A specialized mechanism of translation mediated by FXR1a-associated microRNP in cellular quiescence

MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNP (microRNA-protein complex) that lacks the microRNP repressor, GW182. We conducted global proteomic analysis in THP1 cells depleted of FXR1 to globally identify activation targets of more than one microRNA, since FXR1 is required for microRNAmediated translation activation in THP1 G0 cells by FXR1-microRNPs.Since proteomic data changes could also be due to changes at the RNA level, total RNA levels in FXR1knockdown compared to control shRNA cells were examined in parallel by microarray analysis using Affymetrix Human GeneChip 2.0 ST. We used microarrays to get total RNA levels from FXR1 knockdown and control shRNA cells, to normalize the mass spectrometry data in order to provide normalized protein data or translation efficiency. FXR1 knockdown and shRNA control cells were induced with 1ug/ml of doxycycline continually to cause knockdown of FXR1; the cells were grown in serum for 3 days to induce the shRNAs, followed by 2 days of serum-starvation to induce G0. After harvesting, the cells were lysed in proteinase K buffer (Truesdell et al., 2012) and total RNA was isolated using Trizol (Invitrogen) and hybridized on Affymetrix Human GeneChip 2.0 ST microarray.