HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [Molm13_ADA_RRM_controls]

Understanding RBPs’ molecular functions as well as their cell-type specific activity requires identification of RBPs’ direct mRNA targets. However, efforts to identify RNA targets of RNA binding proteins in stem cells have been hindered by limited cell numbers. Here we adapt the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to allow for global mapping of mRNA targets of the RBP MSI2 in rare mammalian adult stem cells. We first tested to see if this method is applicable in mammalian systems. We overexpressed MSI2 fusion with the catalytic domain of the Hyperactive ADAR mutant (MSI2-ADA) or with the dead catalytic mutant (MSI2-DCD) in MOLM-13 human leukemia cells. As ADAR converts A to I (G), the fusion leaves a "finger-print" where MSI2 binds. MSI2-DCD and empty vector controls were used to normalize the background editing. MOLM-13 cells overexpressing ADA only, RRM(del)MSI2-ADA or RRM(mut)MSI2-ADA fusions for 48 hours were harvested for RNA-seq to look for A-G editing events.