Identification of trans regulators of ADAR and A-to-I RNA editing using mmPCR-seq. Identification of trans regulators of ADAR and A-to-I RNA editing using mmPCR-seq

A-to-I RNA editing levels differ across tissues and cell types, but regulators of the editing process are largely unknown. We performed mmPCRseq on H239T and H293TAdar2OE cells with overexpression of different candidate Adar regulators and GFP as a control to assay editing level differences between overexpression and control. Overall design: To assay the effects of overexpressing candidate Adar regulators on A-to-I RNA editing levels, we transiently overexpressed ILF2, ILF3, and STRBP in H293T cells and H293T cells stably overexpressing Adar2. We extracted RNA and made mmPCR-seq libraries to measure editing levels at targeted sites. We sequenced two technical replicates of two biological replicates of GFP overexpression (OE) controls and of ILF2, ILF3, and STRBP overexpression. We then overexpressed an ILF3 mutant lacking double stranded RNA binding domains (ILF3RBDOE) and compared two biological replicates to two biological replicates of matched GFP overexpression controls (GFPOE reps 3 and 4).