Y65C missense mutation in the WW domain of the Golabi-Ito-Hall syndrome protein PQBP1 affects its binding activity and deregulates pre-mRNA 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 Golabi-Ito-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.

Structure and function of the two tandem WW domains of the pre-mRNA splicing factor FBP21 (formin-binding protein 21).

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.

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases.

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.

Docking motif-guided mapping of the interactome of protein phosphatase-1.

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.

Nucleocytoplasmic shuttling of the splicing factor SIPP1.

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 UV- or 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.