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， 被识别。下一代测序方法的激增 确定了数千个受调节的剪接事件（RNA-seq）， 已经鉴定了许多调节可变剪接的蛋白质， CLIPseq.该领域的一个重要问题是：规则是什么？ 控制选择性剪接决定的行为例如， 决定是否选择的RNA元件（序列和结构） 受调控的外显子将在低浓度或高浓度下响应于 剪接因子，以及剪接反应是否表现出合作行为， 什么都没有？解决这些问题对于提供一个框架， 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 精确地滴定一种选择性剪接调节因子的水平， 1（MBNL 1）蛋白。MBNL 1已被证明可以调节数千种替代品， 剪接事件，是重要的骨骼肌，心脏， 和中枢神经系统。这一规定突出了首要作用 MBNL蛋白在强直性肌营养不良症（DM）中发挥作用，其中 MBNL 1及其旁系同源物（MBNL 2和MBNL 3）通过扩增的 CUG或CCUG重复RNA，导致RNA加工异常。错误的- MBNL靶点的剪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. .