Development of aDrosophila melanogasterspliceosensor system forin vivohigh-throughput screening in myotonic dystrophy type 1

Abstract

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1–MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.

(CCUG)n RNA toxicity in a Drosophila model for myotonic dystrophy type 2 (DM2) activates apoptosis

“…1 B,C), demonstrating that 106 CCUG repeats are sufficient to cause biochemical changes. The average fraction of nuclei with ribonuclear foci in DM2 - 106 muscle cells is similar to that observed in a DM1 fly model expressing 480 CTG repeats ( García-Alcover et al, 2014 ).…”

“…The suitability of our Drosophila DM2 system as a disease model was further demonstrated by the observation that different spliceosensor luciferase reporters, which express specific mammalian reporter mini-genes for identified mis-splicing events in DM1 and DM2 (human INSR exon 11 and mouse Tnn t 3 fetal exon) ( Savkur et al, 2004 ; Vihola et al, 2010 ; García-Alcover et al, 2014 ), were also responsive to the presence of expanded CCUG repeats ( Fig. 2 F,H).…”

“…To verify that the significant changes in luciferase luminescence were due to mis-splicing, we examined the splicing pattern of the INSR spliceosensor reporter directly by RT-PCR in our DM2 fly model. In the Drosophila DM1 model, two splice isoforms of the INSR spliceosensor were detectable due to the inclusion (isoform B) or exclusion (isoform A) of an alternative exon between exons 11 and 12 ( García-Alcover et al, 2014 ). Isoform A is preferentially observed in the disease state ( García-Alcover et al, 2014 ).…”

“…In the Drosophila DM1 model, two splice isoforms of the INSR spliceosensor were detectable due to the inclusion (isoform B) or exclusion (isoform A) of an alternative exon between exons 11 and 12 ( García-Alcover et al, 2014 ). Isoform A is preferentially observed in the disease state ( García-Alcover et al, 2014 ). In the N-16 controls expressing (CCUG) 16 , isoform A was present at 42.5% ( Fig.…”

A Potent Inhibitor of Protein Sequestration by Expanded Triplet (CUG) Repeats that Shows Phenotypic Improvements in a Drosophila Model of Myotonic Dystrophy

“…In contrast, the fly line expressing 60 uninterrupted CTG repeats, (CTG) 60 , does not exhibit severe pathologies. Several small molecules were reported to improve the CUG‐induced phenotypes in the DM1 Drosophila model, including our previous studies of 1 a . The comparative effectiveness of 1 a and 2 a in suppressing the external eye degeneration phenotype and improving larva mobility was examined in the DM1 Drosophila model.…”