Determinng global implications of the ADAR3 R-domain on mediating ADAR3-RNA interactions
Although R-domain has been implicated in RNA binding in vitro, whether ADAR3 uses the arginine-rich R-domain to mediate ADAR3-RNA interactions in vivo remain to be explored. Here, an unbiased, high-throughput approach was undertaken to understand if mutating the R-domain would affect the ability of ADAR3 to either bind and/or recognize target transcripts. Although R-domain doesn't confer target specificity to ADAR3, the R-domain mutant is associated with decreased protein stability and might reduce the ability of ADAR3 to bind target RNAs. Overall design: RNA Immunoprecipitation-Sequencing (RIP-Seq) for FLAG-tagged ADAR3 Wild type and R-domain mutant protein exogenously expressed in U373 glioblastoma cells. To achieve exogenous expression of wild type or mutant protein, U373 cells were retrovirally transduced with either empty vector encoding for antiobiotic resistance (Control), or ADAR3 wild type (ADAR3 WT) or R-domain (ADAR3 R-Mutant) mutant protein.