RNA decay profile for eRF1 and UPF1 depletion in HCT116 cell lines
We had found that non-optimal codon induced frameshifting translation coupled with NMD may play a crucial role in maintaining delicate need of mRNA from yeast to human. Our data suggests that NCFT also exist in human. We constructed eRF1-KD and UPF1-KD cell line and resort a-amanitin to treat cells in different time point, then transcriptionally profiled them to get the landscape of mRNA stability. Overall design: The HCT116-AID2 cell lines were induced by 1 µg/ml of doxycycline for 24h, and wereadded or not added (as the control) to 10 µM 5-Ph-IAA (MCE, HY-134653) for several hours. Then transferring cultures to fresh medium supplemented with 10µg/mLactinomycin D (HY-17559, MCE). Samples were harvested at time points of 0, 4, 8 and 12h followingactinomycin Dtreatment. Three replicates were collected at each time point.To determine decay rate of RNA, 500ng HCT116-AID2 cellRNA and 50ngNeurospora RNA were input RT reaction. For tested genes, random RT primers were employed in RT reaction.