Project description:To identify which genes were regulated by mRNA helicase activity, the effect of eIF4A1 knockdown on the MCF7 cell transcriptome and translatome was determined. eIF4A1-dependent mRNAs were highly enriched for several classes of genes with oncogenic potential, which leads to a model whereby dysregulation of mRNA unwinding contribues to the malignant phenotype in breast cancer cells via preferential translation of a subset of genes.
Project description:To identify which genes were regulated by mRNA helicase activity, the effect of eIF4A1 knockdown on the MCF7 cell transcriptome and translatome was determined. eIF4A1-dependent mRNAs were highly enriched for several classes of genes with oncogenic potential, which leads to a model whereby dysregulation of mRNA unwinding contribues to the malignant phenotype in breast cancer cells via preferential translation of a subset of genes.
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
Project description:EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5’ untranslated regions but very little is known of their roles in non-malignant cells. Using mouse models to delete Eif4a1, Eif4b or Eif4h in B cells we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. Following activation, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable whereas Hippuristanol treatment induces cell death.
