was explored in MM patients. Clinically, PTBP1 expression was present at all stages; it increased with disease progression and poor prognosis, which was even stronger elevated in patients with high tumor burden and drug resistance. Mechanistically, PTBP1 modulated AS of PKM2 and aerobic glycolysis-related genes in MM patients, which play synergistic or additive effects in clinical outcome. PTBP1 may be a novel marker for prognostic prediction and a promising therapeutic target for the development of anti-MM treatments.
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Lei, Jianzhen, Xiaoxin Liu, Miaomiao Song, Yingying Zhou, Jia Fan, Xiaowei Shen, Xiaohan Xu, Isha Kapoor, Guoqing Zhu, and Juejin Wang. "Aberrant Exon 8/8a Splicing by Downregulated PTBP (Polypyrimidine Tract-Binding Protein) 1 Increases Ca V 1.2 Dihydropyridine Resistance to Attenuate Vasodilation." Arteriosclerosis, Thrombosis, and Vascular Biology 40, no. 10 (October 2020): 2440–53. http://dx.doi.org/10.1161/atvbaha.120.315010.
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Objective: Calcium channel blockers, such as dihydropyridines, are commonly used to inhibit enhanced activity of vascular Ca V 1.2 channels in hypertension. However, patients who are insensitive to such treatments develop calcium channel blocker-resistant hypertension. The function of Ca V 1.2 channel is diversified by alternative splicing, and the splicing factor PTBP (polypyrimidine tract-binding protein) 1 influences the utilization of mutually exclusive exon 8/8a of the Ca V 1.2 channel during neuronal development. Nevertheless, whether and how PTBP1 makes a role in the calcium channel blocker sensitivity of vascular Ca V 1.2 channels, and calcium channel blocker-induced vasodilation remains unknown. Approach and Results: We detected high expression of PTBP1 and, inversely, low expression of exon 8a in Ca V 1.2 channels (Ca V 1.2 E8a ) in rat arteries. In contrast, the opposite expression patterns were observed in brain and heart tissues. In comparison to normotensive rats, the expressions of PTBP1 and Ca V 1.2 E8a channels were dysregulated in mesenteric arteries of hypertensive rats. Notably, PTBP1 expression was significantly downregulated, and Ca V 1.2 E8a channels were aberrantly increased in dihydropyridine-resistant arteries compared with dihydropyridine-sensitive arteries of rats and human. In rat vascular smooth muscle cells, PTBP1 knockdown resulted in shifting of Ca V 1.2 exon 8 to 8a. Using patch-clamp recordings, we demonstrated a concomitant reduction of sensitivity of Ca V 1.2 channels to nifedipine, due to the higher expression of Ca V 1.2 E8a isoform. In vascular myography experiments, small interfering RNA-mediated knockdown of PTBP1 attenuated nifedipine-induced vasodilation of rat mesenteric arteries. Conclusions: PTBP1 finely modulates the sensitivities of Ca V 1.2 channels to dihydropyridine by shifting the utilization of exon 8/8a and resulting in changes of responses in dihydropyridine-induced vasodilation.
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Lorenzi, P., A. Sangalli, S. Fochi, A. Dal Molin, G. Malerba, D. Zipeto, and M. G. Romanelli. "RNA-binding proteins RBM20 and PTBP1 regulate the alternative splicing of FHOD3." International Journal of Biochemistry & Cell Biology 106 (January 2019): 74–83. http://dx.doi.org/10.1016/j.biocel.2018.11.009.
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Zhu, H., X. Li, X. Zheng, J. Wang, H. Tang, L. Wang, Y. Xia, J. Liang, W. Xu, and J. Li. "PTBP1 REGULATES ALTERNATIVE SPLICING OF APOPTOTIC PROTEIN: IMPLICATIONS IN CLL AND IBRUTINIB RESISTANCE." Hematological Oncology 37 (June 2019): 373–74. http://dx.doi.org/10.1002/hon.41_2631.
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Wang, Zhi-na, Dan Liu, Bin Yin, Wen-yi Ju, Hui-zhong Qiu, Yi Xiao, Yuan-jia Chen, Xiao-zhong Peng, and Chong-mei Lu. "High expression of PTBP1 promote invasion of colorectal cancer by alternative splicing of cortactin." Oncotarget 8, no. 22 (March 3, 2017): 36185–202. http://dx.doi.org/10.18632/oncotarget.15873.
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Calabretta, S., P. Bielli, I. Passacantilli, E. Pilozzi, V. Fendrich, G. Capurso, G. Delle Fave, and C. Sette. "Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells." Oncogene 35, no. 16 (August 3, 2015): 2031–39. http://dx.doi.org/10.1038/onc.2015.270.
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Izaguirre, Daisy I., Wen Zhu, Tao Hai, Hannah C. Cheung, Ralf Krahe, and Gilbert J. Cote. "PTBP1-dependent regulation of USP5 alternative RNA splicing plays a role in glioblastoma tumorigenesis." Molecular Carcinogenesis 51, no. 11 (October 4, 2011): 895–906. http://dx.doi.org/10.1002/mc.20859.
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Fuentes-Fayos, Antonio C., Mari C. Vázquez-Borrego, Juan M. Jiménez-Vacas, Leire Bejarano, Sergio Pedraza-Arévalo, Fernando L.-López, Cristóbal Blanco-Acevedo, et al. "Splicing machinery dysregulation drives glioblastoma development/aggressiveness: oncogenic role of SRSF3." Brain 143, no. 11 (November 2020): 3273–93. http://dx.doi.org/10.1093/brain/awaa273.
