Determining the factors that control dose-dependent splicing regulation by a master regulator

许多控制选择性剪接的反式和顺式作用因子 已被确认身份。下一代测序方法的爆炸性增长 确定了数千个受调控的剪接事件(RNA-seq)，以及 许多调节选择性剪接的蛋白质已经通过 CLIPSEQ.留给该领域的一个重要问题是：规则是什么 管理替代剪接决定的行为？例如，什么是 决定是否可替换的RNA元件(序列和结构 受调控的外显子在低浓度或高浓度下都会对 剪接因子，以及剪接反应是否表现出协同行为或 不?解决这些问题对于为 了解剪接因子浓度的变化如何导致疾病。 为了解决这些问题，我们创建了细胞模型，使我们能够 精确滴定另一种剪接调节剂的水平，类似于肌肉盲人 1(MBNL1)蛋白。MBNL1已被证明可调节数千种替代方案 剪接事件，对骨骼肌、心脏的发育很重要 和中枢神经系统。这一规定的主要作用是突出 MBNL蛋白在强直性肌营养不良(DM)中的作用 MBNL1及其同源基因(MBNL2和MBNL3)通过扩展隔离 CuG或CCUG重复RNA，导致RNA加工异常。失踪者- MBNL靶标的拼接已被证明是导致一些 与糖尿病相关的症状，包括标志性症状肌强直。 。

Many trans and cis acting factors that control alternative splicing have been identified. The explosion of next-generation sequencing approaches have identified thousands of regulated splicing events (RNA-seq), and binding sites of many proteins which regulate alternative splicing have been identified via CLIPseq. An important question remaining for the field is: What are the rules governing the behavior of alternative splicing decisions? For example, what are the RNA elements (sequence and structure) that determine if alternatively regulated exons will respond at low concentrations or at high concentrations to a splicing factor, and will the splicing responses exhibit cooperative behavior or not? Addressing these questions is important for providing a framework for understanding how changes in splicing factor concentration can lead to disease. To address these questions, we have created cellular models that allow us to precisely titrate the level of an alternative splicing regulator, the Muscleblind-like 1 (MBNL1) protein. MBNL1 has been shown to regulate thousands of alternative splicing events and is important for the development of skeletal muscle, heart and the central nervous system. This regulation is highlighted by the primary role that MBNL proteins play in the disease myotonic dystrophy (DM), in which MBNL1 and its paralogs (MBNL2 and MBNL3) are sequestered by expanded CUG or CCUG repeat RNAs, resulting in aberrant RNA processing. The mis- splicing of MBNL targets has been shown to be responsible for causing some of the symptoms associated with DM, including the hallmark symptom myotonia. .