Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

Abstract

Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type–specific gene and splice isoform expression, notably loss of an indirect flight muscle–specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3′-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type–specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type–specific splicing and expression dynamics of identity genes and structural proteins.

To RNA‐binding and beyond: Emerging facets of the role of Rbfox proteins in development and disease

“…Rbfox1 expression strongly increases until 90 h, suggesting a possible role in late myogenesis stage, corresponding to myofibre growth, as well (Spletter et al, 2018). Indeed, the Rbfox1 protein levels follow the same trend (Nikonova et al, 2022).…”

“…Furthermore, Rbfox1 is differentially expressed among muscle fiber types, and, in concert with the RNA-binding protein Bru1, the Drosophila orthologue of CELF1/2, contributes to the generation of adult muscle diversity in Drosophila. Consistent with its second peak in expression during 72-90 h, the knockdown of Rbfox1 causes detached or torn myofibres, a "hypercontraction" muscle phenotype, that leads to behavioral and eclosion defects (Kao et al, 2021;Nikonova et al, 2022;Table 3). It is known that hypercontraction of the IFM is associated with specific mutations in the genes for the major sarcomeric proteins (Nongthomba et al, 2003).…”

“…These findings have been corroborated by the report that knockdown of Rbfox1 , driven by Mef2‐GAL4 , and, after 24 h APF, using Act88F‐GAL4 , a driver specific to the indirect flight muscle (IFM), resulted in severe phenotypes: flightlessness, or even lethality (Nikonova et al, 2019; Table 3). By virtue of its role as a splicing factor and its ability to bind to the 3′‐UTR of its target transcripts, Rbfox1 regulates the expression of myogenic transcription factors Mef2 and Salm (Nikonova et al, 2022). Furthermore, Rbfox1 is differentially expressed among muscle fiber types, and, in concert with the RNA‐binding protein Bru1, the Drosophila orthologue of CELF1/2, contributes to the generation of adult muscle diversity in Drosophila .…”

“…It is known that hypercontraction of the IFM is associated with specific mutations in the genes for the major sarcomeric proteins (Nongthomba et al, 2003). As was shown by Nikonova et al (2022), the structural defects due to the knockdown of Rbfox1 are to the mis-regulation of fiber type-specific genes, and splice isoform expression, particularly the loss of an IFM-specific isoform of wings up A (wupA), orthologous to human Troponin-I, that is involved in muscle contraction.…”

“…Given that both families of proteins have been implicated in the development and maintenance, Gazzara et al (2017) found physiologically relevant targets, including proteins involved in calcium signaling and members of the Myocyte enhancer factor 2 (MEF2) family of transcription factors. Importantly, suggesting that this antagonism is conserved through evolution, molecular evidence from Drosophila shows that Rbfox1 modulates the expression of myogenic transcription factors Mef2 and Spalt major (Salm), and both modulate expression of and genetically interacts with the CELF orthologue Bruno1 (Bru1) (Nikonova et al, 2022). Together, other splicing factors involved in brain‐ and/or muscle‐specific splicing, such as PTBP1, MBNL1, FRG1, and CELF proteins, may regulate the splicing of RBFOX itself.…”