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Abstract Glioblastomas remain the deadliest brain tumour, with a dismal ∼12–16-month survival from diagnosis. Therefore, identification of new diagnostic, prognostic and therapeutic tools to tackle glioblastomas is urgently needed. Emerging evidence indicates that the cellular machinery controlling the splicing process (spliceosome) is altered in tumours, leading to oncogenic splicing events associated with tumour progression and aggressiveness. Here, we identify for the first time a profound dysregulation in the expression of relevant spliceosome components and splicing factors (at mRNA and protein levels) in well characterized cohorts of human high-grade astrocytomas, mostly glioblastomas, compared to healthy brain control samples, being SRSF3, RBM22, PTBP1 and RBM3 able to perfectly discriminate between tumours and control samples, and between proneural-like or mesenchymal-like tumours versus control samples from different mouse models with gliomas. Results were confirmed in four additional and independent human cohorts. Silencing of SRSF3, RBM22, PTBP1 and RBM3 decreased aggressiveness parameters in vitro (e.g. proliferation, migration, tumorsphere-formation, etc.) and induced apoptosis, especially SRSF3. Remarkably, SRSF3 was correlated with patient survival and relevant tumour markers, and its silencing in vivo drastically decreased tumour development and progression, likely through a molecular/cellular mechanism involving PDGFRB and associated oncogenic signalling pathways (PI3K-AKT/ERK), which may also involve the distinct alteration of alternative splicing events of specific transcription factors controlling PDGFRB (i.e. TP73). Altogether, our results demonstrate a drastic splicing machinery-associated molecular dysregulation in glioblastomas, which could potentially be considered as a source of novel diagnostic and prognostic biomarkers as well as therapeutic targets for glioblastomas. Remarkably, SRSF3 is directly associated with glioblastoma development, progression, aggressiveness and patient survival and represents a novel potential therapeutic target to tackle this devastating pathology.
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Li, Yang I., Luis Sanchez-Pulido, Wilfried Haerty, and Chris P. Ponting. "RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts." Genome Research 25, no. 1 (December 18, 2014): 1–13. http://dx.doi.org/10.1101/gr.181990.114.
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Vernes, Sonja C., and Simon E. Fisher. "Unravelling neurogenetic networks implicated in developmental language disorders." Biochemical Society Transactions 37, no. 6 (November 19, 2009): 1263–69. http://dx.doi.org/10.1042/bst0371263.
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Childhood syndromes disturbing language development are common and display high degrees of heritability. In most cases, the underlying genetic architecture is likely to be complex, involving multiple chromosomal loci and substantial heterogeneity, which makes it difficult to track down the crucial genomic risk factors. Investigation of rare Mendelian phenotypes offers a complementary route for unravelling key neurogenetic pathways. The value of this approach is illustrated by the discovery that heterozygous FOXP2 (where FOX is forkhead box) mutations cause an unusual monogenic disorder, characterized by problems with articulating speech along with deficits in expressive and receptive language. FOXP2 encodes a regulatory protein, belonging to the forkhead box family of transcription factors, known to play important roles in modulating gene expression in development and disease. Functional genetics using human neuronal models suggest that the different FOXP2 isoforms generated by alternative splicing have distinct properties and may act to regulate each other's activity. Such investigations have also analysed the missense and nonsense mutations found in cases of speech and language disorder, showing that they alter intracellular localization, DNA binding and transactivation capacity of the mutated proteins. Moreover, in the brains of mutant mice, aetiological mutations have been found to disrupt the synaptic plasticity of Foxp2-expressing circuitry. Finally, although mutations of FOXP2 itself are rare, the downstream networks which it regulates in the brain appear to be broadly implicated in typical forms of language impairment. Thus, through ongoing identification of regulated targets and interacting co-factors, this gene is providing the first molecular entry points into neural mechanisms that go awry in language-related disorders.
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Williams, Allison Lesher, Vedbar Khadka, Mingxin Tang, Abigail Avelar, Kathryn J. Schunke, Mark Menor, and Ralph V. Shohet. "HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction." Physiological Genomics 50, no. 7 (July 1, 2018): 479–94. http://dx.doi.org/10.1152/physiolgenomics.00130.2017.
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Alternative splicing of RNA is an underexplored area of transcriptional response. We expect that early changes in alternatively spliced genes may be important for responses to cardiac injury. Hypoxia inducible factor 1 (HIF1) is a key transcription factor that rapidly responds to loss of oxygen through alteration of metabolism and angiogenesis. The goal of this study was to investigate the transcriptional response after myocardial infarction (MI) and to identify novel, hypoxia-driven changes, including alternative splicing. After ligation of the left anterior descending artery in mice, we observed an abrupt loss of cardiac contractility and upregulation of hypoxic signaling. We then performed RNA sequencing on ischemic heart tissue 1 and 3 days after infarct to assess early transcriptional changes and identified 89 transcripts with altered splicing. Of particular interest was the switch in Pkm isoform expression (pyruvate kinase, muscle). The usually predominant Pkm1 isoform was less abundant in ischemic hearts, while Pkm2 and associated splicing factors (hnRNPA1, hnRNPA2B1, Ptbp1) rapidly increased. Despite increased Pkm2 expression, total pyruvate kinase activity remained reduced in ischemic myocardial tissue. We also demonstrated HIF1 binding to PKM by chromatin immunoprecipitation, indicating a direct role for HIF1 in mediating this isoform switch. Our study provides a new, detailed characterization of the early transcriptome after MI. From this analysis, we identified an HIF1-mediated alternative splicing event in the PKM gene. Pkm1 and Pkm2 play distinct roles in glycolytic metabolism and the upregulation of Pkm2 is likely to have important consequences for ATP synthesis in infarcted cardiac muscle.
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Yamazaki, Takashi, Lizhi Liu, and James L. Manley. "TCF3 mutually exclusive alternative splicing is controlled by long-range cooperative actions between hnRNPH1 and PTBP1." RNA 25, no. 11 (August 7, 2019): 1497–508. http://dx.doi.org/10.1261/rna.072298.119.
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Georgilis, Athena, Sabrina Klotz, Christopher J. Hanley, Nicolas Herranz, Benedikt Weirich, Beatriz Morancho, Ana Carolina Leote, et al. "PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells." Cancer Cell 34, no. 1 (July 2018): 85–102. http://dx.doi.org/10.1016/j.ccell.2018.06.007.
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Vadlamudi, Yellamandayya, Debasish K. Dey, and Sun C. Kang. "Emerging Multi-cancer Regulatory Role of ESRP1: Orchestration of Alternative Splicing to Control EMT." Current Cancer Drug Targets 20, no. 9 (September 28, 2020): 654–65. http://dx.doi.org/10.2174/1568009620666200621153831.
