title: Suggestive evidence on the involvement of polypyrimidine-tract binding protein in regulating alternative splicing of MAP/microtubule affinity-regulating kinase 4 in glioma.
abstract: MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase that phosphorylates microtubule-associated proteins taking part in the regulation of microtubule dynamics. MARK4 is expressed in two spliced isoforms characterized by inclusion (MARK4S) or exclusion (MARK4L) of exon 16. The distinct expression profiles in the central nervous system and their imbalance in gliomas point to roles of MARK4L and MARK4S in cell proliferation and cell differentiation, respectively. Having ruled out mutations and transcription defects, we hypothesized that alterations in the expression of splicing factors may underlie deregulated MARK4 expression in gliomas. Bioinformatic analysis revealed four putative polypyrimidine-tract binding (PTB) protein binding sites in MARK4 introns 15 and 16. Glioma tissues and glioblastoma-derived cancer stem cells showed, compared with normal brain, significant overexpression of PTB, correlated with high MARK4L mRNA expression. Splicing minigene assays revealed a functional intronic splicing silencer in MARK4 intron 15, but mutagenesis of the PTB binding site in this region did not affect minigene splicing, suggesting that PTB may bind to a splicing silencer other than the predicted one and synergistically acting with the other predicted PTB sites. Electrophoretic mobility shift assays coupled with mass spectrometry confirmed binding of PTB to the polypyrimidine tract of intron 15, and thus its involvement in MARK4 alternative splicing. This finding, along with evidence of PTB overexpression in gliomas and glioblastoma-derived cancer stem cells and differentiated progeny, merged in pointing out the involvement of PTB in the switch to MARK4L, consistent with its established role in driving oncogenic splicing in brain tumors.
authors.affiliation: Department of Health Sciences, Medical Genetics, Università degli Studi di Milano, via Antonio di Rudinì 8, 20142 Milan, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Fontana
authors.affiliation: Department of Health Sciences, Medical Genetics, Università degli Studi di Milano, via Antonio di Rudinì 8, 20142 Milan, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Rovina
authors.affiliation: Department of Health Sciences, Medical Genetics, Università degli Studi di Milano, via Antonio di Rudinì 8, 20142 Milan, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Novielli
authors.affiliation: Department of Animal Pathology, Hygiene and Veterinary Public Health, Università degli Studi di Milano, Via Celoria, 10, 20133 Milan, Italy; Fondazione Filarete, Viale Ortles 22/4, 20139 Milan, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Maffioli
authors.affiliation: Department of Animal Pathology, Hygiene and Veterinary Public Health, Università degli Studi di Milano, Via Celoria, 10, 20133 Milan, Italy; Fondazione Filarete, Viale Ortles 22/4, 20139 Milan, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Tedeschi
authors.affiliation: Department of Health Sciences, Medical Genetics, Università degli Studi di Milano, via Antonio di Rudinì 8, 20142 Milan, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Magnani
authors.affiliation: Department of Health Sciences, Medical Genetics, Università degli Studi di Milano, via Antonio di Rudinì 8, 20142 Milan, Italy; Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Zucchi, 18, 20095 Cusano Milanino, Italy. Electronic address: lidia.larizza@unimi.it.
authors.forename: L
authors.initials: L
authors.lastname: Larizza
mesh_terms: D017398:Alternative Splicing; D001665:Binding Sites; D001932:Brain Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D019295:Computational Biology; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D057809:HEK293 Cells; D006801:Humans; D007438:Introns; D007527:Isoenzymes; D009154:Mutation; D060787:Neoplasm Grading; D014411:Neoplastic Stem Cells / Q000201:enzymology* / Q000473:pathology; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein; C483047:MARK4 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Alternative splicing; Glioma; MARK4; PTB; Splicing minigene