Mechanisms of Developmentally Regulated Splicing

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DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand the mechanisms by which endogenous alternative splicing regulators modulate natural splicing transitions. Many proteins have been shown to bind to specific pre-mRNAs and regulate splicing in experiments using exogenous proteins and RNA, however, very little is known about how natural splicing transitions are regulated in vivo. Striated muscle development provides an ideal system to discover networks of coordinately regulated alternative splicing transitions, identify the determinative regulators of specific sets of splicing transitions, determine how the activities of endogenous splicing factors are modulated to drive these transitions, and identify the signaling pathways that control the activities of the determinative splicing regulators. We will perform a systematic large scale screen to identify alternative splicing events that are robustly regulated during differentiation of skeletal muscle in cell culture and during fetal to adult development of heart and skeletal muscle tissues. We will establish correlations between splicing transitions and the expression of a panel of known splicing regulators. Comparisons of mouse and chicken striated muscle development will be used to identify conserved transitions of splicing patterns and regulator expression. Bioinformatic analyses will be used to identify putative factor binding sites as well as novel motifs among regulated pre-mRNAs. Sets of robustly regulated alternative splicing transitions will be used to identify splicing factors that are determinative for specific splicing transitions and coordinated regulation. Cause:effect relationships between splicing transitions and regulator expression will be tested, in cell culture and in transgenic and knockout mouse models. We will also determine the mechanisms by which the activities of splicing regulators are modulated during development. Understanding developmentally regulated splicing is crucial for understanding the mechanisms of gene expression in normal and pathological states and will ultimately lead to approaches to correct or circumvent disease processes at the molecular level.