A hyper-active APOBEC1 mutant boosted profiling of RNA-binding protein (RBP) targets

RNA deaminase-based protein-RNA interaction mapping tools TRIBE/HyperTRIBE and STAMP became mainstream in recent years. These methods explore RBP's target RNAs with the editing events on targets mediated by the expressed RBP-deaminase fusion proteins. However, the activities of deaminases in different species are varied greatly, and thus these techniques were applicable to a few species so farfor example, STAMP was only applied in mammals. In this study, we successfully developed highly-efficient HyperSTAMP in Saccharomyces cerevisiae by introducing the H122L/D124N double mutations to rAPOBEC1 and profiled the targets of BFR1 and NAB2. The comparison of STAMP with HyperSTAMP revealed that even though STAMP could mark some targets in yeast cells, its low editing efficiency resulted in its poor consistency in target identification here. On the contrary, HyperSTAMP solved the problem of insufficient efficiency and low editing sequence compatibility existed in STAMP in certain species, and displayed excellent sensitivity, specificity and reliability. Furthermore, as APOBEC1 possibly edits both ssRNA and ssDNA, we investigated the genomic DNA editing in STAMP/HyperSTAMP and concluded that the DNA editing was more likely a random event and did not affect RBP's target identification. In summary, the newly-developed HyperSTAMP is characterized by low background, high sensitivity and reliability, and can be hired to explore RBP's targets in yeasts.