Poster abstracts
Poster number 43 submitted by Thomas Gallagher
Delineating Rbfox-regulated splicing networks critical for vertebrate muscle development
Thomas L. Gallagher (Molecular Genetics, The Ohio State University), Zachary T. Morrow (Molecular Genetics, The Ohio State University), Swanny A. Lamboy Rodriguez (Department 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 (Molecular Genetics, The Ohio State University)
Abstract:
Rbfox RNA binding proteins are regulators of phylogenetically-conserved alternative splicing events important for neuromuscular function. To investigate rbfox gene function, we knocked down two zebrafish rbfox genes expressed in muscle, rbfox1l and rbfox2, and showed that double morphant embryos have skeletal and cardiac muscle defects and 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 mutant embryos have disorganized myofibrils coupled with complete paralysis. Despite complete loss of contractility in double mutants, neuromuscular junctions and fiber type specification appear normal. Splicing analysis reveals that predicted Rbfox target exons are down-regulated in rbfox1l-/-; rbfox2-/- double mutants. Interestingly, compound mutant combinations have different effects: rbfox1l-/-; rbfox2+/- mutants have wavy myofibers and undergo seizures upon touch-evoked stimulation, whereas rbfox1l+/-; rbfox2-/- mutants are indistinguishable from single mutants. Together, these data 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 the Rbfox-regulated splicing program, we used RNA-Seq to identify Rbfox target exons that are misregulated in Rbfox-deficient muscle and have identified compelling downstream candidates. 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 predict and evaluate therapeutic approaches for splicing defects that lead to human disease.
References:
Gallagher TL, Arribere JA, Geurts PA, Exner CR, McDonald KL, Dill KK, Marr HL, Adkar SS, Garnett AT, Amacher SL, Conboy JG. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions. Dev Biol 2011, Nov 15;359(2):251-61.
Keywords: rbfox, splicing, muscle