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RNA binding proteins (RBPs) associate with nascent and mature RNAs to perform biological functions such as alternative splicing and RNA stability. Having unique RNA recognition binding motifs, RBPs form complexes with RNA in a sequence- and structure-based manner. Aberrant expressions of several RBPs have been identified in tumorigenesis and cancer progression. These uncontrolled RBPs affect several mechanisms, including cell proliferation, tumor growth, invasion, metastasis and chemoresistance. Epithelial splicing regulatory protein 1 (ESRP1) is a member of the hnRNP family of proteins that play a crucial role in regulating numerous cellular processes, including alternative splicing and translation of multiple genes during organogenesis. Abnormal expression of ESRP1 alters the cell morphology, and leads to cell proliferation and tumor growth during cancer progression. ESRP1 mediated alternative splicing of target genes, including CD44, FGFR, PTBP1, LYN, ENAH, SPAG1 and ZMYND8, results in cancer progression. In addition, ESRP1 also regulates circularization and biogenesis of circular RNAs such as circUHRF1, circNOL10 and circANKS1B, whose expressions have been identified as key factors in various cancers. This multi-functional protein is also involved in imposing stability of target mRNAs such as cyclin A2, and thereby cell cycle regulation. The scope of this review is to examine recent scientific data, outcomes of the up- and down-regulated proteins, and the role of ESRP1 in various cancers. We conclude by summarizing ESRP1 dysregulation and its consequences on target genes in various human cancers. Collectively, the consequences of ESRP1 mediated splicing in cancer cells suggest the role of ESRP1 in cell proliferation and chemoresistance via apoptosis and autophagy modulation, which could, therefore, be potential targets for cancer therapeutics.
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Iwamori, Naoki, Kaoru Tominaga, Tetsuya Sato, Kevin Riehle, Tokuko Iwamori, Yasuyuki Ohkawa, Cristian Coarfa, Etsuro Ono, and Martin M. Matzuk. "MRG15 is required for pre-mRNA splicing and spermatogenesis." Proceedings of the National Academy of Sciences 113, no. 37 (August 29, 2016): E5408—E5415. http://dx.doi.org/10.1073/pnas.1611995113.
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Splicing can be epigenetically regulated and involved in cellular differentiation in somatic cells, but the interplay of epigenetic factors and the splicing machinery during spermatogenesis remains unclear. To study these interactions in vivo, we generated a germline deletion of MORF-related gene on chromosome 15 (MRG15), a multifunctional chromatin organizer that binds to methylated histone H3 lysine 36 (H3K36) in introns of transcriptionally active genes and has been implicated in regulation of histone acetylation, homology-directed DNA repair, and alternative splicing in somatic cells. Conditional KO (cKO) males lacking MRG15 in the germline are sterile secondary to spermatogenic arrest at the round spermatid stage. There were no significant alterations in meiotic division and histone acetylation. Specific mRNA sequences disappeared from 66 germ cell-expressed genes in the absence of MRG15, and specific intronic sequences were retained in mRNAs of 4 genes in the MRG15 cKO testes. In particular, introns were retained in mRNAs encoding the transition proteins that replace histones during sperm chromatin condensation. In round spermatids, MRG15 colocalizes with splicing factors PTBP1 and PTBP2 at H3K36me3 sites between the exons and single intron of transition nuclear protein 2 (Tnp2). Thus, our results reveal that MRG15 is essential for pre-mRNA splicing during spermatogenesis and that epigenetic regulation of pre-mRNA splicing by histone modification could be useful to understand not only spermatogenesis but also, epigenetic disorders underlying male infertile patients.
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Tahmasebi, Soroush, Seyed Mehdi Jafarnejad, Ingrid S. Tam, Thomas Gonatopoulos-Pournatzis, Edna Matta-Camacho, Yoshinori Tsukumo, Akiko Yanagiya, et al. "Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2." Proceedings of the National Academy of Sciences 113, no. 44 (October 24, 2016): 12360–67. http://dx.doi.org/10.1073/pnas.1615540113.
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Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-β (Esrrb). Importantly, overexpression of YY2 directs the differentiation of mESCs into cardiovascular lineages. We show that the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) promotes the retention of an intron in the 5′-UTR of Yy2 mRNA that confers sensitivity to 4E-BP–mediated translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and document a multilayer regulatory mechanism that controls its expression.
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Fuentes-Fayos, A. C., M. C. Vázquez-Borrego, J. M. Jiménez-Vacas, L. Bejarano, C. Blanco-Acevedo, R. Sánchez-Sánchez, J. Solivera, M. A. Blasco, J. P. Castaño, and R. M. Luque. "P11.17 Splicing dysregulation drives glioblastoma malignancy: SRSF3 as a potential therapeutic target to impair glioblastoma progression." Neuro-Oncology 21, Supplement_3 (August 2019): iii46. http://dx.doi.org/10.1093/neuonc/noz126.163.
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Abstract Glioblastomas (GBMs) remain the deadliest human brain tumors, with poor prognosis despite years of research. Currently, standard therapeutic strategies to treat GBM are not efficient and common survival from diagnosis is ~12–16 months. Thus, identification of new diagnostic/prognostic/therapeutic tools to tackle GBMs is crucial. Emerging evidence indicates that the cellular machinery controlling alternative splicing is altered in tumor pathologies, leading to oncogenic splicing events linked to tumor progression. Accordingly, we aimed to determine the expression pattern of the spliceosome components (SCs) and splicing factors (SFs) in high-grade astrocytomas (HGAs), mostly GBMs, and to ascertain the potential consequences of its dysregulation on GBM development. To this end, expression levels of SCs core and selected SFs were measured using a customized-microfluidic qPCR array in a well-characterized cohort of HGAs (n=33). Our results unveiled a profound alteration in the expression of multiple SCs and SFs in HGAs compared to healthy brain control-samples, wherein levels of particular elements (SRSF3/RBM22/PTBP1/RBM3) enabled perfect discrimination between non-pathological vs. tumor human-tissues, and between proneural and mesenchymal-like GBMs vs. control samples in mouse-models. Results were confirmed in an independent validation-cohort (n=49) and available Microarray dataset (Murat), which revealed that the expression of these splicing elements was correlated with relevant tumor markers and with survival. Remarkably, SRSF3/RBM22/PTBP1/RBM3 silencing (using specific siRNAs) decreased several aggressiveness parameters in vitro (e.g. proliferation, migration, tumorsphere formation, VEGFA secretion, etc.) and induced apoptosis, being SRSF3 the most relevant element affecting these parameters. Hence, a preclinical mouse model (U87MG-xenografts) with SRSF3 silencing drastically decreased in vivo tumor development/progression (i.e. tumor size, %MKI67, mitosis number, etc.) likely through a molecular/cellular mechanism involving the regulation of PDGFRB expression and its associated oncogenic signaling pathways. Overall, our results demonstrate that there is a profound dysregulation of the splicing machinery (spliceosome core and SFs) in HGAs/GBMs, which is directly associated to the development/progression of GBMs. Furthermore, this study reveals that SRSF3 can be a novel biomarker of malignancy and a potential therapeutic target to impair GBMs progression.
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Campagne, Sébastien, Tebbe de Vries, Florian Malard, Pavel Afanasyev, Georg Dorn, Emil Dedic, Joachim Kohlbrecher, Daniel Boehringer, Antoine Cléry, and Frédéric H.-T. Allain. "An in vitro reconstituted U1 snRNP allows the study of the disordered regions of the particle and the interactions with proteins and ligands." Nucleic Acids Research 49, no. 11 (March 2, 2021): e63-e63. http://dx.doi.org/10.1093/nar/gkab135.
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Abstract U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon–intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.
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Babenko, Vladimir, Olga Redina, Dmitry Smagin, Irina Kovalenko, Anna Galyamina, and Natalia Kudryavtseva. "Elucidation of the Landscape of Alternatively Spliced Genes and Features in the Dorsal Striatum of Aggressive/Aggression-Deprived Mice in the Model of Chronic Social Conflicts." Genes 14, no. 3 (February 27, 2023): 599. http://dx.doi.org/10.3390/genes14030599.
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Both aggressive and aggression-deprived (AD) individuals represent pathological cases extensively studied in psychiatry and substance abuse disciplines. We employed the animal model of chronic social conflicts curated in our laboratory for over 30 years. In the study, we pursued the task of evaluation of the key events in the dorsal striatum transcriptomes of aggression-experienced mice and AD species, as compared with the controls, using RNA-seq profiling. We evaluated the alternative splicing-mediated transcriptome dynamics based on the RNA-seq data. We confined our attention to the exon skipping (ES) events as the major AS type for animals. We report the concurrent posttranscriptional and posttranslational regulation of the ES events observed in the phosphorylation cycles (in phosphoproteins and their targets) in the neuron-specific genes of the striatum. Strikingly, we found that major neurospecific splicing factors (Nova1, Ptbp1, 2, Mbnl1, 2, and Sam68) related to the alternative splicing regulation of cAMP genes (Darpp-32, Grin1, Ptpn5, Ppp3ca, Pde10a, Prkaca, Psd95, and Adora1) are upregulated specifically in aggressive individuals as compared with the controls and specifically AD animals, assuming intense switching between isoforms in the cAMP-mediated (de)phosphorylation signaling cascade. We found that the coding alternative splicing events were mostly attributed to synaptic plasticity and neural development-related proteins, while the nonsense-mediated decay-associated splicing events are mostly attributed to the mRNA processing of genes, including the spliceosome and splicing factors. In addition, considering the gene families, the transporter (Slc) gene family manifested most of the ES events. We found out that the major molecular systems employing AS for their plasticity are the ‘spliceosome’, ‘chromatin rearrangement complex’, ‘synapse’, and ‘neural development/axonogenesis’ GO categories. Finally, we state that approximately 35% of the exon skipping variants in gene coding regions manifest the noncoding variants subject to nonsense-mediated decay, employed as a homeostasis-mediated expression regulation layer and often associated with the corresponding gene expression alteration.
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SENOO, Manami, Hiroshi HOZOJI, Yu ISHIKAWA-YAMAUCHI, Takashi TAKIJIRI, Sho OHTA, Tomoyo UKAI, Mio KABATA, et al. "RNA-binding protein Ptbp1 regulates alternative splicing and transcriptome in spermatogonia and maintains spermatogenesis in concert with Nanos3." Journal of Reproduction and Development 66, no. 5 (2020): 459–67. http://dx.doi.org/10.1262/jrd.2020-060.
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Mironov, Aleksei, Stepan Denisov, Alexander Gress, Olga V. Kalinina, and Dmitri D. Pervouchine. "An extended catalogue of tandem alternative splice sites in human tissue transcriptomes." PLOS Computational Biology 17, no. 4 (April 7, 2021): e1008329. http://dx.doi.org/10.1371/journal.pcbi.1008329.
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Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 46k miSS with sufficient read support, 9k (20%) are significantly expressed above the expected noise level, and among them 2.5k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP), tissue-specific expression of miSS, and miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. The analysis of human primary cell transcriptomes suggested that both tissue-specific and cell-type-specific factors contribute to the regulation of miSS expression. More than 20% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 15% of maSS and not more than 54% of tissue-specific miSS are noisy, while the proportion of noisy splice sites among non-significantly expressed miSS is above 63%.
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Bielli, Pamela, Matteo Bordi, Valentina Di Biasio, and Claudio Sette. "Regulation of BCL-X splicing reveals a role for the polypyrimidine tract binding protein (PTBP1/hnRNP I) in alternative 5′ splice site selection." Nucleic Acids Research 42, no. 19 (October 7, 2014): 12070–81. http://dx.doi.org/10.1093/nar/gku922.
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Qiao, Lu, Ning Xie, Yuru Bai, Yan Li, Yongquan Shi, Jinhai Wang, and Na Liu. "Identification of Upregulated HNRNPs Associated with Poor Prognosis in Pancreatic Cancer." BioMed Research International 2019 (July 4, 2019): 1–11. http://dx.doi.org/10.1155/2019/5134050.
