Role of RNA Binding Protein RBM15 in m 6 A RNA Methylation During Megakaryocytic Differentiation

N 6 ‐methyladenosine (m 6 A) modification of RNA regulates RNA splicing, localization, export and degradation. Loss of m 6 A RNA is associated with defects in stem cell differentiation and gain of m 6 A RNA is associated with leukemogenesis. The RNA binding protein RBM15 was recently discovered to be involved in the m 6 A methylation of RNA. We have shown previously that RBM15 inhibits differentiation of myeloid cell lines, and its expression is decreased in megakaryocytes compared to bone marrow hematopoietic stem cells. We hypothesize that RBM15 m 6 A methylates RNAs that are involved in megakaryocyte fate specification and maturation. We tested whether RBM15 overexpression affects global m 6 A RNA levels during megakaryocytic maturation using two human erythroleukemia (HEL) cell lines: parental control (HEL‐pcDNA) cells carrying only the Tet‐repressor gene and HEL‐RBM15 cells, which encode the RBM15 transgene tagged with hemagglutinin (HA), under the control of a tetracycline‐inducible promoter. Megakaryocytic differentiation was induced by addition of 12‐Otetradecanoylphorbol‐13‐acetate (TPA) with and without doxycycline induction of the transgene for 24 hours. Immunoprecipitation using an anti‐HA antibody to pull down the HA‐tagged RBM15 followed by a Western blot revealed that WTAP (Wilms' tumor associated protein), which is part of the N 6 ‐methyltransferase complex, binds to HA‐tagged RBM15. Global m 6 A RNA methylation levels were analyzed by ELISA and confirmed by LC‐MS/MS. HEL‐pcDNA (control) m 6 A RNA levels did not change with any of the treatments using the ELISA or LC‐MS/MS. In contrast, overexpression of RBM15 during megakaryocytic differentiation increased m 6 A RNA significantly when measured by ELISA (HEL‐RBM15= 307.5±83.0% increase in m 6 A RNA vs. untreated (no doxycycline, no TPA); N=3]. Confirmation by LC‐MS/MS however, showed no significant changes in m 6 A RNA levels (HEL‐RBM15= 7.14±7.26% increase in m 6 A RNA compared to untreated; N=3). These data show that RBM15 associates with WTAP, one of the methyltransferase complex proteins, in a megakaryocyte cell line, and that antibody assays to detect m 6 A RNA should be validated by other methods such as mass spectrometry. We are currently performing rigorous identification of the factors responsible for discordance of ELISA and LC‐MS/MS measures. Additionally, we will test the hypothesis that RBM15‐induced RNA methylation is a mechanism by which RBM15 regulates megakaryocytic maturation with the ultimate goal of identifying the mechanisms of translational control at the RNA m 6 A methylation level that can be targeted in megakaryocytic leukemia. Support or Funding Information NIH 2T32HL007974‐16 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .