The mitotic phosphorylation of p54nrbmodulates its RNA binding activity

Abstract

The RNA-binding protein p54nrbis involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54nrblocalizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54nrbbecomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54nrbphosphorylation 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-p54nrbconstruct 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 p54nrb, is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54nrbin either interphase or mitotic cells, suggesting that p54nrb–Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54nrbstill interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54nrb. In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54nrbconstruct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54nrbinteraction with RNA could be selectively modulated by phosphorylation during mitosis.