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Heterogeneous nuclear ribonucleoproteins (HNRNPs) are reported to play a crucial role in the pathogenic process of multiple malignancies. However, the expression patterns and prognostic values of HNRNPs in pancreatic cancer (PC) are lacking. In this study, several public databases were explored to identify the commonly upregulated HNRNPs in PC. The clinical significance of HNRNPL (heterogeneous nuclear ribonucleoproteins L) in PC was analyzed. We further performed a series of experiments to elucidate the biological functions of HNRNPL. Bioinformatics analysis including pathway enrichment and interactors with HNRNPL was used to explain the potential mechanisms of HNRNPL in PC pathogenesis. Herein, we reported that HNRNPL was commonly overexpressed in public databases and that high expression of HNRNPL in PC was positively associated with aggressive disease and poor overall survival. Downregulation of HNRNPL suppressed the abilities of migration and epithelial mesenchymal transition of PC cells in vitro, while depletion of HNRNPL did not affect the proliferation rate of PC cells. We further showed that HNRNPL might combine with RNA-binding protein, PTBP1, and function as a part of the spliceosome to regulate alternative splicing of target genes in the occurrence and development of PC. HNRNPL could be employed as an innovative prognostic biomarker and therapeutic target for PC.
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Hershberger, Courtney, James Hiznay, Rosemary Dietrich, Xiaorong Gu, Cassandra M. Hirsch, Amy Graham, Bartlomiej P. Przychodzen, et al. "LUC7L2 Is a Novel RNA-Splicing Regulatory Factor Mutated in Myelodysplastic Syndromes." Blood 132, Supplement 1 (November 29, 2018): 3073. http://dx.doi.org/10.1182/blood-2018-99-112838.
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Abstract Myelodysplastic syndromes (MDS) are unique among cancers because of the frequent occurrence of somatic mutations impacting spliceosome machinery. At least 65% of MDS patients harbor a mutation in one of several splicing factors including U2AF1, SF3B1 and SRSF2. Whole exome sequencing of MDS bone marrow uncovered somatic frameshift mutations in LUC7L2, the mammalian ortholog of a yeast splicing factor. LUC7L2 is located in the most commonly deleted region of chromosome 7. Deletions and frameshifts lead to haploinsufficient expression and therefore it can be approximated that a combined 14% of MDS patients have low expression of LUC7L2. Restoring expression of LUC7L2 in del(7q)-iPSCs partially rescues the differentiation of iPSCs into CD45+ myeloid progenitors. Although perhaps partly due to associated losses of other genes on chromosome 7, low expression of LUC7L2 correlates with a poorer patient prognosis, so its haploinsufficiency may play an important role in bone marrow failure. While U2AF1, SF3B1, and SRSF2 are well-characterized splicing factors, the function of LUC7L2 in pre-mRNA splicing is unexamined and its role in the MDS pathogenesis is undefined. We hypothesize that low expression of LUC7L2 results in the aberrant splicing of oncogenes and tumor suppressor gene transcripts thus reducing expression or altering function and contributing to the pathogenesis of MDS. We have characterized LUC7L2 as an alternative splicing regulatory protein that plays a repressive role in the regulation of alternative RNA splicing. We generated HEK-293 cells overexpressing V5-tagged LUC7L2 for immunoprecipitation-mass spectrometry, to ascertain protein interactions with LUC7L2. LUC7L2 co-immunoprecipitated with splicing regulators which are involved in splice site recognition. We performed cross-linking-IP-high-throughput-sequencing (CLIP-seq) to identify LUC7L2 binding sites on RNA. We identified 301 LUC7L2 RNA-binding sites as well as binding sites on U1 and U2 which is common for splicing regulatory proteins. Metagene analysis of these binding sites showed that LUC7L2 bound near splice sites in exonic sequences. We knocked down LUC7L2 expression in HEK293 and K562 cells to phenocopy the frameshifts and deletions observed in MDS patients. We used a PCR-based assay to measure the splicing efficiency of introns near LUC7L2-binding sites. Knockdown of LUC7L2 increased the splicing efficiency of 8/13 selected introns; this suggests that LUC7L2 represses selective splice site usage. We also performed RNA-seq to characterize global mis-splicing events. Analysis of RNA transcripts revealed a multitude of splicing changes, including enhanced exclusion of alternative introns. Knockdown LUC7L2 cells exhibited-altered expression of other splicing factors; this could have further contributed to the vast number of splicing changes observed. To identify specific splicing changes that could contribute to the pathogenesis of MDS, we compared the splicing profiles of LUC7L2-knockdown in K562 cells with RNA-seq data from K562 cells expressing U2AF1S34F, SRSF2P95H or SF3B1K700E. This analysis yielded several exon-skipping splicing patterns in cancer-relevant transcripts, such as oncogene PRC1, splicing factor PTBP1 and MRPL33. Additionally, we noticed commonly mis-spliced transcripts among the four datasets in which the missplicing events occurred in the functional domain, potentially conferring a functional change. Surprisingly, we observed missplicing of U2AF1 in LUC7L2-knockdown, SRSF2P95H, and SF3B1K700E K562 cells, which altered the length of the RNA-recognition UHM domain by inclusion of a mutually exclusive exon or retention of an intron. In this way, low expression of LUC7L2, or point mutants U2AF1S34F, SRSF2P95H, and SF3B1K700E,could alter U2AF1 function as a distal convergence point. In summary, we identified a novel splicing factor implicated in the pathogenesis of MDS. We characterized LUC7L2 as a splicing repressor and discovered many splicing changes caused by low expression of LUC7L2. Several genes were also mis-spliced in U2AF1S34F, SRSF2P95H and SF3B1K700E K562 cells targeting these for further study. Commonly mis-spliced targets such as U2AF1 may indicate that some of the novel therapeutics may have spliceosome mutation agnostic effects. If this applies to the LUC7L2 mutations, then they may also be effective in del7/del7q cases. Disclosures Carraway: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.
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Wu, Haili, Jin’e Du, Chenglu Li, Hanqing Li, Huiqin Guo, and Zhuoyu Li. "Kaempferol Can Reverse the 5-Fu Resistance of Colorectal Cancer Cells by Inhibiting PKM2-Mediated Glycolysis." International Journal of Molecular Sciences 23, no. 7 (March 24, 2022): 3544. http://dx.doi.org/10.3390/ijms23073544.
