Loss of Lamin A leads to nuclear translocation of AGO2 mediated by EGFR signaling and inactivation of RNAi
In mammals, RNA interference (RNAi) is mostly studied as a cytoplasmic event, however, numerous reports convincingly showed nuclear localization of AGO proteins. Nevertheless, the mechanism of the nuclear entry remains to be fully elucidated, and the extent of nuclear RNAi explored. The nuclear import of large proteins requires active transport through the nuclear envelope (NE). Therefore, we hypothesized that the NE, and its composition, is important for nuclear AGO2 translocation. Lamin A knockout significantly induced nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cells. We unravelled that the loss of Lamin A leads to EGFR and Src kinase activation, which regulates the turnover and stability of cytoplasmic specifically AGO2. Furthermore, EGFR mediated phosphorylation at Y393 inhibits the activity of nuclear RNAi. This was evident by AGO fPAR-CLIP in WT and KO cells, where we observed a decrease in RNAi potential. Mass spectrometry of AGO interactome, from the nuclear fraction, indicated that AGO2 is in complex with FAM120A, which may further reduced the activity of RNAi by competing with AGO2 transcript binding. Therefore, loss of Lamin A starts a signalling cascade that mediates nuclear AGO2 translocation to rapidly inhibit RNAi. Overall design: We performed a gene expression profiling of A375 and SHSY5Y WT and Lamin A KO cells via RNA sequencing in 3 biological replicates, and carried out differential expression analysis between WT and Lamin A KO cells of respective cell line.