Identification and analysis of RNA binding proteins interacting with the myotonic dystrophy protein kinase 3' untranslated region mRNA

The mutation responsible for Myotonic Dystrophy is a CTG expansion in the 3′UTR of DMPK, a serine-threonine kinase of uncertain function. The mechanism by which CTG expansion causes the disease is unknown. The gain of function hypothesis of DM pathogenesis states that the disorder occurs due to the presence of the mutation as a CUG expansion in the DMPK mRNA, presumably mediated by altered interactions of RNA binding proteins with the DMPK 3 ′UTR. One effect of the DM mutation is to cause nuclear retention of mRNAs carrying CUG expansions. We have used a UV crosslinking assay to search for RNA binding proteins capable of interaction with any part of the DMPK 3′UTR. We identified four binding proteins known to be involved in several aspects of mRNA metabolism: hnRNP C (heterogeneous ribonucleoprotein C), U2AF65 (U2 auxiliary factor), PTB (polypyrimidine tract binding protein) and PSF (PTB associated splicing factor). These proteins bind to two short regions (named R1 and R2) 3′ of the (CUG)_n that approximately flank a novel, alternatively spliced 3′ DMPK exon (named E16). Isoforms using this alternative 3′splice site result in a mRNA lacking the CUG repeats. We demonstrated the functionality of the E16 3′ splice site in vivo and detected isoforms incorporating E16 in human tissues by RT-PCR. We presented evidence that even large CUG expansions have a deleterious effect but do not abolish E16 3′splice site use. Due to the lack of CUG repeats in DMPK mRNAs resulting from this alternative splicing pathway and in contrast to mRNAs containing CUG expansions, the novel isoform is not retained in the nucleus, suggesting an unforeseen effect of the DM mutation: an imbalance of relative levels of cytoplasmic DMPK mRNA isoforms. We have characterized the interactions of PTB and hnRNP C with R1 and R2. Mutation of R2 significantly increases E16 exon incorporation, while over-expression of PTB results in repression of E16 incorporation. Splicing assays in hnRNP C −/− cells do not suggest a role for hnRNP C in use of the E16 3′ splice site. Our results indicate that PTB is involved in cell type specific regulation of E16 incorporation.