IGF2BP proteins Enhance mRNA stability

To evaluate the effect of IGF2BPs on mRNA stability and gene expression output, we conducted RNA-seq in individual IGF2BP knockdown and control HepG2 cells with or without actinomycin D treatment. Our RNA-seq and RNA stability profiling revealed that IGF2BPs were involved in RNA stability regulation and contributed to the stabilization of the transcriptome. Overall design: HepG2 cells were infected with individual lentiviral IGF2BP shRNA and non-specific control (shNS), and selected by puromycin to generate stable knockdown lines. We treated HepG2 cells with actinomycin D to inhibit transcription and collected cells at indicated time points (i.e., 0h, 1h, 3h, 6h). The total RNA was extracted by miRNeasy Kit (Qiagen) and sequenced by Illumina. For IGF2BP-dependent gene expression, untreated cells (i.e., 0h samples) were sequenced in triplicate and analyzed. For RNA stability profiling, RNA half-life was calculated by comparing the gene expression at 1, 3, 6 hours with actinomycin treatment to that in un-treated samples, with two biological replicates for each group.