Physiological Insulin Regulates Alternative Mrna Splicing In Liver And Muscle At Multiple Levels

Alternative splicing of mRNAs contributes importantly to proteome diversity and tissue identity. Work by us and others have suggested that alternative splicing and the spliceosome are dysregulated in diabetes, but the role of this under physiological conditions is not known. To gain more insight into alternative splicing regulation by physiological insulin in vivo, we performed RNA-seq of mRNAs in skeletal muscle and liver from mice undergoing euglycemic clamp with physiological insulin for 3h. In both tissues, insulin significantly regulated over 1000 transcripts, including known pathways related to glucose and fatty acid metabolism. In addition, insulin also regulated many components involved in mRNA splicing, including mRNAs of ribonucleoprotein particles (RNP) spliceosome components, splicing factors and CLK splicing kinases, many of which were down-regulated by insulin. This was associated with significant changes in 102 and 63 alternatively spliced mRNAs in muscle and liver in a variety of gene classes including those encoding proteins involved in tissue-structure, mitochondrial function, autophagy and transcription. Insulin also regulated the spliceosome at the post-translational level, with a 1.5- to 2-fold increase in the phosphorylation of the U2-RNP component SF3B1 and the splicing factor SRSF5. Phosphorylation of the splicing factors SRSF4, SRSF5 and SRSF6 was also increased by insulin in C2C12 myotubes, and this was prevented by pretreatment with the CLK inhibitor TG003. Myotubes treated with TG003 also showed dose-dependent decrease of insulin-stimulated glucose uptake and increased FCCP-uncoupled respiration. Taken together, these data demonstrate that regulation of alternative splicing at multiple mechanistic levels integrates the physiological response to insulin. Disruption of insulin signaling to the spliceosome could potentially shift isoform balance of metabolically relevant proteins and contribute to insulin resistance. Disclosure T.M. Batista: None. W. Cai: None. B. Brandao: None. C. Kahn: Advisory Panel; Self; MedImmune. Board Member; Self; Kaleido Biosciences. Consultant; Self; AntriaBio, Inc., Cobalt Therapeutics, Flagship Pioneering. Research Support; Self; Alnylam. Funding National Institutes of Health