Mechanisms of Developmentally Regulated Splicing

描述(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自然剪接转变的机制。在使用外源蛋白质和RNA的实验中，许多蛋白质被证明与特定的前mRNAs结合并调节剪接，然而，对于体内自然剪接过渡是如何调控的知之甚少。横纹肌发育提供了一个理想的系统来发现协调调控的选择性剪接过渡网络，识别特定剪接过渡集合的决定性调控因子，确定如何调节内源性剪接因子的活性来驱动这些过渡，并确定控制决定性剪接调控因子活性的信号通路。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有力调控的选择性剪接事件。我们将在剪接转换和一组已知剪接调控因子的表达之间建立关联。老鼠和鸡的横纹肌发育的比较将被用来确定剪接模式和调控基因表达的保守转变。生物信息学分析将被用来确定推测的因子结合位点以及受调控的前mRNAs中的新基序。将使用一组严格调控的备选剪接过渡来确定对特定剪接过渡和协调调控起决定性作用的剪接因素。原因：将在细胞培养以及转基因和基因敲除小鼠模型中测试剪接转变和调节子表达之间的效应关系。我们还将确定剪接调控因子在发育过程中调节活动的机制。了解发育调控的剪接对于理解正常和病理状态下基因表达的机制至关重要，并最终将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.