title: The mitotic phosphorylation of p54(nrb) modulates its RNA binding activity.
abstract: The RNA-binding protein p54(nrb) is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54(nrb) localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54(nrb) becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54(nrb) phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-p54(nrb) construct by CDK1 abolishes the interaction with 5' splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of p54(nrb), is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54(nrb) in either interphase or mitotic cells, suggesting that p54(nrb)-Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54(nrb) still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54(nrb). In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54(nrb) construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54(nrb) interaction with RNA could be selectively modulated by phosphorylation during mitosis.
authors.affiliation: PROTEO Research Center and Département de biologie moléculaire, biochimie médicale et pathologie, Université Laval, Québec, Canada.
authors.forename: Céline
authors.initials: C
authors.lastname: Bruelle
authors.forename: Mikaël
authors.initials: M
authors.lastname: Bédard
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Blier
authors.forename: Martin
authors.initials: M
authors.lastname: Gauthier
authors.forename: Abdulmaged M
authors.initials: AM
authors.lastname: Traish
authors.forename: Michel
authors.initials: M
authors.lastname: Vincent
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007399:Interphase; D008938:Mitosis; D046912:Multiprotein Complexes / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D050810:Octamer Transcription Factors / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000378:metabolism*; D022661:RNA, Untranslated / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins