Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1

In disease‐associated genes, understanding the functional significance of deep intronic nucleotide variants represents a difficult challenge. We previously reported that an NF1 intron 30 exonization event is triggered from a single correct nomenclature is ‘c.293‐279 A>G’ mutation [Raponi M, Upadhyaya M & Baralle D (2006) Hum Mutat 27, 294–295]. In this paper, we investigate which characteristics play a role in regulating inclusion of the aberrant pseudoexon. Our investigation shows that pseudoexon inclusion levels are strongly downregulated by polypyrimidine tract binding protein and its homologue neuronal polypyrimidine tract binding protein. In particular, we provide evidence that the functional effect of polypyrimidine tract binding protein is proportional to its concentration, and map the cis‐acting elements that are principally responsible for this negative regulation. These results highlight the importance of evaluating local sequence context for diagnostic purposes, and the utility of developing therapies to turn off activated pseudoexons.

[1]  Christopher W. J. Smith,et al.  Expression of Human nPTB Is Limited by Extreme Suboptimal Codon Content , 2008, PloS one.

[2]  M. Ugarte,et al.  Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA. , 2007, American journal of human genetics.

[3]  S. Chew,et al.  Pre-spliceosomal binding of U1 small nuclear ribonucleoprotein (RNP) and heterogenous nuclear RNP E1 is associated with suppression of a growth hormone receptor pseudoexon. , 2007, Molecular endocrinology.

[4]  T. Miyashita,et al.  U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites , 2007, Journal of Human Genetics.

[5]  C. Smith,et al.  Crossregulation and Functional Redundancy between the Splicing Regulator PTB and Its Paralogs nPTB and ROD1 , 2007, Molecular cell.

[6]  E. Buratti,et al.  RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events , 2007, Nucleic acids research.

[7]  D. Baralle,et al.  NF1 mRNA biogenesis: Effect of the genomic milieu in splicing regulation of the NF1 exon 37 region , 2006, FEBS letters.

[8]  E. Buratti,et al.  Defective splicing, disease and therapy: searching for master checkpoints in exon definition , 2006, Nucleic acids research.

[9]  E. Buratti,et al.  Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon. , 2006, RNA.

[10]  S. Grellscheid,et al.  An Apparent Pseudo-Exon Acts both as an Alternative Exon That Leads to Nonsense-Mediated Decay and as a Zero-Length Exon , 2006, Molecular and Cellular Biology.

[11]  D. Baralle,et al.  Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization , 2006, Human mutation.

[12]  Christopher W. J. Smith,et al.  Novel modes of splicing repression by PTB. , 2006, Trends in biochemical sciences.

[13]  Robert Castelo,et al.  Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. , 2005, Molecular cell.

[14]  A. Krainer,et al.  Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a , 2005, Human mutation.

[15]  Gene W. Yeo,et al.  Systematic Identification and Analysis of Exonic Splicing Silencers , 2004, Cell.

[16]  L. Chasin,et al.  Computational definition of sequence motifs governing constitutive exon splicing. , 2004, Genes & development.

[17]  M. Garcia-Blanco,et al.  Alternative splicing in disease and therapy , 2004, Nature Biotechnology.

[18]  N. Bresolin,et al.  Silencer elements as possible inhibitors of pseudoexon splicing. , 2004, Nucleic acids research.

[19]  M. Claustres,et al.  Pseudoexon activation in the DMD gene as a novel mechanism for Becker muscular dystrophy , 2003, Human mutation.

[20]  I. Eperon,et al.  HnRNP G and Tra2beta: opposite effects on splicing matched by antagonism in RNA binding. , 2003, Human molecular genetics.

[21]  T. Dörk,et al.  A new type of mutation causes a splicing defect in ATM , 2002, Nature Genetics.

[22]  M. Caulfield,et al.  Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity. , 2001, American journal of human genetics.

[23]  E. Wagner,et al.  Polypyrimidine Tract Binding Protein Antagonizes Exon Definition , 2001, Molecular and Cellular Biology.

[24]  L. Chasin,et al.  Multiple Splicing Defects in an Intronic False Exon , 2000, Molecular and Cellular Biology.

[25]  M. Hattori,et al.  The DNA sequence of human chromosome 21 , 2000, Nature.

[26]  Melanie E. Goward,et al.  The DNA sequence of human chromosome 22 , 1999, Nature.

[27]  D. Helfman,et al.  Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon , 1999, Molecular and Cellular Biology.

[28]  R. C. Chan,et al.  The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream , 1997, Molecular and cellular biology.

[29]  I. Pérez,et al.  Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo. , 1997, RNA.

[30]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[31]  Y Sakaki,et al.  The DNA sequence of human chromosome 21. , 2000, Nature.