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Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development.

ABSTRACT: Dystrophin transcripts were shown to be alternatively spliced in a pattern characteristic of both tissue type and developmental stage. Multiple novel spliced forms of dystrophin mRNA were identified in murine brain tissue, skeletal and cardiac muscle, diaphragm, and human cardiac Purkinje fibers. The transcript diversity was greatest in adult, non-skeletal muscle tissues. Sequence analysis revealed that four tandem exons of the murine gene are differentially spliced in at least 11 separate patterns to generate distinct isoforms. Two of these forms were observed in all tissues examined, while several others were uniquely observed in cardiac muscle and brain. Cardiac Purkinje fibers express an isoform primarily observed in brain tissue. Several spliced transcripts were observed only in postnatal development. Differential utilization of a fifth exon results in two mRNA splice forms that encode separate embryonic and adult C-termini of dystrophin. Comparison of murine with human dystrophin mRNAs showed that similar isoform expression patterns exist across species. These observations suggest that functionally distinct isoforms of the dystrophin protein are expressed in separate tissues and at different stages of development. These isoforms may be of significance in understanding the various tissue-specific effects produced by dystrophin gene mutations in Duchenne and Becker muscular dystrophy patients.

SUBMITTER: Bies RD

PROVIDER: S-EPMC312263 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development.

Bies R D RD Phelps S F SF Cortez M D MD Roberts R R Caskey C T CT Chamberlain J S JS

Nucleic acids research 19920401 7

Dystrophin transcripts were shown to be alternatively spliced in a pattern characteristic of both tissue type and developmental stage. Multiple novel spliced forms of dystrophin mRNA were identified in murine brain tissue, skeletal and cardiac muscle, diaphragm, and human cardiac Purkinje fibers. The transcript diversity was greatest in adult, non-skeletal muscle tissues. Sequence analysis revealed that four tandem exons of the murine gene are differentially spliced in at least 11 separate patte ...[more]

PMID: 1579466

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Project description:BackgroundWe are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells.AimsOur aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells.MethodsSkeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression.ResultsCell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs.ConclusionsEctopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

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Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development.

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Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development.

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