Abstract
We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.
Highlights
There is no clear picture of the global splicing profile of the muscle transcript
A literature survey supports this common assumption with many reports describing alternative splicing events in the DMD gene since its discovery in 1986 (Supplementary Table S2)
These data derive from studies that are very heterogeneous in terms of the techniques used, specificity of the transcript isoform analysed (Dp427m and/or other isoforms), biological samples analysed, and gene regions explored, that makes qualitative and quantitative comparisons between these different datasets difficult
Summary
There is no clear picture of the global splicing profile of the muscle transcript. Available data are not readily comparable because techniques of different sensitivity were used in previous studies that generally focused only on specific regions of the gene[7,8,9,10]. Besides allowing for the comparison of gene expression changes in response to cellular differentiation, environmental factors or disease conditions, RNA-Seq can be used to accurately identify novel isoforms, assess relative transcript abundances and detect alternative exon and splice site usage in tissues or cells[15]. This approach has not yet provided information for all genes uniformly. We designed a suitable strategy that relies on a DMD-targeted RNA-Seq protocol and the 454 technology to allow accurate and comprehensive inventory of physiological ASEs of the DMD transcript in human skeletal muscle
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