Two Isoforms of Eya3 Influence Muscle Cell Differentiation and Become Dysregulated in Striated Muscle Diseases

Abstract

Alternative splicing is a ubiquitous gene regulatory mechanism by which diverse transcripts are derived from a finite genome. During development, complex splicing transitions occur, leading to varied transcript profiles in fetal and adult tissues. Like gene expression, splicing often becomes dysregulated in cancer, neurological, and muscular diseases. One barrier to therapeutic targeting of alternative splicing is that the function of splice isoforms, both physiologically and pathologically, are vastly understudied. Here, we begin to address this knowledge gap by describing an evolutionarily conserved alternative splicing event in eyes absent homolog 3 (Eya3) gene for the first time. The short isoform of Eya3 is a dual threonine- and tyrosine-phosphatase that is known to promote cell growth and chemotherapeutic resistance. By analyzing RNA-sequencing data from various mouse tissues, we identified the long isoform of Eya3, which contains an additional 138nt, representing a 46 amino acid in-frame insertion immediately following the threonine phosphatase domain. Using reverse transcription-PCR (RT-PCR) of mouse, human, and rabbit tissues, we discovered that Eya3 splicing is tissue-specific, with the long isoform expressed exclusively in the heart, skeletal muscle, and brain. Additionally, we found that during heart and skeletal muscle development, expression of the long Eya3 isoform gradually increases relative to the expression of the short isoform. In myotonic dystrophy type 1 patient hearts, and failing mouse hearts, the short Eya3 isoform is re-expressed, illustrating that Eya3 splicing is dysregulated in striated muscle diseases. To understand Eya3 splicing in further molecular detail, we utilized the well-established C2C12 murine myoblast cell line. Differentiation of myoblasts to myotubes robustly recapitulates the Eya3 splicing transition observed during tissue development, making them an appropriate cell culture system for our work. Depletion of Eya3 via siRNA transfection inhibited myoblast proliferation and fusion. Interestingly, blocking the endogenous Eya3 alternative splicing transition with morpholino antisense oligonucleotides also suppressed normal differentiation as determined by immunohistochemical staining and confocal microscopy. These results indicate that Eya3, and its splice isoforms, play an important role in early muscle development. In summary, our studies uncover a developmentally-regulated, tissue-specific, and disease-relevant splicing event in Eya3. Moving forward, we seek to determine the molecular mechanism by which Eya3 isoforms regulate normal muscle development and to evaluate Eya3 as a potential therapeutic target.