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.

NONO and tumorigenesis: More than splicing

“…CDK1 also can phosphorylate T15 in the N‐terminal of NONO in vitro. Two independent studies found that NONO could be tyrosine‐phosphorylated; however, they could not exclude that the p‐Tyr antibodies could non‐specifically bind NONO, and a p‐Tyr antibody was found having non‐specific binding affinity to NONO in another study later . Furthermore, crystal structure of NONO shows that the five Tyr residues of NONO are not in favourable positions to be phosphorylated because of steric hindrance .…”

“…Structural and biological data suggest DBHS proteins rarely play their biological roles alone, their interactions with various proteins are regulated by post‐translational modifications . NONO were proved to be phosphorylated in mitosis in some independent studies . CDK1 phosphorylates T412, T430 and T452 in the C‐terminal extremity of NONO, subsequently the prolyl isomerase Pin1 interacts with the phosphorylated NONO.…”

“…Furthermore, crystal structure of NONO shows that the five Tyr residues of NONO are not in favourable positions to be phosphorylated because of steric hindrance . Even though, the tyrosine residues still regulate NONO’s multifarious nuclear functions …”

Alternative RNA splicing of the GIT1 gene is associated with neuroendocrine prostate cancer

“…Real‐time qPCR and immunoblot assays were carried out as previously described . Antibodies and primers used are listed in Tables and , respectively.…”

“…Real-time qPCR and immunoblot assays were carried out as previously described. [26][27][28] Antibodies and primers used are listed in Tables S2 and S3, respectively. Three biological repeats were carried out for each experiment.…”