The long-term goal of this project is to determine the extent, regulatory mechanisms, and functional consequences of regulated pre-mRNA processing in skeletal muscle. A large number of genes express pre- mRNAs that undergo alternative splicing to produce conserved muscle-specific protein isoforms the functions of which are unknown. These isoforms often appear during late fetal or early postnatal development. Disruption of alternative splicing is a common feature of diseases affecting skeletal muscle, often involving reversion to fetal isoforms, yet little is known about the contributions of these changes to pathogenesis. An underlying hypothesis of this proposal is that a focus on genes with conserved fetal and adult protein isoforms, including adult isoforms that are muscle-specific, will discover not only previously unknown isoform-specific functions but also previously unknown gene functions critical for adult muscle homeostasis. To identify the functions of alternative splicing events in vivo we are using CRISPR-mediated removal of exons that undergo muscle- specific and/or postnatally regulated inclusion. In the first aim, we will determine the functions of a striated muscle-specific isoform of the Map4 microtubule-binding protein in which deletion of the muscle-specific exon significantly disrupts microtubule architecture in skeletal muscle myofibers and impacts muscle force generation. In the second aim we will determine the function of the skeletal muscle-specific Limch1 isoform, the absence of which also decreases skeletal muscle function in vivo. In the third aim we will use CRISPR- mediated introduction of epitope tags into endogenous genes to monitor the temporal and spatial details of the alternative splicing transitions and adult muscle-specific isoforms at the level of individual cells in vivo. This study is expected to identify previously unknown gene functions, increase understanding of adult skeletal muscle homeostasis and the impact of its disruption in disease.
Many genes express muscle-specific protein isoforms generated by alternative splicing for which functions are unknown. This proposal will identify the functions of muscle-specific isoforms with a strong potential to reveal previously unknown roles in maintaining healthy muscle. The knowledge gained will expand understanding of skeletal muscle homeostasis for recognition of previously unknown features in disease.
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|Brinegar, Amy E; Xia, Zheng; Loehr, James Anthony et al. (2017) Extensive alternative splicing transitions during postnatal skeletal muscle development are required for calcium handling functions. Elife 6:|
|Manning, Kassie S; Rao, Ashish N; Castro, Miguel et al. (2017) BNANC Gapmers Revert Splicing and Reduce RNA Foci with Low Toxicity in Myotonic Dystrophy Cells. ACS Chem Biol 12:2503-2509|
|Sharpe, Joshua J; Cooper, Thomas A (2017) Unexpected consequences: exon skipping caused by CRISPR-generated mutations. Genome Biol 18:109|
|Morriss, Ginny R; Cooper, Thomas A (2017) Protein sequestration as a normal function of long noncoding RNAs and a pathogenic mechanism of RNAs containing nucleotide repeat expansions. Hum Genet 136:1247-1263|
|Manning, Kassie S; Cooper, Thomas A (2017) The roles of RNA processing in translating genotype to phenotype. Nat Rev Mol Cell Biol 18:102-114|
|Cox, Diana C; Cooper, Thomas A (2016) Non-canonical RAN Translation of CGG Repeats Has Canonical Requirements. Mol Cell 62:155-156|
|Giudice, Jimena; Loehr, James A; Rodney, George G et al. (2016) Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology. Cell Rep 17:1923-1933|
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