Project description:We studied translation factor eIF4E paralogs that regulate germline mRNAs. Translational control of mRNAs is essential for germ cell differentiation and embryogenesis. Messenger ribonucleoprotein (mRNP) complexes assemble on mRNAs in the nucleus, as they exit via perinuclear germ granules, and in the cytoplasm. Bound mRNP proteins including eIF4Es exert both positive and negative post-transcriptional regulation. In C. elegans, germ granules are surprisingly dynamic mRNP condensates that remodel during development. Two eIF4E paralogs (IFE-1 and IFE-3), their cognate eIF4E-Interacting Proteins (4EIPs), and polyadenylated mRNAs are present in germ granules. Affinity purification of IFE-1 and IFE-3 mRNPs allowed mass spectrometry and mRNA-Seq to identify other proteins and the mRNAs that populate stable eIF4E complexes. We find translationally repressed mRNAs (e.g. pos-1, mex-3, spn-4, etc.) enriched with IFE-3, but excluded from IFE-1. Identified mRNAs overlap substantially with mRNAs previously described to be IFE-1-dependent for translation. The findings suggest that oocytes and embryos utilize the two eIF4Es for opposite purposes on critically regulated germline mRNAs. Biochemical composition of isolated mRNPs indicates opposing yet cooperative roles for the two eIF4Es. We propose that the IFEs accompany controlled mRNAs in the repressed or activated state during transit to the cytoplasm. Copurification of IFE-1 with IFE-3 suggests they may interact to move repressed mRNAs to ribosomes.
Project description:Relative polysomal loading changes for wild type (N2) versus ife-1(bn127) C. elegans strains Strain ife-1(bn127) is null for the gene encoding one of five eIF4E isoforms in the worm, IFE-1. Only this isoform associates with P granules. Resolution of translating and non-translating mRNAs from WT and mutant worms on 10-45% sucrose gradients. Comparision was by ratios of polysomal content. Please note that each sample data table contains raw signal directly imported from the Affymetrix files and the ratio of signals, P/(NP+P), and fold-changes were further calculated. The TSAA_overview.xls contains more datails about the TSAA procedure and 'complete_data.xlsx' contains complete TSAA data output.
Project description:Relative polysomal loading changes for wild type (N2) versus ife-1(bn127) C. elegans strains Strain ife-1(bn127) is null for the gene encoding one of five eIF4E isoforms in the worm, IFE-1. Only this isoform associates with P granules.
Project description:This project aims to identify novel RNA binding proteins in the nematode, Caenorhabditis elegans. Since interactions between RNAs and proteins may be transient, these animals were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringent conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples: i) samples digested with RNase one; ii) samples where we performed competition assays with polyadenylic acid