title: Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb.
abstract: The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein-protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 232: 852-861, 2017. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.
authors.forename: Ahn R
authors.initials: AR
authors.lastname: Lee
authors.affiliation: Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.
authors.forename: Wayne
authors.initials: W
authors.lastname: Hung
authors.affiliation: Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.
authors.forename: Ning
authors.initials: N
authors.lastname: Xie
authors.affiliation: Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Urology, Third Xiangya Hospital, Institute of Prostate Disease, Central South University, Changsha, China.
authors.forename: Leye
authors.initials: L
authors.lastname: He
authors.affiliation: Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada.|Department of Urology, Third Xiangya Hospital, Institute of Prostate Disease, Central South University, Changsha, China.
authors.forename: Xuesen
authors.initials: X
authors.lastname: Dong
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D012326:RNA Splicing / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D011944:Receptors, Androgen; D012097:Repressor Proteins; D019000:Phosphotyrosine; D014443:Tyrosine