High-Throughput Assay for Profiling Alternative Splicing and Splicing Regulators

总结 该I期项目将开发TempO-Splice，这是一种高度多重靶向测序分析监测技术， RNA结合蛋白（RBP）剪接/表达和选择性剪接（AS）mRNA代表共- 调节剪接模块（用于高通量筛选的TempO-Seq™测定的新变体）， 旨在解决这一假设，即有可能提供整个转录组的替代测定 的AS事件，以测量已知的共调节网络，使高通量化合物筛选， 鉴定通过调节AS来调节特定RNA靶标的化合物，以确定AS在 化合物的功效，并解决假设，有新的化合物调制共调节AS 在组织中未识别的网络。该测定将解决目前正在发展的领域中未满足的需求。 由于高成本和测量所有~ 100，000 AS mRNA亚型所需的信息工作， 转录组失调的AS是几种疾病的特征，从癌症到免疫、神经和神经系统疾病。 虽然AS影响>95%的人类基因，但异常剪接变体在肌肉疾病中的作用， 对这种疾病的了解还不够，部分原因是测量问题的规模。 已知暴露于化合物会改变体内和体外AS，并在JSL 1中得到充分表征 T细胞系，其中PMA刺激通过MAPK途径的信号转导引起AS变化。 我们将设计针对约1,100种已知RBP、约350种JSL 1细胞中确认的AS事件的亚型特异性探针， 和约500个组织特异性AS事件，代表已知的共调节模块。性能将 使用来自JSL 1细胞和组织RNA的总RNA和细胞裂解物证明。为了证明TempO-拼接 可以监测AS的动态变化，JSL 1细胞将用一组44种MAPK抑制剂治疗， 在PMA刺激之前的不同途径。对于抑制剂，RBP表达和AS特征应该相似 靶向相同的途径，定义信号转导中的共调节剪接， 基于组织或疾病状态的公开的共同调节。我们还将测试一系列抑制剂 浓度，以评估每种抑制剂的每种RBP和AS事件的敏感性， 反应剂量，将用于识别每种途径的最敏感RBP和AS事件。如果 假设剪接是由指导AS事件的RBP组合调节的，那么大多数 一个途径中敏感的RBP应该直接调节该途径中最敏感的AS事件。这个项目 将检验这一假设，剖析信号转导对AS的控制，并证明克里思- 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- 靶向药物开发。

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.