High-Throughput Assay for Profiling Alternative Splicing and Splicing Regulators

SummaryThis Phase I program will develop TempO Splicea highly multiplexed targeted sequencing assay monitoring RNA Binding ProteinRBPsplicing expression and alternatively splicedASmRNA representatives of coregulated splicing modulesa novel variation of the TempO Seqassay used for high throughput screeningdesigned 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 networksenabling high throughput compound screening to identify compounds that regulate specific RNA targets through modulating ASto define the role of AS in compound efficacyand address the hypothesis that there are novel compound modulated co regulated AS networks not identified in tissuesThis assay will address an unmet need in a growing field that is currently stymied by the high costs and informatic effort needed to measure allAS mRNA isoforms in the transcriptomeDysregulated AS is characteristic of several conditionsfrom cancers to immuneneural and muscular diseasesand although AS affects andgtof human genesthe role of abnormal splice variants in disease is not well understoodpartly due to the scale of the measurement problemExposure to compounds is known to change AS both in vivo and in vitro and is well characterized in the JSLT cell linewhere stimulation with PMA causes changes in AS via signal transduction through MAPK pathwaysWe will design isoform specific probes targetingknown RBPsconfirmed AS events in JSLcellsandtissue specific AS events that represent known co regulated modulesPerformance will be demonstrated using total RNA and cell lysates from JSLcells and tissue RNAsTo show that TempO Splice can monitor dynamic changes in ASJSLcells will be treated with a panel ofMAPK inhibitors targeting different pathways prior to PMA stimulationRBP expression and AS profiles should be similar for inhibitors targeting the same pathwaysdefining co regulated splicing in signal transduction that could be distinct from published co regulation based on tissues or disease statesWe will also test a range of inhibitor concentrationsto assess the sensitivity of each RBP and AS event for each inhibitorderiving a minimum responsive dose that will be used to identify the most sensitive RBP and AS events for each pathwayIf the hypothesis that splicing is regulated by the combination of RBPs directing AS events is truethen the most sensitive RBPs in a pathway should directly regulate the most sensitive AS events in that pathwayThis project will test that hypothesisdissect the control of AS by signal transductionand demonstrate the utility of TempOSplice for monitoring changes in AS that will enable high throughput small molecule screening and novel RNAtargeted drug development NarrativeRNA targeted drug discovery is a promising new areawith recent FDA clearance of treatments for diseases caused by defects in alternative pre mRNA splicingbut the cost and informatic effort needed to survey alternative splicing in the whole transcriptome remains a major barrier to developing such drugsThis Phase I program will develop TempO Splicea highly multiplexed targeted sequencing assay monitoring RNA Binding ProteinRBPsplicing expression and alternatively splicedASmRNA representatives of co regulated splicing modulesto enable high throughput small molecule compound screening for RNA targetsand much like surrogate assays for monitoring pathwaysmonitor all known splicing modulesIn developing TempO Splicewe will be able to dramatically reduce costs and simplify data analysiswhile addressing the hypothesis that compound responsive co regulated AS events are distinct from co regulation observed in static tissuesthat the efficacy of drug compounds is associated with modulation of alternative splicingdemonstrating the assay s utility for monitoring changes in AS in response to compoundsdissecting how signal transduction regulates alternative splicing through distinct biochemical pathwaysand testing the hypothesis that RNA binding proteins drive alternative splicing choices