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. Author manuscript; available in PMC: 2022 Jan 2.
Published in final edited form as: Sci Immunol. 2021 Jul 2;6(61):eabd1287. doi: 10.1126/sciimmunol.abd1287

Fig 4. IMP2 binds to Cebpd and Cebpb mRNAs and mediates post-transcriptional regulation.

Fig 4.

(A) Imp2+/+ MEFs were treated ± IL-17 for 3 h and subjected to RIP with anti-IMP2 or IgG Abs. qPCR of the indicated mRNAs was normalized to input. Data show mean ± SEM of 4 independent experiments. Inset; IMP2 immunoprecipitates from Imp2+/+ and Imp2−/− MEFs was assessed by immunoblotting. (B) MEFs were treated with IL-17 for 3 h and subjected to RIP-seq with anti-IMP2 Abs. Diagram indicates the intersection between mRNAs that are IMP2-occupied based on RIPSeq compared to mRNAs that are IMP2-influenced at baseline or after IL-17 treatment. (C) Top: Diagram of Cebpd 3’UTR and predicted m6A site mutants (sequence, fig S8). Dashed line indicates sequence in the biotinylated synthetic mRNA. Bottom: HEK293T cells were co-transfected with IMP2-FLAG and a Luc reporter fused to WT Cebpd-3’UTR or sequences in which putative m6A sites were mutated. Lysates were subjected to RIP with anti-FLAG Abs and Luc mRNA assessed by qPCR. Data are normalized to input and show mean ± SEM of 3 independent experiments. (D) Lysates from HEK293T cells transfected with FLAG-tagged IMP2 were incubated with the biotinylated mRNAs corresponding to the Cebpd 3’ UTR (863-982, dashed line in panel C) in which indicated adenosines were m6A-modified or unmodified. RNA pulldowns were performed with streptavidin beads. FLAG-tagged IMP2 analyzed by immunoblotting. Blots are representative of 3 experiments. Pooled band intensity values with mean ± SEM. (E) MEFs were treated with TNFα for 3 h, given actinomycin D ± IL-17 for the indicated times, and Cebpd assessed by qPCR. Left: Data normalized to time=0 (100%), representative of 3 independent experiments. Right: Half-life was estimated by linear regression (17). (F) MEFs were treated ± IL-17 for 3 h and subjected to RIP with anti-eIF4G Abs. mRNAs were assessed by qPCR. Data normalized to untreated Imp2+/+ samples precipitated with IgG. Data are representative of 2 independent experiments. Inset; eIF4G in cytoplasmic RIP fractions from Imp2+/+ MEFs. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 by ANOVA with post hoc Tukey’s test or t-test.