Investigating the role and regulation of Polypyrimidine tract binding protein (PTB) in isoform switching during muscle development in Drosophila melanogaster

Abstract

Muscles are an excellent system to dissect the development, physiology and evolution of cell heterogeneity and isoform switching. The vertebrate skeletal muscles are composed of heterogeneous muscle fibres. These fibres are classified as Type I (slow-twitch) and Type IIA and IIB (fast-twitch- oxidative & glycolytic) based on their distinct contractile and physiological properties. Fibre specific properties can be directly attributed to the differential expression of their protein isoforms. Thus, fibres can switch from one type to another based on developmental, physiological, and environmental cues by coordinated regulation of gene expression, co-integrated with protein isoform transitions. Alternative splicing is one of the major contributors to isoform transitions. Extensive investigations over the years have shown the involvement of several RNA Binding Proteins (RBPs) like MBNL, RBFOX1, PTBP, CELF etc., in establishing, refining, and maintaining fibre specific properties. However, the process by which isoform switching is fine-tuned remains largely unexplored. In this study, we investigated the process of developmentally regulated isoform switching mediated by the polypyrimidine-tract binding protein (PTB)- an hnRNP (heterogeneous ribonucleoprotein), in the developing Indirect Flight Muscles (IFMs) of Drosophila melanogaster. In mammals, PTB proteins have been shown to affect the alternative splicing of neuronal and muscle genes and the mutations in PTB are implicated in several cancers. Nevertheless, the role and genetic interactions of PTBs in muscle development remain largely unexplored. Drosophila IFMs are an excellent model to study isoform switching as it expresses distinct isoforms of sarcomeric proteins, which give rise to their characteristic fibrillar type of muscle fibres. During IFM development, the sarcomeric genes undergo pupal 🡪 adult isoform transition during 55-95 hours after puparium formation, which impart properties like stretch activation, enabling the fly to attain a wingbeat frequency of ~200 Hz. We observed that PTB is upregulated explicitly during this window of isoform switching. To further investigate the role of PTB in isoform switching during IFM development, we adopted an RNAi based approach. PTB in Drosophila is encoded by a single gene, hephaestus (heph). Knockdown of heph in IFMs leads to loss of flight function and defective muscle fibres. We show that the sarcomere maturation phase is affected, resulting in thinning, and shortening of sarcomeres accompanied by actin blob formation at Z-discs of the sarcomeres. Through RNA sequencing analysis, we identified that heph is required for inclusion of fibrillar exons of several sarcomeric genes like Mhc (Myosin heavy chain), wupA, up (Troponin I & T) etc., Several RBPs and channel proteins have also been differentially spliced upon heph knockdown. Subsequent bio-informatic analyses coupled with functional experiments, helped us extend our search to identify the network of other RNA Binding Proteins that work in tandem with PTB. This led to the identification of how (Held out wings) and Sm (Smooth) as potential candidates involved in the isoform switching stage. This presentation, thus, shall focus on the pathways and molecular players along with a brief mention of the role of calcium signalling in the regulation of isoform switching during Drosophila IFM development.