Myotonic dystrophy (DM) is caused by the expansion of a trinucleotide repeat, CTG, in the 3 untranslated region of a protein kinase gene, DMPK. We set out to determine what effect this expanded repeat has on RNA processing. The subcellular fractionation of RNA and the separate analysis of DMPK transcripts from each allele reveals that transcripts from expanded DMPK alleles are retained within the nucleus and are absent from the cytoplasm of DM cell lines. The nuclear retention of DMPK transcripts occurs above a critical threshold between 80 and 400 CTGs. Further analysis of the nuclear RNA reveals an apparent reduction in the proportion of expansion-derived DMPK transcripts after poly(A) ؉ selection. Quantitative analysis of RNA also indicates that although the level of cytoplasmic DMPK transcript is altered in DM patients, the levels of transcripts from 59 and DMAHP, two genes that immediately f lank DMPK, are unaffected in DM cell lines.Myotonic dystrophy (DM) is the most common form of muscular dystrophy affecting adults. It is dominantly inherited and involves many systems, including endocrine, heart, and brain, though principally it is a disease of muscle characterized by myotonia with muscle weakness and wasting (1). DM is associated with the expansion of a CTG repeat located in the 3Ј untranslated region (UTR) of a protein kinase gene, DMPK (2-4) (See Fig. 1). It is not known how this mutation, in a noncoding part of the gene, exerts an effect at the cellular level, and conf licting data have been published about the effect of repeat expansion on DMPK RNA levels in DM tissues and cell lines (5-8). Three models have been proposed to explain the molecular mechanism of DM (9). First, the mutation may directly effect the level of DMPK protein. Second, the mutation may affect the higherorder structure of DNA around the repeat, altering the level of expression of neighboring genes in a field effect (10 -13). Third, the repeat expansion may produce a gain-of-function mutation at either the DNA or RNA level. Recently, foci of DMPK transcripts containing expanded repeats have been reported in the nuclei of DM muscle specimens and fibroblast cell lines (14), and other studies have suggested that a disruption of RNA processing may be critical for the development of DM (15,16). In view of these findings, we have performed a series of experiments to compare the distribution of expanded and wild-type DMPK alleles in RNA from nuclear and cytoplasmic fractions of DM cells. We have also examined the expression of genes 59 (10, 17) and DMAHP (12), which f lank DMPK, to establish whether this is affected by repeat expansion.
Myotonic dystrophy is caused by the expansion of a CTG repeat sequence. The mechanism by which this expanded repeat produces the pathophysiology of myotonic dystrophy is not clear. It has been shown previously that expansion of the repeat produces allele-specific effects on transcripts from two genes, DMPK and SIX5. We have examined the effect of repeat expansion on the level of RNA from a third gene, DMWD. We have identified a polymorphism in this gene and developed a quantitative allele-specific assay for DMWD RNA levels, which we have applied to nuclear and cytoplasmic fractions of RNA from DM cell lines. We have found that the level of the DM-associated allele in the cytoplasm of DM cell lines is reduced by 20-50% compared with the wild-type allele, similar to the level of reduction found for SIX5 in allele-specific analysis. However, no such reduction is observed in RNA from the nuclear fraction of DM cell lines. This may reflect the complex nature of processing transcriptional units at the DM locus.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.