High-Throughput Assay for Profiling Alternative Splicing and Splicing Regulators

摘要 该第一阶段计划将开发Tempo-Splice，这是一种高度多元化的靶向测序分析监测 RNA结合蛋白(RBP)剪接/表达和选择性剪接(AS)mRNA的代表 调节剪接模块(用于高通量筛选的TEMPO-SEQ™分析的一种新变体)， 旨在解决这样一种假设，即有可能提供整个转录组的替代分析 作为事件来测量已知的共同调节网络，使高通量化合物筛选能够 识别通过调节AS来调节特定RNA靶标的化合物，以定义AS在 复合药效，并解决了这样的假设，即存在新的复合调控共调节AS 未在组织中识别的网络。这项测试将解决一个不断增长的领域中尚未得到满足的需求，目前 受阻于高昂的成本和所需的信息工作，以测量所有~100,000作为mRNA异构体在 转录组。失调的AS是几种疾病的特征，从癌症到免疫，神经和 肌肉疾病，虽然AS影响95%的人类基因，但异常剪接变异在 疾病还没有被很好地理解，部分原因是测量问题的规模。 已知在体内和体外接触化合物都会发生变化，这在JSL1中是很好的特征 T细胞系，其中PMA刺激通过MAPK通路的信号转导引起AS的变化。 我们将设计针对约1,100个已知限制性商业惯例的异构体特异性探针，其中约350个已确认为JSL1细胞中的事件， 和~500个组织特异性AS事件，代表已知的共同调节模块。性能将会是 使用JSL1细胞和组织RNA的总RNA和细胞裂解产物进行了演示。来展示那个节奏-拼接 可以监测AS的动态变化，JSL1细胞将被一组44个MAPK抑制剂靶向处理 PMA刺激前的不同通路。抑制剂的RBP表达和AS图谱应该相似 靶向相同的通路，定义信号转导中的共同调控剪接，这可能与 公布了基于组织或疾病状态的共同调节。我们还将测试一系列的抑制剂 浓度，以评估每个RBP的敏感性，并作为每个抑制剂的事件，得出最小 反应剂量将用于识别最敏感的RBP，并作为每个途径的事件。如果 剪接受限制性商业惯例的组合调控的假设是正确的，那么大多数 一个途径中的敏感限制性商业惯例应该直接调节该途径中最敏感的AS事件。这个项目 将检验这一假说，剖析信号转导对AS的控制，并演示节奏的效用。 用于监测AS变化的剪接，这将使高通量小分子筛选和新型RNA- 有针对性的药物开发。

Summary This Phase I program will develop TempO-Splice, a highly multiplexed targeted sequencing assay monitoring RNA Binding Protein (RBP) splicing/expression and alternatively spliced (AS) mRNA representatives of co- regulated splicing modules (a novel variation of the TempO-Seq™ assay used for high-throughput screening), designed to address the hypothesis that it is possible to provide a surrogate assay of the whole transcriptome of AS events to measure known co-regulated networks, enabling high-throughput compound screening to identify compounds that regulate specific RNA targets through modulating AS, to define the role of AS in compound efficacy, and address the hypothesis that there are novel compound modulated co-regulated AS networks not identified in tissues. This assay will address an unmet need in a growing field that is currently stymied by the high costs and informatic effort needed to measure all ~100,000 AS mRNA isoforms in the transcriptome. Dysregulated AS is characteristic of several conditions, from cancers to immune, neural and muscular diseases, and although AS affects >95% of human genes, the role of abnormal splice variants in disease is not well understood, partly due to the scale of the measurement problem. Exposure to compounds is known to change AS both in vivo and in vitro and is well characterized in the JSL1 T-cell line, where stimulation with PMA causes changes in AS via signal transduction through MAPK pathways. We will design isoform-specific probes targeting ~1,100 known RBPs, ~350 confirmed AS events in JSL1 cells, and ~500 tissue-specific AS events that represent known co-regulated modules. Performance will be demonstrated using total RNA and cell lysates from JSL1 cells and tissue RNAs. To show that TempO-Splice can monitor dynamic changes in AS, JSL1 cells will be treated with a panel of 44 MAPK inhibitors targeting different pathways prior to PMA stimulation. RBP expression and AS profiles should be similar for inhibitors targeting the same pathways, defining co-regulated splicing in signal transduction that could be distinct from published co-regulation based on tissues or disease states. We will also test a range of inhibitor concentrations, to assess the sensitivity of each RBP and AS event for each inhibitor, deriving a minimum responsive dose that will be used to identify the most sensitive RBP and AS events for each pathway. If the hypothesis that splicing is regulated by the combination of RBPs directing AS events is true, then the most sensitive RBPs in a pathway should directly regulate the most sensitive AS events in that pathway. This project will test that hypothesis, dissect the control of AS by signal transduction, and demonstrate the utility of TempO- Splice for monitoring changes in AS that will enable high-throughput small molecule screening and novel RNA- targeted drug development.