Identify QKI-bound transcripts via RIP-seq

N7-methylguanosine (m7G) modification, routinely occurring at the 5' cap of mRNA or within tRNA and rRNA, also exists internally in mRNA. Although essential for mRNA translation as well as stress response, the “reader” protein for mRNA internal m7G modification is still unrevealed. Here, we reported that Quaking protein (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. We identified over 1000 confident m7G-modified and QKI binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, by sequestering mRNA within SGs, QKI7 modulates the translation efficiency of selected transcripts. Moreover, in line with the observation that doxorubicin triggers the assembly of SGs, QKI7 mediates the sensitivity of cancer cells to chemotherapy drug treatment. Overall design: To identify the directly bound transcripts of QKI (QKI5, QKI6, and QKI7), RNA immunoprecipitation sequencing (RIP-seq) was conducted in HepG2 cells stable expressing QKI proteins. Briefly, HepG2 cells were infected with lentivirus, pmiRNA1-3 x Flag-QKI. Only the GFP-positive cells were used for study and expanded in DMEM medium. RIP assay was performed with Flag antibody (F3165, Sigma).