Y65C Missense Mutation in the WW Domain of the Golabi-Ito-Hall Syndrome Protein PQBP1 Affects Its Binding Activity and Deregulates Pre-mRNA Splicing

Victor E Tapia,Emilia Nicolaescu,Caleb B Mcdonald,Valeria Musi,Tsutomu Oka,Yujin Inayoshi,Adam C Satteson,Virginia Mazack,Jasper Humbert,Christian J Gaffney,Monique Beullens,Charles E Schwartz,Christiane Landgraf,Rudolf Volkmer,Annalisa Pastore,Amjad Farooq,Mathieu Bollen,Marius Sudol

doi:10.1074/jbc.m109.084525

Abstract

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.

Highlights

London NW7 1AA, United Kingdom, the **Laboratory of Signal Transduction and Proteomic Profiling, Weis Center for Research, Danville, Pennsylvania 17822, the ‡‡J
Binding Profiles of the Mutated and WT WW Domains on Peptide Arrays—To assess the effect of the Y65C mutation within the WW domain of PQBP1 on the binding function of the domain, we screened the repertoire of PPXY motif containing 12-mer peptides that represent the entire human proteome
Of 1958 peptides assayed with WT and Y65C mutant WW domain probes labeled to the same specific activity, 1116 displayed relatively lower binding to the Y65C mutant than to the WT WW domain

Summary

Introduction

London NW7 1AA, United Kingdom, the **Laboratory of Signal Transduction and Proteomic Profiling, Weis Center for Research, Danville, Pennsylvania 17822, the ‡‡J. We could not exclude the existence of cognate, proline-rich ligands or modified CTD repeats, which we did not include in our screens and which could represent still unknown physiological targets of PQBP1 WW domain, we tentatively concluded that the Y65C mutation of the WW domain may have an effect on binding to CTD repeat peptides, but the differences observed for Y65C mutant in screens of PPXY containing peptides were more significant.

Results

Conclusion