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-⌬C) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -⌬C 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.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) 4 and a large number of nonsnRNP proteins. Spliceosome assembly is an ordered process that includes stepwise recruitment of U1, U2, U5, and U4/6 snRNPs on a pre-mRNA and sequential formation of complex E 3 A/B 3 B* 3 C. The activated B* spliceosome catalyzes step I of splicing, whereas the C complex catalyzes step II. During and after splicing, spliceosome components dissociate and are recycled for further rounds of splicing. Spliceosome assembly/disassembly and splicing catalysis are thought to be regulated in part by reversible phosphorylation of spliceosomal proteins (1-3).U2 snRNP includes U2 snRNA and two heteromeric protein complexes, Sf3a and Sf3b. Sap155, also known as Sf3b1 or Sf3b155, is a component of the Sf3b and becomes hyperphosphorylated concomitant with or just after the first catalytic step of splicing in vitro (4). A recent study reveals that Sf3a/b proteins are destabilized and dissociate from the RNP core of the activated spliceosome during the transition from the B to C complex (5). Although Sf3a and Sf3b are essential early in the splicing reaction, they are apparently not required for the second catalytic step. Currently, 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) ar...
Protein phosphatase type-1 (PP1), one of the most abundant Ser/Thr protein phosphatases, plays a central role in the regulation of various cell functions. Almost all the PP1 molecules exist as holoenzymes in vivo consisting of a catalytic subunit (PP1C) and a variable regulatory subunit that regulates substrate specificity and/or subcellular localization. In order to clarify fine regulation of PP1, we overexpressed a nuclear inhibitor of PP1 (NIPP-1) in a Flag-tagged form in mammalian cells. The Flag-tagged NIPP-1 was found to be immunoprecipitated with three isoforms of PP1C, namely, PP1alpha, PP1gamma1, and PP1delta with a similar efficiency, suggesting that NIPP-1 makes a complex with the PP1C through the region conserved among the three isoforms. These results suggested that NIPP-1 can be involved in the regulation of various PP1 holoenzymes in vivo.
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