Abstract
Myotonic Dystrophy Type 1 (DM1) is the most common form of adult muscular dystrophy (~1:8000). In DM1, expansion of CTG trinucleotide repeats in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene results in DMPK mRNA hairpin structures which aggregate as insoluble ribonuclear foci and sequester several RNA-binding proteins. The resulting sequestration and misregulation of important splicing factors, such as muscleblind-like 1 (MBNL1), causes the aberrant expression of fetal transcripts for several genes that contribute to the disease phenotype. Previous work has shown that antisense oligonucleotide-mediated disaggregation of the intranuclear foci has the potential to reverse downstream anomalies. To explore whether the nuclear foci are, to some extent, controlled by cell signalling pathways, we have performed a screen using a small interfering RNA (siRNA) library targeting 518 protein kinases to look at kinomic modulation of foci integrity. RNA foci were visualized by in situ hybridization of a fluorescent-tagged (CAG)10 probe directed towards the expanded DMPK mRNA and the cross-sectional area and number of foci per nuclei were recorded. From our screen, we have identified PACT (protein kinase R (PKR) activator) as a novel modulator of foci integrity and have shown that PACT knockdown can both increase MBNL1 protein levels; however, these changes are not suffcient for significant correction of downstream spliceopathies.
Highlights
IntroductionMyotonic Dystrophy Type 1 (DM1) is the most common form of adult muscular dystrophy with a prevalence of 1:8000 worldwide; in some regions of Quebec, Canada, the prevalence is as high as 1:500
For passaging and plating cells, old media was aspirated, and cells were washed with 1X PBS (Fisher Scientific) to remove residual serum from the FCS; the PBS (Fisher Scientific) was aspirated, cells were incubated in 1X (0.25%) trypsin (Gibco; reconstituted in 15mM NaCl (Fisher Scientific), 0.5mM EDTA (Sigma), and 1X PBS (Fisher Scientific)) for 3-7min at 37 ̊C to lift them from the plate, and trypsin was inhibited using complete growth media containing fetal calf serum (FCS)
We report here the decrease of both the size and number of intranuclear RNA foci in DM1 patient fibroblasts with the small interfering RNA (siRNA)-mediated reduction of the kinase modulator PACT
Summary
Myotonic Dystrophy Type 1 (DM1) is the most common form of adult muscular dystrophy with a prevalence of 1:8000 worldwide; in some regions of Quebec, Canada, the prevalence is as high as 1:500. DM1 is caused by a CTG repeat expansion in the 3’ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene [1]. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript
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