A robust kit, standards, and software for identifying RNA binding protein targets

PROJECT SUMMARYAfter transcriptionRNA molecules undergo a number of processing steps that regulate the spliced sequenceRNA expression levelsubcellular localizationand initiation and rate of translationand are each controlled by the activity of RNA binding proteinsRBPsRNA processing plays critical roles in normal developmentand altered RNA processing has been shown to cause familial Spinal Muscular AtrophyAmyotrophic Lateral Sclerosisand multiple cancer typesIdentifying the RNA targets of the RBP mutated in these diseases is a critical first step towards a mechanistic understanding of the disease andultimatelydevelopment of targeted therapeuticsHoweverstandardized methods for identification of RBP targets transcriptome wide were lackingas they required large sample input amountshad high experimental failure ratesand required high amplification leading to significant wasted sequencing and non quantitative resultsRecently we developed the enhanced CLIP seqeCLIPmethodologywhich showed thousand fold improved efficiency of generating high throughput sequencing libraries from RBP profiling experiments and enabled highly robust and reproducible RBP target profiling through the incorporation of paired size matched inputsHere we will develop the eCLIP method for wide spread use by the academic and commercial biotechnology industry in the following aimsDevelop an eCLIP kitand perform optimization and stability testing to ensure robust datageneration from a standardized kit formatPerform alpha testing of a standardized eCLIP kit and protocol to validate experimental successand identify areas for further optimizationDevelop and validate standard conditions for various cell typestissuesand sample typesincluding flash frozen versus fixed tissuessmall sample amountsand non human samplesDevelop software tools for standard data processing and primary data analysis of eCLIP dataincluding integration with publicly available eCLIP data resourcesEclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and computational biologyparticularly in the area of RNA processingas well as in the starting of early phase biology companiesmake us ideal candidates to perform the research proposed aboveThe four aims above will take eCLIP from an academic research method to one that can be standardly performed by biomedical researchers in academia as well as commercial entitieswithout requiring extensive expertise in RNA biologyThe development of standardized commercial kits to profile DNA binding protein targets by ChIP seq rapidly led to large scale adoption of this techniqueand has provided significant benefits to studies of DNA binding proteins involved in various diseasesdevelopment of an eCLIP kit will similarly enable studies of RNA binding proteinsAdditionallyour location in San Diego proximal to numerous academic research institutesincluding UCSD and the Salk Instituteas well as biotechnology companies will provide scientific and commercialization expertise and assistancecurrentlylines overline limit