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.
RNA-targeted drug discovery is a promising new area, with recent FDA clearance of treatments for diseases caused by defects in alternative pre-mRNA splicing, but the cost and informatic effort needed to survey alternative splicing in the whole transcriptome remains a major barrier to developing such drugs. 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, to enable high-throughput small molecule compound screening for RNA targets, and much like surrogate assays for monitoring pathways, monitor all known splicing modules. In developing TempO-Splice, we will be able to dramatically reduce costs and simplify data analysis, while addressing the hypothesis that compound-responsive co-regulated AS events are distinct from co-regulation observed in static tissues, that the efficacy of drug compounds is associated with modulation of alternative splicing, demonstrating the assay?s utility for monitoring changes in AS in response to compounds, dissecting how signal transduction regulates alternative splicing through distinct biochemical pathways, and testing the hypothesis that RNA binding proteins drive alternative splicing choices.