The mitotic phosphorylation of p54nrbmodulates its RNA binding activity

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.