The m6A reader IMP2 directs autoimmune inflammation through an IL-17- and TNFa-dependent C/EBP transcription factor axis

Using IMP2-deficient mice to study the role of the RNA-binding protein (RBP) IMP2 in models of autoimmunity, namely autoimmune glomerulonephritis Examination of IMP2 Binding proteins using RIP-seq and RNA-seq RNA-seq and RIP-seq were both done on Mouse embryonic fibroblasts (MEFs): For RNA-seq: Mouse embryonic fibroblasts (MEFs) were plated in 6-well tissue culture-treated dishes and placed in a 5% CO2 37C incubator overnight. MEFs were transfected with siRNA control or siRNA targeting IMP2 . After 24 hours, media was changed and cells were left either unstimulated, or stimulated with IL-17 (200 ng/mL) for 8 hours . Cells were trypsinized and RNA was harvested using Rneasy Mini Kit from Qiagen. RNA-seq libraries were prepared using standard Nextera XT Kit. For RIP-seq: Mouse embryonic fibroblasts (MEFs) were plated in 150mm tissue culture-treated dishes and placed in a 5% CO2 37C incubator overnight. Cells were left either unstimulated or stimulated with IL-17 (200 ng/mL) for 3 hours . Cells were trypsinized and extracts were isolated with lysis buffer [100 mM KCl, 5 mM MgCl2, 10 mM Hepes (pH 7.0), 0.5% NP-40, and 1 mM dithiothreitol] with RNaseOUT (100 U/ml; Invitrogen). Buffers included a protease inhibitor cocktail (Sigma-Aldrich). Lysates were precleared with protein A agarose (Roche) and immunoprecipitated with Abs against IMP2 (MBL) or rabbit IgG control (MBL). Beads were washed with NT2 buffer and digested with DNase I (Roche Applied Science) and protease K (Sigma-Aldrich). Total RNA was extracted with acid phenol or TRIzol. RIP-seq libraries were prepared using SMART-Seq v4 Ultra Low Input RNA kit