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)

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-⌬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...

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth

“…The cell lines expressed the fusions in a doxycycline (Dox)-dependent manner and to a similar extent (Figs S1A and S2A). As expected (Tanuma et al, 2008;Van Dessel et al, 2010), EGFP traps of the NIPP1-Wt and NIPP1-Fm fusions showed an association with endogenous PP1, which was not seen with the NIPP1-Pm and NIPP1-Fm+Pm fusions (Figs S1B and S2B). The associated PP1 was inactive with glycogen phosphorylase as substrate but the phosphatase activity could be revealed by trypsinolysis, which destroys NIPP1 but not PP1 (Beullens et al, 1998), confirming that NIPP1-Wt and NIPP1-Fm inhibit PP1 (Fig.…”

“…Full-length NIPP1 binds to PP1 with an extremely high affinity and inhibits the phosphatase towards all tested protein substrates except FHA ligands O'Connell et al, 2012;Tanuma et al, 2008). Given that the mitotic-arrest phenotype induced by the overexpression of NIPP1 was entirely dependent on a functional PP1-binding site, we reasoned that the phenotype could be caused by titrating PP1 away from other PIPs.…”

“…7A) (Tanuma et al, 2008;Van Dessel et al, 2010). This enabled us to study the effect of the selective inhibition of PP1 on colony formation and tumor growth in the absence of an inducing agent.…”

“…Hela Tet-off cell lines expressing Flag-tagged NIPP1 variants in the absence of doxycycline (Dox) and Flip-In T-REx cell lines expressing EGFP or EGFP-tagged NIPP1 variants in the presence of Dox were obtained as described previously (Tanuma et al, 2008;Van Dessel et al, 2015). Transfections were carried out using a commercial kit (Jet-Prime, Polyplus), according to the manufacturer's instructions.…”

SF1 Phosphorylation Enhances Specific Binding to U2AF 65 and Reduces Binding to 3′-Splice-Site RNA

“…Co-transcriptional dephosphorylation could then promote loading of SF1-U2AF 65 on the emerging pre-mRNA during the early stages of splicing. Accordingly, SF1 associates with U2AF 65 in extra-spliceosomal complexes (6), and phosphorylation and dephosphorylation events such as this one are required for pre-mRNA splicing (e.g., (68)(69)(70)). Indeed, the influences of SPSP phosphorylation on any SF1 functions other than UHM and RNA interactions have yet to be examined.…”