Abstract
Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTGexp) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA LR transgenic mouse model which expresses a pathogenic range CTGexp. In the present study, we addressed the possibility that MBNL1 sequestration by CUGexp RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1 ΔE3/ΔE3 knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1 ΔE3/ΔE3 knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1 ΔE3/ΔE3 brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUGexp RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.
Highlights
Myotonic dystrophy type1 (DM1) is a multi-systemic disorder affecting skeletal muscle, heart, ocular lens, testis, and the central nervous system (CNS)
Splicing perturbations in the Mbnl1DE3/DE3 knockout brain To test directly whether loss of muscleblind-like 1 (MBNL1) expression contributes to aberrant splicing in the CNS, RNA was extracted from the brains of age-matched males carrying homozygous Mbnl1DE3/DE3 knockout or wild-type alleles in the same (C57BL/6J) background and analyzed on splicing-sensitive microarrays [9]
MBNL1 is a major factor which regulates exon 25 splicing with nearly complete inclusion in wild-type adult heart but only,60% inclusion in the Mbnl1 knockout heart
Summary
Myotonic dystrophy type (DM1) is a multi-systemic disorder affecting skeletal muscle, heart, ocular lens, testis, and the central nervous system (CNS). Recent evidence suggests that transcripts containing expanded CUG repeats (CUGexp) accumulate in nuclear RNA foci and exert toxic effects on a variety of cellular regulatory pathways including splicing and transcription [6]. One disease model that is supported by considerable experimental evidence proposes that CUGexp RNAs cause sequestration and inhibition of the RNA-binding protein MBNL1 [7]. In support of this model, Mbnl knockout mice develop the muscle, eye, and RNA splicing abnormalities that are characteristic of DM1 disease [8]. We have reported a strong correlation in skeletal muscle splicing changes between two mouse models of DM1, the HSALR transgenic and the Mbnl knockout [9]
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