HyperTRIBE Analysis for DDX41 in human leukemia cells

In HyperTRIBE analysis, a hyperactive mutant of the catalytic domain of ADAR enzyme is fused to an RNA-binding protein of interest in order to identify RNAs that are bound by the protein interest through RNA-sequencing (Rahman et al Nat Protoc. 2018). By discovery of RNAs where adenosines have been edited to iosines, the RNAs that were bound by the protein of interest and then edited by ADAR are identified genome-wide. We conducted this analyis for DDX41, an RNA Helicase protein with multiple described cellular functions, including RNA splicing and innate immune sensing. Because DDX41 mutations have been discovered in leukemia patients, this analysis was conducted in the human lyeloid leukemia cell line THP-1. DDX41 was fused to the hyperactive catalytic domain of ADAR by cloning and this fusion protein was expressed from the pGK-GFP retrovirus in THP-1 cells. Cells transduced with empty retrovirus or retrovirus containing the ADAR hyperactive catalytic domain alone were used as controls. The cells were allowed to grow for one week post-transduction and then were harvested for total RNA.