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首页> 外文期刊>Biochemistry and Cell Biology >The mitotic phosphorylation of p54(nrb) modulates its RNA binding activity.
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The mitotic phosphorylation of p54(nrb) modulates its RNA binding activity.

机译：P54（NRB）的丝分裂磷酸化调节其RNA结合活性。

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摘要

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.
机译：RNA结合蛋白P54（NRB）涉及许多核过程，包括转录，RNA加工和超型RNA的保留。在间胞间细胞中，P54（NRB）通过与非编码RNA Neat1的相互作用使P54（NRB）定位于核状物中并浓缩蛋白质伴侣。在有丝分裂期间，P54（NRB）变得多磷酸化，并且不知道该修饰的效果。在本研究中，我们表明P54（NRB）磷酸化不会影响与其蛋白质合作伙伴的相互作用，而是减少其通用RNA结合能力。生物化学测定表明，通过CDK1的GST-P54（NRB）构建体的体外磷酸化废除了与5'剪接部位RNA序列的相互作用。定向诱变的诱变表明，位于P54（NRB）的RRM串联结构域的N-末端的苏氨酸15残基涉及该抑制。体内分析表明，Neat1 NCRNA在间差异或有丝分裂细胞中用P54（NRB）与P54（NRB）共同免疫沉淀，表明P54（NRB）-Neat1相互作用未通过磷酸化调节。因此，体外磷酸化的GST-P54（NRB）仍然与PIR-1 RNA相互作用，已知与P54（NRB）相互作用。体外RNA结合测定表明，GST-P54（NRB）构建体的CDK1-磷酸化废除其与均麻聚合物聚（a），聚（c）和聚（u）的相互作用，但不具有聚（g）。这些数据表明P54（NRB）与RNA的相互作用可以通过在有丝分裂期间磷酸化选择性地调节。

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