Abstract
BackgroundMuscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons. Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene.Methodology/Principal FindingsWe used evolutionary, genetic and cell culture approaches to study muscleblind (mbl) function in flies. The evolutionary study showed that the MblC protein isoform was readily conserved from nematods to Drosophila, which suggests that it performs the most ancestral muscleblind functions. Overexpression of MblC in the fly eye precursors led to an externally rough eye morphology. This phenotype was used in a genetic screen to identify five dominant suppressors and 13 dominant enhancers including Drosophila CUG-BP1 homolog aret, exon junction complex components tsunagi and Aly, and pro-apoptotic genes Traf1 and reaper. We further investigated Muscleblind implication in apoptosis and splicing regulation. We found missplicing of troponin T in muscleblind mutant pupae and confirmed Muscleblind ability to regulate mouse fast skeletal muscle Troponin T (TnnT3) minigene splicing in human HEK cells. MblC overexpression in the wing imaginal disc activated apoptosis in a spatially restricted manner. Bioinformatics analysis identified a conserved FKRP motif, weakly resembling a sumoylation target site, in the MblC-specific sequence. Site-directed mutagenesis of the motif revealed no change in activity of mutant MblC on TnnT3 minigene splicing or aberrant binding to CUG repeat RNA, but altered the ability of the protein to form perinuclear aggregates and enhanced cell death-inducing activity of MblC overexpression.Conclusions/SignificanceTaken together our genetic approach identify cellular processes influenced by Muscleblind function, whereas in vivo and cell culture experiments define Drosophila troponin T as a new Muscleblind target, reveal a potential involvement of MblC in programmed cell death and recognize the FKRP motif as a putative regulator of MblC function and/or subcellular location in the cell.
Highlights
Human Muscleblind-like 1, 2 and 3 (MBNL1-3) are RNA binding proteins that have been involved in numerous diseases
MBNL1 function is impaired in myotonic dystrophy (DM), a multisystemic disease characterized by myotonia, cataracts and muscle weakness, and a fly model of spinocerebelar ataxia 8 genetically interacted with Drosophila muscleblind mutants [6]
To assess whether Drosophila Muscleblind protein isoforms were evolutionary conserved or were species-specific, we searched the genomes of organisms from representative taxonomic groups for homologous sequences
Summary
Human Muscleblind-like 1, 2 and 3 (MBNL1-3) are RNA binding proteins that have been involved in numerous diseases. MBNL1 function is impaired in myotonic dystrophy (DM), a multisystemic disease characterized by myotonia, cataracts and muscle weakness (reviewed in [4,5]), and a fly model of spinocerebelar ataxia 8 genetically interacted with Drosophila muscleblind mutants [6]. In spite of their biomedical relevance, the processes in which Muscleblind proteins are required and the molecular mechanisms they use to carry out such functions are only beginning to be understood. Muscleblind-like proteins (MBNL) have been involved in a developmental switch in the use of defined cassette exons Such transition fails in the CTG repeat expansion disease myotonic dystrophy due, in part, to sequestration of MBNL proteins by CUG repeat RNA. Four protein isoforms (MblA-D) are coded by the unique Drosophila muscleblind gene
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