A low absolute number of expanded transcripts is involved in myotonic dystrophy type 1 manifestation in muscle

Abstract

Muscular manifestation of myotonic dystrophy type 1 (DM1), a common inheritable degenerative multisystem disorder, is mainly caused by expression of RNA from a (CTG·CAG)n-expanded DM1 locus. Here, we report on comparative profiling of expression of normal and expanded endogenous or transgenic transcripts in skeletal muscle cells and biopsies from DM1 mouse models and patients in order to help us in understanding the role of this RNA-mediated toxicity. In tissue of HSALR mice, the most intensely used ‘muscle-only’ model in the DM1 field, RNA from the α-actin (CTG)250 transgene was at least 1000-fold more abundant than that from the Dmpk gene, or the DMPK gene in humans. Conversely, the DMPK transgene in another line, DM500/DMSXL mice, was expressed ∼10-fold lower than the endogenous gene. Temporal regulation of expanded RNA expression differed between models. Onset of expression occurred remarkably late in HSALR myoblasts during in vitro myogenesis whereas Dmpk or DMPK (trans)genes were expressed throughout proliferation and differentiation phases. Importantly, quantification of absolute transcript numbers revealed that normal and expanded Dmpk/DMPK transcripts in mouse models and DM1 patients are low-abundance RNA species. Northern blotting, reverse transcriptase–quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses showed that they occur at an absolute number between one and a few dozen molecules per cell. Our findings refine the current RNA dominance theory for DM1 pathophysiology, as anomalous factor binding to expanded transcripts and formation of soluble or insoluble ribonucleoprotein aggregates must be nucleated by only few expanded DMPK transcripts and therefore be a small numbers game.

The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells

“…Approximately 100,000 LNA molecules were required for 50% knockdown of this transcript with a copy number of ;2500 per cell (based on literature values). Thus, the stoichiometry was ;40:1, which is strikingly close to the 130:1 ratio that we found for our blocking-type ASO that targeted expanded DMPK transcripts, which are low abundant (39). We hypothesize that the 3-fold difference can be explained by the RNase H-dependent catalytic mode of action of the LNA gapmer.…”

“…Thus, nuclear ASO concentrations in the upper nanomolar range are effective to block MBNL1 binding to the expanded (CUG)n repeat in this DM1 muscle cell model. The DM1 myoblasts contained, on average, 3 foci, which we assume to represent 1 expanded DMPK transcript each on the basis of previous findings of our group (39). Combined with the repeat length of (CUG)2600, this presents an average of 1560 potential binding sites per cell for the CAG5 ASO.…”

“…Thus, areas that showed highly fluorescent spots from other focal planes than the nucleus were distinguished from those nuclei that had a homogeneous fluorescence distribution. For each of the 3 independent uptake experiments, between 57 and 103 nuclei were measured (untreated control [38][39][40][41][42][43][44][45][46][47][48]. A 1-way ANOVA was performed to test whether there was a treatment effect.…”

Analyzing Presymptomatic Tissue to Gain Insights into the Molecular and Mechanistic Origins of Late-Onset Degenerative Trinucleotide Repeat Disease

“…It is likely that each "foci" detected by FISH is a single RNA molecule. A low copy number for a disease-causing RNA has also been observed for myotonic dystrophy type 1(DM1) (45) and C9orf72 ALS/FTD (46). We find that the CUG repeat expansion stabilizes TCF4 intronic RNA in a corneal cell-specific manner (Figure 3).…”

Expanded CUG repeats in DMPK transcripts adopt diverse hairpin conformations without influencing the structure of the flanking sequences

“…A classical hallmark of DM1 is the occurrence of ribonuclear foci in the cell nucleus, visualized by fluorescence in situ hybridization (FISH). These foci are thought to be complexes of one or at most a few expanded DMPK RNA molecules and associated RBPs (Gudde et al 2016;Wojciechowska et al 2018).…”