Poster abstracts
Poster number 17 submitted by Tom Gallagher
Delineating Rbfox-regulated splicing networks critical for vertebrate muscle development
Thomas L. Gallagher, Zachary T. Morrow (Dept. of Molecular Genetics, The Ohio State University), Swanny A. Lamboy Rodriguez (Dept. of Biology, University of Puerto Rico in Humacao), Marcus H. Stoiber, James B. Brown, Susan E. Celniker, John G. Conboy (Life Sciences Division, Lawrence Berkeley National Lab), Sharon L. Amacher (Dept. of Molecular Genetics, The Ohio State University)
Abstract:
Rbfox RNA binding proteins are regulators of phylogenetically-conserved alternative splicing events important for neuromuscular functions. To investigate rbfox gene function in muscle, we knocked down muscle-expressed rbfox1l and rbfox2 in zebrafish embryos using antisense morpholinos and showed that double morphant embryos have skeletal and cardiac muscle defects and exhibit changes in splicing of bioinformatically-predicted Rbfox target exons (Gallagher et al., 2011). Using CRISPR/Cas9 technology, we now have generated rbfox1l and rbfox2 single and compound null mutants. Whereas rbfox1l and rbfox2 single mutant embryos have normal myofibers and contractility, rbfox1l-/-; rbfox2-/- double homozygous mutant embryos have disorganized myofibrils coupled with complete paralysis, very much like double morphant embryos. Despite contractile defects, neuromuscular junctions appear normal in both compound and double mutants, as well as specification of slow- and fast-twitch muscle fiber types. Additionally, splicing analysis reveals that predicted Rbfox target exons are down-regulated in rbfox1l-/-; rbfox2-/- double homozygotes. Interestingly, compound mutant combinations have different effects: rbfox1l-/-; rbfox2+/- mutants have wavy myofibers and exhibit seizure/tremor-like behavior upon touch-evoked stimulation, whereas rbfox1l+/-; rbfox2-/- mutants are indistinguishable from single mutants. Together, these results suggest that levels of Rbfox factors are critical for the coordinated regulation of an alternative splicing program essential for muscle function. In order to globally define this Rbfox-regulated splicing program, we have used RNA-Seq coupled with advanced computational strategies to identify Rbfox target exons that are misregulated in Rbfox-deficient muscle. Our goal is to understand how the network of Rbfox-regulated splicing events impacts muscle development and function that in the long term will allow us to rationally predict and functionally evaluate therapeutic approaches for splicing defects that lead to human muscle disease.
Keywords: Rbfox, alternative splicing, muscle