Nuclear Inhibitor of Protein Phosphatase-1 (NIPP1) Directs Protein Phosphatase-1 (PP1) to Dephosphorylate the U2 Small Nuclear Ribonucleoprotein Particle (snRNP) Component, Spliceosome-associated Protein 155 (Sap155)

Haruhiko Koseki,Kunimi Kikuchi,Masami Sato,Sei-Eun Kim,Miyuki Nomura,Shinya Mitsuhashi,Hiroshi Shima,Takeshi Kawamura,Nobuhiro Tanuma,Yao Tsubaki,Monique Beullens,Mathieu Bollen,Kyoichi Isono

doi:10.1074/jbc.m805468200

Abstract

Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-DeltaC) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -DeltaC resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap155 hyperphosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155.

Highlights

Pre-mRNA splicing is an essential step for expression of most genes in metazoans
We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155, an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation
Intron excision from a nascent transcript is achieved by pre-mRNA splicing catalyzed by the spliceosome, a macromolecular complex consisting of five small nuclear ribonucleoprotein particles4 and a large number of nonsnRNP proteins

Summary

Introduction

Pre-mRNA splicing is an essential step for expression of most genes in metazoans. Intron excision from a nascent transcript is achieved by pre-mRNA splicing catalyzed by the spliceosome, a macromolecular complex consisting of five small nuclear ribonucleoprotein particles (snRNPs) and a large number of nonsnRNP proteins. Sf3a and Sf3b are essential early in the splicing reaction, they are apparently not required for the second catalytic step It is not known what triggers exchange of proteins during spliceosome transitions. Shi et al [6] reported that the protein Ser/Thr phosphatase (PPase) type 1 (PP1) and/or type 2A (PP2A) are essential for splicing in vitro, in particular, at the second catalytic step. They observed that Sap155 and U5-116k are potential substrates of these two PPases, suggesting the importance of dephosphorylation of spliceosomal proteins in spliceosome structural. Studies in yeast and human cells suggest that PP1 phosphorylation is essential for proper cell cycle progression [13, 14]

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