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Resistance to 5-Fluorouracil (5-Fu) chemotherapy is the main cause of treatment failure in the cure of colon cancer. Therefore, there is an urgent need to explore a safe and effective multidrug resistance reversal agent for colorectal cancer, which would be of great significance for improving clinical efficacy. The dietary flavonoid kaempferol plays a key role in the progression of colorectal cancer and 5-Fu resistance. However, the molecular mechanism of kaempferol in reversing 5-Fu resistance in human colorectal cancer cells is still unclear. We found that kaempferol could reverse the drug resistance of HCT8-R cells to 5-Fu, suggesting that kaempferol alone or in combination with 5-Fu has the potential to treat colorectal cancer. It is well known that aerobic glycolysis is related to tumor growth and chemotherapy resistance. Indeed, kaempferol treatment significantly reduced glucose uptake and lactic acid production in drug-resistant colorectal cancer cells. In terms of mechanism, kaempferol promotes the expression of microRNA-326 (miR-326) in colon cancer cells, and miR-326 could inhibit the process of glycolysis by directly targeting pyruvate kinase M2 isoform (PKM2) 3′-UTR (untranslated region) to inhibit the expression of PKM2 or indirectly block the alternative splicing factors of PKM mRNA, and then reverse the resistance of colorectal cancer cells to 5-Fu. Taken together, our data suggest that kaempferol may play an important role in overcoming resistance to 5-Fu therapy by regulating the miR-326-hnRNPA1/A2/PTBP1-PKM2 axis.
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Scholl, Amanda, Alexander Muselman, and Dong-Er Zhang. "An Intronic Suppressor Element Regulates RUNX1 Alternative Polyadenylation." Blood 126, no. 23 (December 3, 2015): 3578. http://dx.doi.org/10.1182/blood.v126.23.3578.3578.
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Abstract Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.
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Patiño-Trives, A. M., C. Perez-Sanchez, A. Ibañez-Costa, P. S. Laura, M. Luque-Tévar, I. Arias de la Rosa, M. C. Ábalos-Aguilera, et al. "OP0038 SPLICEOSOME ALTERATIONS IN LEUCOCYTES FROM APS, SLE AND SLE+APS PATIENTS ARE CLOSELY RELATED TO THEIR MAIN CLINICAL FEATURES." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 20.2–20. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2485.
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Background:To date, although multiple molecular approaches have illustrated the various aspects of Primary Antiphospholipid Syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome plus lupus (APS plus SLE), no study has so far fully characterized the potential role of posttranscriptional regulatory mechanisms such as the alternative splicing.Objectives:To identify shared and differential changes in the splicing machinery of immune cells from APS, SLE and APS plus SLE patients, and their involvement in the activity and clinical profile of these autoimmune disorders.Methods:Monocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS plus SLE) and 50 healthy donors (HD) were purified by immunomagnetic selection. Then, selected elements of the splicing machinery were evaluated using a microfluidic qPCR array (Fluidigm). In parallel, extensive clinical/serological evaluation was performed, comprising disease activity, thrombosis and renal involvement, along with autoantibodies, acute phase reactants, complement and inflammatory molecules. Molecular clustering analyses and correlation/association studies were developed.Results:Patients with primary APS, SLE and APS plus SLE displayed significant and specific alterations in the splicing machinery components in comparison with HD, that were further specific for each leukocyte subset. Besides, these alterations were associated with distinctive clinical features.Hence, in APS, clustering analysis allowed to identify two sets of patients representing different molecular profile groups with respect to the expression levels of splicing machinery components. Principal component analyses confirmed a clear separation between patients. Clinically, cluster 1 characterized patients with higher thrombotic episodes and recurrences than cluster 2 and displayed a higher adjusted global APS score (aGAPSS). Accordingly, these patients showed higher levels of inflammatory mediators than cluster 2.Similarly, in patients with APS plus SLE, clustering analysis allowed to identify two sets of patients showing differential expression of splicing machinery components. Clinical and laboratory profiles showed that cluster 2 characterized patients that had suffered more thrombotic recurrences, most of them displaying an aGAPSS over 12 points and expressing higher levels of inflammatory mediators than cluster 1. The incidence of lupus nephropathy was similarly represented in both clusters.Lastly, in SLE patients, molecular clustering analysis identified two sets of patients showing distinctive clinical features. One cluster characterized most of the patients positive for anti-dsDNA antibodies, further suffering lupus nephropathy, and a high proportion of them also presenting atheroma plaques and high levels of inflammatory mediators.Correlation studies further demonstrated that several deranged splicing machinery components in immune cells (i.e. SF3B1tv1, PTBP1, PRP8 and RBM17) were linked to the autoimmune profile of the three autoimmune diseases, albeit in a specific way on each disorder. Accordingly, in vitro treatment of HD lymphocytes with aPL-IgG or anti-dsDNA-IgG changed the expression of spliceosome components also found altered in vivo in the three autoimmune diseases. Finally, the induced over/downregulated expression of selected spliceosome components in leukocytes modulated the expression of inflammatory cytokines, changed the procoagulant/adhesion activities of monocytes and regulated NETosis in neutrophils.Conclusion:1) The splicing machinery, profoundly altered in leukocytes from APS, APS plus SLE and SLE patients, is closely related to the activity of these diseases, their autoimmune and inflammatory profiles. 2) The analysis of the splicing machinery allows the segregation of APS, APS plus SLE and SLE, with specific components explaining the CV risk and renal involvement in these highly related autoimmune disorders.Acknowledgements:Funded by ISCIII, PI18/00837 and RIER RD16/0012/0015 co-funded with FEDERDisclosure of Interests:None declared
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Miao, Hui, Fan Wu, Yu Li, Chenyu Qin, Yongyun Zhao, Mingfeng Xie, Hongyuan Dai, et al. "MALAT1 modulates alternative splicing by cooperating with the splicing factors PTBP1 and PSF." Science Advances 8, no. 51 (December 23, 2022). http://dx.doi.org/10.1126/sciadv.abq7289.
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Understanding how long noncoding RNAs (lncRNAs) cooperate with splicing factors (SFs) in alternative splicing (AS) control is fundamental to human biology and disease. We show that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-documented AS-implicated lncRNA, regulates AS via two SFs, polypyrimidine tract–binding protein 1 (PTBP1) and PTB-associated SF (PSF). MALAT1 stabilizes the interaction between PTBP1 and PSF, thereby forming a functional module that affects a network of AS events. The MALAT1-stabilized PTBP1/PSF interaction occurs in multiple cellular contexts; however, the functional module, relative to MALAT1 only, has more dominant pathological significance in hepatocellular carcinoma. MALAT1 also stabilizes the PSF interaction with several heterogeneous nuclear ribonucleoparticle proteins other than PTBP1, hinting a broad role in AS control. We present a model in which MALAT1 cooperates with distinct SFs for AS regulation and pose that, relative to analyses exclusively performed for lncRNAs, a comprehensive consideration of lncRNAs and their binding partners may provide more information about their biological functions.
