Mechanisms of Developmentally Regulated Splicing

描述（由申请人提供）：本提案的长期目标是了解内源性选择性剪接调节因子调节天然剪接转换的机制。在使用外源蛋白和RNA的实验中，许多蛋白质已被证明与特定的前体mRNA结合并调节剪接，然而，关于天然剪接转换如何在体内调节的知之甚少。横纹肌发育提供了一个理想的系统，以发现协调调节的选择性剪接转换的网络，确定特定组的剪接转换的决定性调节器，确定内源性剪接因子的活动是如何调制驱动这些转换，并确定控制决定性剪接调节器的活动的信号通路。我们将进行系统的大规模筛选，以确定在细胞培养中骨骼肌分化过程中以及在心脏和骨骼肌组织从胎儿到成人发育过程中受到强烈调控的选择性剪接事件。我们将建立剪接转换和一组已知剪接调节因子表达之间的相关性。小鼠和鸡横纹肌发育的比较将被用来确定剪接模式和调节表达的保守转换。生物信息学分析将用于确定推定的因子结合位点以及受调控的前体mRNA中的新基序。将使用稳健调节的可变剪接转换的集合来鉴定对特定剪接转换和协调调节具有决定性的剪接因子。将在细胞培养物以及转基因和基因敲除小鼠模型中测试剪接转换和调节子表达之间的因果关系。我们还将确定剪接调节剂的活动在发育过程中调制的机制。了解发育调控剪接对于了解正常和病理状态下的基因表达机制至关重要，并最终导致在分子水平上纠正或规避疾病过程的方法。

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.