The ubiquitous protein Ser/Thr phosphatase-1 (PP1) interacts with dozens of regulatory proteins that are structurally unrelated. However, most of them share a short, degenerate "RVxF"-type docking motif. Using a broad in silico screening based on a stringent definition of the RVxF motif, in combination with a multistep biochemical validation procedure, we have identified 78 novel mammalian PP1 interactors. A global analysis of the validated RVxF-based PP1 interactome not only provided insights into the conserved features of the RVxF motif but also led to the discovery of additional common PP1 binding elements, described as the "SILK" and "MyPhoNE" motifs. In addition to the doubling of the known mammalian PP1 interactome, our data contribute to the design of PP1 interaction networks. Notably, an interaction network linking PP1 interactors discloses a pleiotropic role of PP1 in cell polarity.

Human FBP21 (formin-binding protein 21) contains a matrin-type zinc finger and two tandem WW domains. It is a component of the spliceosomes and interacts with several established splicing factors. Here we demonstrate for the first time that FBP21 is an activator of pre-mRNA splicing in vivo and that its splicing activation function and interaction with the splicing factor SIPP1 (splicing factor that interacts with PQBP1 and PP1) are both mediated by the two tandem WW domains of group III. We determined the solution structure of the tandem WW domains of FBP21 and found that the WW domains recognize peptide ligands containing either group II (PPLP) or group III (PPR) motifs. The binding interfaces involve both the XP and XP2 grooves of the two WW domains. Significantly, the tandem WW domains of FBP21 are connected by a highly flexible region, enabling their simultaneous interaction with two proline-rich motifs of SIPP1. The strong interaction between SIPP1 and FBP21 can be explained by the conjugation of two low affinity interactions with the tandem WW domains. Our study provides a structural basis for understanding the molecular mechanism underlying the functional implication of FBP21 and the biological specificity of tandem WW domains.Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Pre-mRNA splicing takes place in the spliceosome, a highly dynamic ribonucleoprotein particle that consists of five small nuclear RNAs and at least 150 proteins. Small nuclear ribonucleoproteins (snRNPs) 3 and numerous protein factors are essential for the formation of the active spliceosome (1, 2). In budding yeast, the splicing factor Prp40 participates in crossintron bridging by interacting with the branch point-binding protein (BBP) and the U5 snRNP component Prp8. Prp40 contacts the 5Ј splice site and interacts with BBP, bringing the 5Ј splice site and the branch point in spatial proximity. These interactions are believed to be conserved in mammals (3-5). FBP21 (formin-binding protein 21), the mammalian Prp40-like protein, colocalizes with splicing factors in nuclear storage sites for pre-mRNA splicing factors. In addition, FBP21 is a component of the mammalian spliceosomal A/B complex and is associated with U2 snRNPs (6). FBP21 interacts directly with the splicing factors U1 snRNP protein U1C, the core snRNP proteins SmB and SmBЈ, and the branch point-binding protein SF1/mBBP, suggesting that it may also play a role in crossintron bridging of U1 and U2 snRNPs in the spliceosomes. FBP21 contains a matrin-type zinc finger and two group III WW domains ( Fig. 1) that are structurally related to those of the established splicing factors U1C and Prp40, respectively (6, 7). The binding of FBP21 to splicing factors is mediated by its tandem WW domains, which represent interaction modules for proline-rich ligands (4, 8, 9). Although the above data strongly suggest that FBP21 has a role in pre-mRNA splicing, there are no in vivo data to support this contention. The splicing...

The PQBP1 (polyglutamine tract-binding protein 1) gene encodes a nuclear protein that regulates pre-mRNA splicing and transcription. Mutations in the PQBP1 gene were reported in several X chromosome-linked mental retardation disorders including Golabi-Ito-Hall syndrome. The missense mutation that causes this syndrome is unique among other PQBP1 mutations reported to date because it maps within a functional domain of PQBP1, known as the WW domain. The mutation substitutes tyrosine 65 with cysteine and is located within the conserved core of aromatic amino acids of the domain. We show here that the binding property of the Y65C-mutated WW domain and the full-length mutant protein toward its cognate proline-rich ligands was diminished. Furthermore, in GolabiIto-Hall-derived lymphoblasts we showed that the complex between PQBP1-Y65C and WBP11 (WW domain-binding protein 11) splicing factor was compromised. In these cells a substantial decrease in pre-mRNA splicing efficiency was detected. Our study points to the critical role of the WW domain in the function of the PQBP1 protein and provides an insight into the molecular mechanism that underlies the X chromosome-linked mental retardation entities classified globally as Renpenning syndrome.

SIPP1 (splicing factor that interacts with PQBP1 and PP1) is a widely expressed protein of 70 kDa that has been implicated in pre-mRNA splicing. It interacts with protein Ser/Thr phosphatase-1 (PP1) and with the polyglutamine-tract-binding protein 1 (PQBP1), which contributes to the pathogenesis of X-linked mental retardation and neurodegenerative diseases caused by polyglutamine tract expansions. We show here that SIPP1 is a nucleocytoplasmic shuttling protein. Under basal circumstances SIPP1 was largely nuclear, but it accumulated in the cytoplasm following UVor X-radiation. Nuclear import was mediated by two nuclear localization signals. In addition, SIPP1 could be piggy-back transported to the nucleus with its ligand PQBP1. In the nucleus SIPP1 and PQBP1 formed inclusion bodies similar to those detected in polyglutamine diseases. SIPP1 did not function as a nuclear targeting subunit of PP1 but re-localized nuclear PP1 to storage sites for splicing factors. The C-terminal residues of SIPP1, which do not conform to a classic nuclear export signal, were required for its nuclear export via the CMR-1 pathway. Finally, SIPP1 activated pre-mRNA splicing in intact cells, and the extent of splicing activation correlated with the nuclear concentration of SIPP1. We conclude that SIPP1 is a positive regulator of pre-mRNA splicing that is regulated by nucleocytoplasmic shuttling. These findings also have potential implications for a better understanding of the pathogenesis of X-linked mental retardation and polyglutamine-linked neurodegenerative disorders. SIPP1,2 also known as SNP70 or Npw38BP, is a polypeptide of 70 kDa that appears to be expressed in all metazoa and higher plants (1-3). It is a largely nuclear protein but has also been reported to be bound to vimentin-containing intermediate filaments in the cytoplasm (2, 3). Within the nucleus SIPP1 is associated with both the speckles (2, 3), which represent storage/assembly sites for splicing factors, and the spliceosomes (3-6), the nuclear protein-RNA complexes that catalyze pre-mRNA splicing. In further agreement with a role in pre-mRNA splicing, SIPP1 was shown to interact directly with single-stranded RNA (2), the polyglutamine-tract-binding protein PQBP1/Npw38 (1), and protein phosphatase-1 (3), all established components of the spliceosomes (Fig. 1A, see also Refs. 6 and 7). RNA binds to the N terminus of SIPP1 (2), the WW-domain of PQBP1 interacts with two proline-rich regions of SIPP1 (1), and the binding of protein phosphatase-1 (PP1) is mediated by two motifs in the central domain of SIPP1 (3). SIPP1 also contains a coiled-coil domain and an Asp-rich domain but the function of these domains is still unknown (1-3).Although the above data strongly suggest that SIPP1 has a role in pre-mRNA splicing, there are no in vivo data to support this contention. A further complication is that the exact function of the protein ligands of SIPP1 in splicing is not yet known and that these ligands have also functions outside pre-mRNA splicing. Indeed, PP1 plays a role in ...

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