High-throughput kinome-RNAi screen identifies protein kinase R activator (PACT) as a novel genetic modifier of CUG foci integrity in myotonic dystrophy type 1 (DM1)

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.

Natural Compound Boldine Lessens Myotonic Dystrophy Type 1 Phenotypes in DM1 Drosophila Models, Patient-Derived Cell Lines, and HSALR Mice

“…Boldine significantly reduced the number of foci in muscle cells ( Figure 2 , Figure 3 and Figure S3 ) from the three disease models used herein (Drosophila, patient-derived cells and mice). While the pathogenicity of the foci is still controversial [ 5 ], focal reduction has been used to screen anti-DM1 drugs or disease modulators [ 37 ]. Importantly, our observations indicate that boldine acted in mammalians’ setting, ruling out any specific effect on Drosophila and confirming its potential to the likelihood of its beneficial effect in humans.…”

Vorinostat Improves Myotonic Dystrophy Type 1 Splicing Abnormalities in DM1 Muscle Cell Lines and Skeletal Muscle from a DM1 Mouse Model

“…Differentiated myoblasts were transfected with ASO using Lipofectamine RNAi MAX (Invitrogen, Burlington, ON, Canada), employing a similar protocol to siRNA transfections [ 58 ]. Upon receipt, the control (Isis No.…”

“…All primers were optimized for use at 60 °C annealing temperature and the SybrGreen signal was acquired after extension at 72 °C. The SERCA1 splicing assay by qPCR has been previously described [ 58 ]. The primer sequences for human qPCR were (5′ to 3′) and they have shown in Table 1 .…”

The Current Situation and Development Prospect of Whole-Genome Screening

“…Therefore, RNAi is suitable for cells with low efficiency in delivering Cas9/dCas9 [ 12 ]. In recent years, RNAi screening has become an important means of genetic screening [ 13 , 14 ]. Recently, Kristijonas et al used a small hairpin (shRNA) to screen modifier regulators of hematopoietic stem and progenitor cells (HSPCs) that regulate self-renewal and differentiation [ 15 ].…”