mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains

Nuclear mRNA transport is often thought of in terms of translocation through the nuclear pore, but mRNA export also requires intranuclear progression of transcripts from the gene to the nuclear pore. In some genetic diseases, failed export of a mutant mRNA is critical to the phenotype, yet typically it is not well understood how nuclear export is impeded or whether mutant mRNA accumulates at a specifi c point within the nuclear structure. In fact, the examination of mRNA blocked at a specifi c step in export may help illuminate the path whereby mRNA normally transits from the gene to the nuclear pore. The analysis of human disease gene mutations that impact nuclear metabolism of the mRNA provides an avenue to study both disease pathogenesis and the interrelationship between nuclear structure and steps in mRNA biogenesis. In addition, the study of naturally occurring disease alleles in patient cells provides the advantage that the mutant mRNA is expressed in a normal structural and physiological context. This study examines the intranuclear fate of normal and triplet repeat–expanded transcripts in myotonic dystrophy type 1 (DM1) to defi ne the point in the nuclear structure where the progression of normal and mutant transcripts diverge, which, in turn, provides insight into the step at which mutant mRNA transport is blocked. In DM1, expansion of a CTG triplet repeat occurs in the 3′ untranslated region of the gene encoding dystrophia myotonica protein kinase (DMPK), a serine/threonine protein kinase (Aslanidis et al., 1992; Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992). Normal alleles have 5–35 repeats, whereas DM1 alleles have 50 to >1,000. It has been shown that the mutant DMPK genes encode mRNAs containing expanded repeat sequences and that these RNAs are sequestered in the nucleus, where they accumulate in discrete nuclear foci (Taneja et al., 1995; Hamshere et al., 1997; Liquori et al., 2001). However, it is not known whether normal DMPK mRNA associates with any defi ned intranuclear nuclear structure after its transcription and whether this differs for the mutant RNA. In addition to the defect in mRNA transport in DM1, it is hypothesized that the expanded repeat RNAs alter the nuclear distri bution or activity of specifi c CUG-binding proteins, which, in turn, affects the alternative splicing of other pre-mRNAs (Day and Ranum, 2005; Osborne and Thornton, 2006). Multiple proteins such as CUG-binding protein (Timchenko et al., 1996, 2001; Philips et al., 1998), heterogeneous nuclear RNP H (hnRNP H; Kim et al., 2005), double-stranded RNA–binding proteins Defi ning early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains