HyperTRIBE identifies hepatic IGF2BP2/IMP2 targets <i>in vivo</i> and links IMP2 to autophagy

Abstract Targets of RNA-binding proteins (RBPs) are often investigated by implementing variants of cross-linking and immunoprecipitation methodology, which can yield several disadvantages in target detection. The RBP and N6-methyladenosine (m6A) reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2/IMP2) exerts an essential pathophysiological role as a metabolic regulator and tumor promoter, impacting the stability, localization, and translation of its targets. Here, we employed HyperTRIBE as a method to identify RBP targets in native cells in vivo and identified targets of IMP2 in murine hepatocytes. IMP2-associated adenosine-to-inosine editing sites were identified by hydrodynamic transfection of mouse livers using an IMP2-ADAR (adenosine deaminase acting on RNA) construct. Functional enrichment and motif analysis results suggest IMP2-facilitated target stabilization and confirm presence of m6A binding motifs. In addition, the overlap with data of a TRIBE experiment employing murine embryonic fibroblasts and with those of differential gene expression, was investigated. Comparative transcriptomics between IMP2, wild-type, and control samples (mCherry-ADAR) revealed an enrichment of IMP2-bound mRNAs associated with autophagy, which could be validated by RNA immunoprecipitation in a human liver cancer cell line. A functional knockdown of IMP2 demonstrated an increased autophagic flux, providing further evidence for the involvement of IMP2 in autophagy.