Transcriptional abnormality in myotonic dystrophy affects DMPK but not neighboring genes

Abstract

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.

A Family of Human RNA-binding Proteins Related to theDrosophila Bruno Translational Regulator

“…This RNA interference hypothesis includes a cis defect, the shuttling of DMPK mRNA with expanded repeats, and a trans defect, the shuttling of other mRNAs that are bound by Bruno proteins. Consistent with this hypothesis, the DMPK mRNA with expanded CUG repeats accumulates in the nucleus in foci and does not accumulate in the cytoplasm to the same levels as mRNA without expanded repeats (70,71,77). The mRNA retained in the nucleus is properly spliced and polyadenylated (70,72).…”

Muscleblind-Like Proteins

“…The expanded repeats in DM1 and DM2 accumulate in the nuclei as discrete foci. [13][14][15][16] The relationship between these ribonuclear inclusions, which may consist of double stranded hairpin loop structures, 17,18 and DM pathogenesis is not entirely clear. 19 Mutant DMPK mRNA in nuclear foci of DM1 cells appears to be spliced and polyadenylated normally, 15 whereas DM2 foci appear to consist of spliced-out introns.…”

Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats

“…CUG-repeat tracts form hairpins that could affect the processing of the mutant DMPK mRNA and render it incompetent for export. However, it is thought that DMPK mutant mRNA is properly spliced, capped and polyadenylated (Davis et al, 1997;Hamshere et al, 1997). DM1 foci were found to be adjacent to SC35 speckles, suggesting that DMPK mutant mRNA could be blocked from entry into speckles and restricted at this step of nuclear export (Holt et al, 2007;Smith et al, 2007).…”