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Choksi, Arpankumar, Apoorva Parulekar, Richa Pant, Vibhuti Kumar Shah, Ramakrishna Nimma, Priyanka Firmal, Smriti Singh, Gopal C. Kundu, Sanjeev Shukla, and Samit Chattopadhyay. "Tumor suppressor SMAR1 regulates PKM alternative splicing by HDAC6-mediated deacetylation of PTBP1." Cancer & Metabolism 9, no. 1 (April 16, 2021). http://dx.doi.org/10.1186/s40170-021-00252-x.
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Abstract Background Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1. Methods Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed. Results The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner. Conclusions SMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.
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Lan, Chungen, Huikun Zhang, Kezhen Wang, Xiaoli Liu, Yawen Zhao, Zhifang Guo, Ning Zhang, et al. "The alternative splicing of intersectin 1 regulated by PTBP1 promotes human glioma progression." Cell Death & Disease 13, no. 9 (September 28, 2022). http://dx.doi.org/10.1038/s41419-022-05238-1.
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AbstractIntersectin 1 (ITSN1) contains two isoforms: ITSN1-S and ITSN1-L, which are highly regulated by alternative splicing. Our previous results showed that the two isoforms of ITSN1 displayed opposite functions: ITSN1-S promoted glioma development, while ITSN1-L exerted an inhibitory role in glioma progression. In this study, our transcriptome analysis using a large glioma cohort indicated that the ratio of ITSN1-S/ITSN1-L was positively correlated with glioma grading and poor prognosis. We identified the RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1) as an ITSN1 pre-mRNA interaction protein through RNA pull-down assay and RNA immunoprecipitation assay. Knockdown of PTBP1 decreased the ratio of ITSN1-S/ITSN1-L. Minigene reporter assay and mutation analyses further confirmed PTBP1 targeted polypyrimidine sequences on ITSN1 exon 30 (TTGCACTTCAGTATTTT) and promoted the inclusion of ITSN1 exon 30. Subsequently, silencing PTBP1 inhibited glioma cell proliferation, migration, and invasion by down-regulating the ratio of ITSN1-S/ITSN1-L. Taken together, our study provides a novel mechanism that PTBP1 modulates the alternative splicing of ITSN1 and promotes glioma proliferation and motility by up-regulating the ratio of ITSN1-S/ITSN1-L, thereby highlighting that PTBP1 may be an attractive therapeutic target for gliomas.
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Linares, Anthony J., Chia-Ho Lin, Andrey Damianov, Katrina L. Adams, Bennett G. Novitch, and Douglas L. Black. "The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation." eLife 4 (December 24, 2015). http://dx.doi.org/10.7554/elife.09268.
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The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.
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Taylor, William, Stephane Deschamps, David Reboutier, Luc Paillard, Agnes Mereau, and Yann Audic. "The Splicing Factor PTBP1 Represses TP63γ Isoform Production in Squamous Cell Carcinoma." Cancer Research Communications, December 5, 2022. http://dx.doi.org/10.1158/2767-9764.crc-22-0350.
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Abstract The TP63 gene encodes the p63 transcription factor. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, β, γ and δ. The regulatory functions of p63 are isoform-specific. The α isoform inhibits the epithelial to mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Using TCGA data, we observed that a higher proportion of the TP63γ isoform is a detrimental factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC) and is accompanied by the downregulation of desmosomal genes. By a correlation-based approach we investigated the regulation of the production of the TP63γ isoform. According to our analysis of GTEx data, the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ in several tissues. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines, keratinocyte or Xenopus embryos leads to an increase in TP63γ isoform abundance. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. Intronic regions around the TP63γ-specific exon were sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Together, these results identify TP63γ as an unfavorable prognostic marker in HNSCC, and identify PTBP1 as the first direct splicing regulator of TP63γ production and a potential route toward TP63 isoform control.
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Zhang, Song, Haitao Wan, and Xiaobo Zhang. "LncRNA LHFPL3-AS1 contributes to tumorigenesis of melanoma stem cells via the miR-181a-5p/BCL2 pathway." Cell Death & Disease 11, no. 11 (November 2020). http://dx.doi.org/10.1038/s41419-020-03141-1.
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Abstract Long noncoding RNAs (lncRNAs) are recognized as a new area for cancer therapy. B-cell lymphoma-2 (Bcl-2)-mediated suppression of apoptosis is an important molecular hallmark of cancer. However, the influence of lncRNA on the regulation of oncogenic Bcl-2 in cancer stem cells has not been explored. In this study, our findings revealed that the lncRNA LHFPL3-AS1-long, generated from the polypyrimidine tract binding protein 1 (PTBP1)-mediated splicing of the LHFPL3-AS1 precursor, upregulated BCL2 protein to contribute to tumorigenesis of melanoma stem cells. The in vitro and in vivo results showed that LHFPL3-AS1-long directly interacted with miR-181a-5p to inhibit the mRNA degradation of Bcl-2 (the target of miR-181), thus suppressing apoptosis of melanoma stem cells. The splicing factor PTBP1 regulated the alternative splicing of LHFPL3-AS1 transcript by preferentially binding to the motifs located in exon3 of LHFPL3-AS1 precursor, leading to the biogenesis of LHFPL3-AS1-long in melanoma stem cells. In patients with melanoma, the expressions of PTBP1 and LHFPL3-AS1 were significantly upregulated compared with the healthy donors. Therefore, our study revealed a mechanistic crosstalk among an onco-splicing factor, lncRNA and tumorigenesis of melanoma stem cells, enabling PTBP1 and LHFPL3-AS1 to serve as the attractive therapeutic targets for melanoma.
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Monzón-Casanova, Elisa, Louise S. Matheson, Kristina Tabbada, Kathi Zarnack, Christopher WJ Smith, and Martin Turner. "Polypyrimidine tract-binding proteins are essential for B cell development." eLife 9 (February 21, 2020). http://dx.doi.org/10.7554/elife.53557.
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Polypyrimidine tract-binding protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle.
