Integrative Genomics Positions MKRN1 as a Novel Ribonucleoprotein within the Embryonic Stem Cell Gene Regulatory Network

For identification of proteins that associate with Makorin1 (MKRN1) in RNA-dependent and RNA-independent manners, we affinity purified FLAG-tagged Makorin1 (MKRN1) from mouse embryonic stem cells constitutively expressing FLAG:MKRN1. Anti-FLAG control immunoprecipitations were performed from a FLAG vectrol control (FLAG:Ctrl) mouse embryonic stem cell line that did not express FLAG:MKRN1. Following FLAG immunoprecipitation, anti-FLAG beads from FLAG:MKRN1 and FLAG:Ctrl immunoprecipitations were split into separate tubes such that half of the beads were digested with 200Ã∞â≈ Ã≠µg/mL RNase A while the other half of the beads were undigested. RNase A-digested and undigested immunoprecipitates were subjected to LC-MS/MS analysis. Of the 48 RNA-related proteins previously identified to associate with FLAG:MKRN1, L1TD1, PABPC1, PABPC4, YBX1, IGF2BP1 and UPF1 were found to remain associated with FLAG:MKRN1 in the presence of RNase A.