Project description:Gastrocnemius muscle from Eif6 heterozygous mice and wild-type mice was used in a polysome profiling experiment. Total RNA was extracted from the whole muscle and from monosome/polysome fractions and profiled using Agilent gene expression arrays.
Project description:Tibialis anterior and gastrocnemius muscle from Eif6 heterozygous mice and wild-type mice profiled using Agilent gene expression arrays
Project description:A cohort of age-matched mice (eIF6+/+ and eIF6+/-) were fed with High-Fat Diet. All experimental mice were sacrificed after 16 weeks and the livers were recovered. RNA was isolated from liver biopsies of eIF6+/+ and eIF6+/- mice and RNAseq analysis was performed. The aim of the analysis is the investigation of the transcriptional changes driven by chronic depletion of eIF6 in the liver of mice upon High Fat Diet (HFD) feeding.
Project description:Purpose: mRNA translation into protein is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants has yet to be systematically studied. Using high-throughput sequencing (RNA-seq), we have measured cellular levels of mRNAs and ncRNAs, and their isoforms, in lymphoblast cell lines (LCL) and in polysomal fractions, the latter shown to yield strong correlations of mRNAs with expressed protein levels. Analysis of allelic RNA ratios at heterozygous SNPs served to reveal genetic factors in ribosomal loading. Methods: RNA-seq was performed on cytosolic extracts and polysomal fractions (3 ribosomes or more) from three lymphoblastoid cell lines. As each RNA fraction was amplified (NuGen kit), and relative contributions from various RNA classes differed between cytosol and polysomes, the fraction of any given RNA species loaded onto polysomes was difficult to quantitate. Therefore, we focused on relative recovery of the various RNA classes and rank order of single RNAs compared to total RNA. Results: RNA-seq of coding and non-coding RNAs (including microRNAs) in three LCLs revealed significant differences in polysomal loading of individual RNAs and isoforms, and between RNA classes. Moreover, correlated distribution between protein-coding and non-coding RNAs suggests possible interactions between them. Allele-selective RNA recruitment revealed strong genetic influence on polysomal loading for multiple RNAs. Allelic effects can be attributed to generation of different RNA isoforms before polysomal loading or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Several variants and genes identified by this approach are also associated with RNA expression and clinical phenotypes in various databases. Conclusions: These results provide a novel approach using complete transcriptome RNA-seq to study polysomal RNA recruitment and regulatory variants affecting protein translation.
Project description:Analysis of the global variability of gene expression between the Eif6 WT control genotype (CTRL) and the Eif6 S235A mutated (SA) MEF lines. To analyze the effects over time of the lack of Eif6 phosphorylation, we transduced MEFs with tamoxifen-inducible Cre, so that treatment leads to excision of wild-type exons and the generation of cells bearing the S235A mutation. The analysis is done on three replicates per genotype.