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van Bergeijk, Petra, Uthpala Seneviratne, Estel Aparicio-Prat, Robert Stanton, and Samuel A. Hasson. "SRSF1 and PTBP1 Aretrans-Acting Factors That Suppress the Formation of a CD33 Splicing Isoform Linked to Alzheimer’s Disease Risk." Molecular and Cellular Biology 39, no. 18 (June 17, 2019). http://dx.doi.org/10.1128/mcb.00568-18.
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ABSTRACTA single nucleotide polymorphism (SNP) in exon 2 of the CD33 gene is associated with reduced susceptibility to late-onset Alzheimer’s disease (AD) and causal for elevated mRNA lacking exon 2. In contrast to full-length CD33, transcripts lacking exon 2 result in CD33 protein unable to suppress activation responses in myeloid cells, including microglia. Currently, little is known about the regulation of CD33 exon 2 splicing. Using functional genomics and proteomic approaches, we found that SRSF1 and PTBP1 act as splicing enhancers to increase CD33 exon 2 inclusion in mRNA. Binding of PTBP1 to RNA sequences proximal to the intron 1-exon 2 splice junction is altered by the SNP and represents a potential mechanism behind the SNP-genotype dependent alternative splicing. Our studies also reveal that binding of SRSF1 to the CD33 RNA is not altered by the SNP genotype. Instead, a putative SRSF1 binding sequence at the 3′ end of exon 2 directs CD33 exon 2 inclusion into the mRNA, indicating that PTBP1 and SRSF1 promote full-length isoform expression through different mechanisms. Our findings shed light on molecular interactions that regulate CD33 exon 2 splicing, ultimately impacting receptor expression on the cell surface. These data aid in the understanding of CD33’s regulation of microglial signaling underpinning the AD genetic associations.
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Hensel, Jessica A., Sarah-Anne E. Nicholas, Amy L. Kimble, Arjun S. Nagpal, Omar M. F. Omar, Jordan D. Tyburski, Evan R. Jellison, et al. "Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions." Proceedings of the National Academy of Sciences 119, no. 30 (July 18, 2022). http://dx.doi.org/10.1073/pnas.2122227119.
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NF-κB–mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.
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D, Dhananjaya, Kuan-Yang Hung, and Woan-Yuh Tarn. "RBM4 Modulates Radial Migration via Alternative Splicing of Dab1 during Cortex Development." Molecular and Cellular Biology 38, no. 12 (March 26, 2018). http://dx.doi.org/10.1128/mcb.00007-18.
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ABSTRACT The RNA-binding motif 4 (RBM4) protein participates in cell differentiation via its role in regulating the expression of tissue-specific or developmentally regulated mRNA splice isoforms. RBM4 is expressed in embryonic brain during development; it is initially enriched in the ventricular zone/subventricular zone and subsequently distributed throughout the cerebral cortex. Rbm4a knockout brain exhibited delayed migration of late-born neurons. Using in utero electroporation, we confirmed that knockdown of RBM4 impaired cortical neuronal migration. RNA immunoprecipitation with high-throughput sequencing identified Disabled-1 ( Dab1 ), which encodes a critical reelin signaling adaptor, as a potential target of RBM4. Rbm4a knockout embryonic brain showed altered Dab1 isoform ratios. Overexpression of RBM4 promoted the inclusion of Dab1 exons 7 and 8 (7/8), whereas its antagonist polypyrimidine tract-binding protein 1 (PTBP1) acted in an opposite manner. RBM4 directly counteracted the effect of PTBP1 on exon 7/8 selection. Finally, we showed that the full-length Dab1, but not exon 7/8-truncated Dab1, rescued neuronal migration defects in RBM4-depleted neurons, indicating that RBM4 plays a role in neuronal migration via modulating the expression of Dab1 splice isoforms. Our findings imply that RBM4 is necessary during brain development and that its deficiency may lead to developmental brain abnormality.
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Vaquero-Garcia, Jorge, Alejandro Barrera, Matthew R. Gazzara, Juan González-Vallinas, Nicholas F. Lahens, John B. Hogenesch, Kristen W. Lynch, and Yoseph Barash. "A new view of transcriptome complexity and regulation through the lens of local splicing variations." eLife 5 (February 1, 2016). http://dx.doi.org/10.7554/elife.11752.
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Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.
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Cheng, Shanshan, Debleena Ray, Raymond Teck Ho Lee, Kishore Babu Naripogu, Permeen Akhtar Bt Mohamed Yusoff, Pamela Bee Leng Goh, Yujing Liu, et al. "A functional network of gastric-cancer-associated splicing events controlled by dysregulated splicing factors." NAR Genomics and Bioinformatics 2, no. 2 (February 28, 2020). http://dx.doi.org/10.1093/nargab/lqaa013.
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Abstract Comprehensive understanding of aberrant splicing in gastric cancer is lacking. We RNA-sequenced 19 gastric tumor–normal pairs and identified 118 high-confidence tumor-associated (TA) alternative splicing events (ASEs) based on high-coverage sequencing and stringent filtering, and also identified 8 differentially expressed splicing factors (SFs). The TA ASEs occurred in genes primarily involved in cytoskeletal organization. We constructed a correlative network between TA ASE splicing ratios and SF expression, replicated it in independent gastric cancer data from The Cancer Genome Atlas and experimentally validated it by knockdown of the nodal SFs (PTBP1, ESRP2 and MBNL1). Each SF knockdown drove splicing alterations in several corresponding TA ASEs and led to alterations in cellular migration consistent with the role of TA ASEs in cytoskeletal organization. We have therefore established a robust network of dysregulated splicing associated with tumor invasion in gastric cancer. Our work is a resource for identifying oncogenic splice forms, SFs and splicing-generated tumor antigens as biomarkers and therapeutic targets.
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Mochizuki, Yasushi, Ryo Funayama, Matsuyuki Shirota, Yuna Kikukawa, Masahiro Ohira, Hideaki Karasawa, Minoru Kobayashi, Shinobu Ohnuma, Michiaki Unno, and Keiko Nakayama. "Alternative microexon splicing by RBFOX2 and PTBP1 is associated with metastasis in colorectal cancer." International Journal of Cancer, August 16, 2021. http://dx.doi.org/10.1002/ijc.33758.
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