Uncovering isoform-specific functions of RNA-binding protein Bruno1 in Drosophila muscle development - RNA regulation plays a critical role in muscle development and function. Fine-tuning isoform expression ratios of sarcomere proteins enables muscles to acquire mature, fiber-type specific contractile properties and to adapt to physiological demand, although the regulatory mechanisms that direct fiber-type specific splicing are still not well understood. In diseases such as myotonic dystrophy, mis-regulation of RNA-binding proteins including CELF1 leads to altered isoform expression patterns and muscle malfunction. The development of new therapies will rely on a better understanding of how aberrant RNA regulation leads to muscle disease. Our work has helped establish Drosophila melanogaster, a tractable model organism with a powerful genetic toolbox, as a model to study conserved mechanisms of RNA-binding protein function in myogenesis. We have shown that the CELF1 homolog Bruno1 (Bru1) regulates fiber-type specific splice events in indirect flight muscle. Loss or gain of Bru1 disrupts cytoskeletal rearrangements during early stages of differentiation that are necessary for myofibrillogenesis and blocks a developmental transition in alternative splicing to mature sarcomere protein isoforms necessary for proper regulation of sarcomere growth and myosin activity. Bru1 as well as CELF homologs in vertebrates are themselves alternatively spliced, but little is known about their isoform-specific functions. Here we propose to elucidate the function of individual Bru1 isoforms during muscle development using a multi-pronged approach integrating genetic, molecular and biochemical techniques. Based on preliminary data demonstrating Bru1 isoform-specific phenotypes, we will expand our isoform-specific toolbox to monitor isoform localization dynamics and test which isoforms are required by manipulating developmental-stage dependent isoform expression. We will uncover Bru1 isoform-specific regulatory logic by analyzing transcriptome-wide splicing signatures in RNA-Seq complemented with Nanopore sequencing to assemble full-length transcripts. We will verify alternative regulatory events in loss and gain of function backgrounds with splicing reporters. We will demonstrate Bru1 protein isoform-specific binding affinity and motif preference using EMSA and eCLIP, and test domain-specific function with structure-function constructs. Successful completion of these aims will enable us to evaluate Bru1 isoform-specific function in alternative splicing and translation, and demonstrate how alternative splicing of CELF proteins fine-tunes regulatory capacity. Given the strong conservation in muscle structure and function between flies and vertebrates, our work has the potential to identify conserved and disease-relevant genetic principles governing RNA-binding protein function.
