A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

Abstract

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here, we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

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

“…We observe this effect on bru1, exd, and salm expression levels in Rbfox1 knockdown flies (Figs 5, 7, and S7), and on Rbfox1 expression in bru1-IR or bru1 mutant flies (Fig 5). We have previously reported a similar variation in phenotypic strength after knockdown with multiple RNAi hairpins targeting other RBPs, notably SF1, Hrb87F, snf, Prp19, and others (Kao et al, 2021). These results illustrate the experimental importance of testing multiple, independent RNAi constructs to distinguish hypomorphic from null phenotypes, beyond merely addressing off-target effects (Mohr & Perrimon, 2012;Kaya-Çopur & Schnorrer, 2019;Neumeier & Meister, 2020).…”

“…We also saw defective jumping in Act88F-Gal4–driven Rbfox1 knockdown, and phenotypic severity was dependent on the strength of knockdown ( Fig 2E ). This reflects weak expression of the driver in jump muscle ( Kao et al, 2021 ). Together, these data indicate that a decrease in Rbfox1 levels results in behaviour defects associated with impaired tubular muscle function.…”

“…An integral part of this regulatory program is muscle type–specific alternative splicing, and notably, the splicing regulator Bru1 is up-regulated downstream of Salm ( Oas et al, 2014 ; Spletter et al, 2015 ). We have previously shown that Bru1 promotes many, but not all, fiber type–specific splice events in IFMs, whereas in parallel factors such SF1 and Hrb87F regulate splicing independently of the fiber identity pathway ( Spletter et al, 2015 ; Kao et al, 2021 ). The identity and function of other RNA regulatory components and their interaction with the fiber differentiation pathway is still under active investigation.…”

Myosin Transducer Inter-Strand Communication Is Critical for Normal ATPase Activity and Myofibril Structure

“…These defects include myofibril degeneration and splitting, as well as accumulation of abnormal Z-line structures. Such abnormalities have been noted to arise in Drosophila from reductions in thin filament proteins [ 28 ], reduced levels of proteins required for tension generation [ 29 ], as well as from decreases in proteins that alter transcription [ 30 ] or alternative RNA splicing of myofibrillar components [ 31 ]. Interestingly, a model of human β-myosin-based HCM displays thickened Z-discs in myofibrils of stem-cell-derived cardiomyocytes [ 32 ].…”

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function

“…Functional screening of genetic perturbations with high-throughput phenotypic measurements have identified novel regulators of muscle biology and disease. These include forward genetic mutagenesis screens to identify regulators of skeletal muscle development and locomotion in zebrafish ( Birely et al, 2005 ; Horstick et al, 2013 ; Johnson et al, 2013 ; Bennett et al, 2018 ) and worms ( Beron et al, 2015 ), RNAi screening of muscle size and function in fruit fly ( Kao et al, 2021 ; Graca et al, 2021 ), and CRISPR/Cas9 screening of muscle cells to identify regulators of myogenesis and cell survival in dystrophy models ( Bi et al, 2017 ; Lek et al, 2020 ; Ashoti et al, 2022 ). Here, we combined forward genetics via proteomic analysis of a diverse mouse panel with a targeted reverse genetics screen via AAV6 vector mediated expression of shRNAs to knockdown specific genes in bioengineered skeletal muscle to identify candidate regulators of skeletal muscle function.…”