Abstract
Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUGexp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSALR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUGexp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.
Highlights
Studies on myotonic dystrophy type 1 (DM1) have led to the RNAmediated disease model for hereditary disorders caused by noncoding microsatellite expansions
We proposed a gene therapy strategy for DM type 1 (DM1) that involved MBNL overexpression, leading to an increase in the nonsequestered nuclear pool of MBNL protein and reversal of DM1-associated RNA missplicing and pathology. To test this therapeutic approach, we demonstrated that MBNL1 was overexpressed following intramuscular (i.m.) injection of rAAV2/1-mycMbnl1 virus, and this led to the reversal of myotonia and missplicing of DM1 target RNAs in the HSALR poly(CUG) model for DM1 [16]
MBNL2 was chosen for overexpression, since this paralog compensates for loss of MBNL1 due to sequestration by CUG RNA expansions (CUGexp) RNA in skeletal muscle and is the major MBNL protein expressed in the brain [9]
Summary
Reduced Lifespan and Muscle Pathology Following HNRNPA1 Overexpression in a Mouse DM1 Model. Contrary to MBNL2, HNRNPA1 overexpression led to a striking shift of these RNAs to a more fetal pattern in all muscles examined, similar to the splicing changes observed previously in CELF1 overexpression mice [8] These results suggested that in addition to CELF1, the splicing activities of other nuclear RNA-binding proteins might be affected by expression of CUGexp RNAs. These results suggested that in addition to CELF1, the splicing activities of other nuclear RNA-binding proteins might be affected by expression of CUGexp RNAs To confirm that these splicing shifts were not the result of systemic pathology and compromised lifespan following neonatal systemic delivery of AAV, DM1-relevant splicing shifts were subsequently evaluated in adult skeletal muscle following direct i.m. injections. Clcn exon 7a cFHonVrntBnrpola FVB cHonrntnrpola Ψ (percent spliced in) fetal Mbnl adult exon 7 adult Pdlim fetal exon 5 fetal Tnnt adult exon F adult myc fetal fetal adult
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