9723935 Cooper The long-range goal of this project is to understand the mechanisms regulating physiological responses to insulin. Studies from this laboratory demonstrate that peptide hormones, such as insulin and IGF-1, influence alternative splicing of PKC(II mRNA within minutes. The switch in mRNA is accompanied by altered metabolic and mitogenic responses. Peptide-regulated RNA splicing may be an important feature of the response to physiological stimuli. In response to regulation of PKC(II splicing, glucose uptake is altered in skeletal muscle cells by insulin. RNA splicing is governed by the regulation of factors that inhibit or activate the use of 3' or 5' splice sites. For PKC(II to be encoded, an additional exon is included in the mRNA. If this exon is skipped, PKC(II is encoded. Activation of an additional 5' splice site was identified for PKC(II in insulin-responsive skeletal muscle cells. The sequential activation of more than one splice site by a hormonal stimulus could allow for the unmasking of other regulatory features for gene expression. To examine the mechanisms of hormone-induced alternative splice, a minigene corresponding to regions spanning the last exon common to both PKC(I and (II, (c, the intervening intron, the (II exon with its 3' flanking intron, and its 3'untranslated region will be constructed. The construct would contain the genetic information necessary for regulation of exon inclusion by insulin following its expression in L6 rat skeletal muscle cells. Cis-elements such as pyrimidine tracts, exon splicing enhancers (ESE), stem loops, and serine-arginine rich (SR) protein association sequences will be included in the minigene. Development of an insulin responsive minigene will provide a valuable model for examining splicing of mRNA by hormone and growth factor signaling cascades. Previous reported minigenes were constructed from simpler genes and were not regulated by extracellular signaling receptors. The regulation of mRNA splicing by pepti de hormones is an emerging field that links signals from the cell surface to the nucleus to alter post-transcriptional regulation of gene expression. This report of insulin altering PKC(II expression is the first characterization of hormone-regulated alternative pre-mRNA splicing. The development of an insulin-responsive minigene will provide a valuable tool for examining the effects of insulin on physiological processes since unknown critical elements can be identified in future studies through reduction and condensation of portions of the minigene not essential for insulin regulation of exon inclusion and mutational analysis of known regulatory sequences. The lone-range goal of this project is to understand the mechanisms regulating physiological responses to insulin. Development of an insulin responsive minigene will provide a valuable model for examining splicing of mRNA by hormone and growth factor signaling cascades, and a valuable tool for examining the effects of insulin on physiological processes.
