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 myot onic dystrophy, the mechanism whereby this genetic defect induces the disease remains unknown. To detect proteins binding to CTG triplet rep eats, we performed bandshift analysis using as probes double-stranded DNA fragments having CTG repeats [ds(CTG)(6-10)] and single-stranded o ligonucleotides having CTG repeats ss(CTG)(8) or RNA CUG triplet repea ts (CUG)(8). The source of protein was nuclear and cytoplasmic extract s of HeLa cells, fibroblasts and myotubes. Proteins binding to the dou ble-stranded DNA repeat [ds(CTG)(6-10)], were inhibited by nonlabeled ds(CTG)(6-10), but not by a non-specific DNA fragment (USF/AD-ML). Ano ther protein binding to ssCTG probe and RNA CUG probe was inhibited by nonlabeled (CTG)(8) and (CUG)(8). Nonlabeled oligos with different tr iplet repeat sequences, ss(CAG)(8) or ss(CGG)(8), did not inhibit bind ing to the ss(CTG)(8) probe. However, when labeled as probes, the (CAG )(8) and (CGG)(8) bound to proteins distinct from the CTG proteins and binding was inhibited by nonlabeled (CAG)(8) or (CGG)(8) respectively . The protein binding only to the RNA repeat (CUG)(8) was inhibited by nonlabeled (CUG)(8) but not by nonlabeled single- or double-stranded CTG repeats. Furthermore, the CUG-BP exhibited no binding to an RNA ol igonucleotide of triplet repeats of the same length but having a diffe rent sequence, CGG. The CUG binding protein was localized to the cytop lasm, whereas dsDNA binding proteins were localized to the nuclear ext ract. Thus, several trinucleotide binding proteins exist and their spe cificity is determined by the triplet sequence. The novel protein, CUG -BP, is particularly interesting since it binds to triplet repeats kno wn to be present in myotonin protein kinase mRNA which is responsible for myotonic dystrophy.