Identification of Rbfox-regulated transcripts important for muscle function
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Date
2018-12
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The Ohio State University
Abstract
Rbfox RNA-binding proteins are important regulators of muscle-specific splicing. Rbfox
proteins contain an RNA recognition motif that is conserved from flies to human and binds to (U)GCAUG motifs that are enriched in introns next to muscle-specific alternative exons. Many muscle-specific splicing events are conserved among vertebrates, and we use the zebrafish model to understand how Rbfox-mediated splicing regulates muscle development and function. In zebrafish, Rbfox1l and Rbfox2 are both expressed in muscle. Double knockdown of rbfox1l and rbfox2 using antisense morpholinos leads to splicing changes of muscle-specific alternative exons, coupled with skeletal muscle paralysis and reduced heart rate. To confirm whether splicing changes in morphants are indeed due to Rbfox depletion and not morpholino-induced side effects, I compared alternative splicing changes in rbfox genetic mutants versus morphants.
Using our unpublished RNA-seq data, I identified candidate Rbfox-regulated exons with
substantial splicing changes between wild-type versus double morphant embryos. To enrich for direct targets, I selected those candidates flanked by intronic (U)GCAUG motifs for further analysis. Seven out of ten tested candidates were similarly affected in double mutants and double morphants. These results indicate that splicing is similarly affected in morphants and mutants, consistent with the observation that rbfox double mutants and double morphants have the same morphological phenotypes. To identify Rbfox-regulated transcript(s) that are sufficient to rescue muscle function in Rbfox-deficient larvae, I set out to clone these seven validated Rbfox-regulated transcripts for mRNA synthesis and injection into double morphant embryos. Thus far, I have found that an Rbfox-regulated tpm3 isoform is not sufficient to rescue contractility in 3 rbfox double morphants. Because it is unlikely that a single Rbfox-regulated transcript is sufficient to restore muscle function in Rbfox-depleted larvae, future rescue experiments with pooled Rbfox-regulated mRNAs will be conducted. Our long-term goal is to identify Rbfox-regulated splicing events that are required for muscle function. The insight gained from this work will improve our understanding of Rbfox-regulated muscle splicing and may provide clues for the development of therapies for human muscle diseases.