Abstract
Circular RNAs (circRNAs) are a class of RNA molecule formed by a process called backsplicing, where a downstream splice-site joins with an upstream splice-site forming a covalently closed loop structure. CircRNAs are highly expressed and evolutionarily conserved across species. The gene locus of muscleblind (mbl) undergoes alternative splicing to form many mRNA isoforms. In addition, the second exon of mbl is circularized to generate circMbl the most abundant circular RNA in fly heads which can translate into a peptide in the fly synapse. MBL is known to regulate the biogenesis of circMbl. Additionally, MBL protein strongly binds to circMbl likely leading to regulation of the protein by sequestration and/or transport of the protein. This constitutes an auto-regulatory loop between circMbl and MBL protein.Although circMbl is highly expressed in Drosophila melanogaster, its physiological functions are still not known. Also, it is unclear whether these functions are different from the functions of MBL, the protein produced from the mbl mRNAs. To address these questions in Drosophila, the Kadener lab generated fly lines expressing short-hairpin RNA specifically targeting the backsplice junction of circRNAs. Using this tool, we specifically knocked down (KD) circMbl in vivo without affecting the linear counterparts (as the KD occurs in the cytoplasm). We also generated shRNAs targeting the highly expressed linear mbl isoforms (called mbl-C and mbl-O/P).
In this study, we demonstrated the roles of circMbl in Drosophila development and behavior. Knockdown of circMbl led to several phenotypes: male-specific developmental lethality, locomotor defects in larvae and an increase in bouton number at the larval neuromuscular junction (NMJ), wing posture defect and hyperactivity in adults. We experimentally proved that these phenotypes are exclusive for circMbl. Also, we showed the mbl-C specific knockdown exhibits different phenotypes. We further illustrated the cell types responsible for the larval locomotion defects in circMbl knockdown and established NMJ as a system to demonstrate the role of circMbl in synaptic growth. Overall, we displayed first-of-its-kind experimental data that showcases the role of circRNA in the development and behavior of an invertebrate model.