Novel proteins with binding specificity for DNA CTG repeats and RNA CUG repeats: implications for myotonic dystrophy

While an unstable CTG triplet repeat expansion is responsible for myotonicdystrophy, the mechanism whereby this genetic defect induces the diseaseremains unknown. To detect proteins binding to CTG triplet repeats, weperformed bandshift analysis using as probes double- stranded DNA fragmentshaving CTG repeats [ds(CTG)6-10] and single- stranded oligonucleotideshaving CTG repeats ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The sourceof protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblastsand myotubes. Proteins binding to the double-stranded DNA repeat[ds(CTG)6-10], were inhibited by nonlabeled ds(CTG)6-10, but not by anon-specific DNA fragment (USF/AD-ML). Another protein binding to ssCTGprobe and RNA CUG probe was inhibited by nonlabeled (CTG)8 and (CUG)8.Nonlabeled oligos with different triplet repeat sequences, ss(CAG)8 orss(CGG)8, did not inhibit binding to the ss(CTG)8 probe. However, whenlabeled as probes, the (CAG)8 and (CGG)8 bound to proteins distinct fromthe CTG proteins and binding was inhibited by nonlabeled (CAG)8 or (CGG)8respectively. The protein binding only to the RNA repeat (CUG)8 wasinhibited by nonlabeled (CUG)8 but not by nonlabeled single- ordouble-stranded CTG repeats. Furthermore, the CUG-BP exhibited no bindingto an RNA oligonucleotide of triplet repeats of the same length but havinga different sequence, CGG. The CUG binding protein was localized to thecytoplasm, whereas dsDNA binding proteins were localized to the nuclearextract. Thus, several trinucleotide binding proteins exist and theirspecificity is determined by the triplet sequence. The novel protein,CUG-BP, is particularly interesting since it binds to triplet repeats knownto be present in myotonin protein kinase mRNA which is responsible formyotonic dystrophy.