Consis tent with the polysome profiles, the rate of total protein synthesis, measured by incorporation of radioactive methionine into acid insoluble material, was reduced in the eIF4G1 td mutant to 30% of the WT value after 8 h in the non permissive condition, whereas the eIF3 degron mutant displayed no detectable Met incorporation selelck kinase inhibitor under these conditions. Thus, in accordance with our previous conclusions, depletion of eIF4G1 in cells lacking eIF4G2 leads to a marked reduction in the rate of translation initiation, but one less severe than that provoked by a comparable depletion of eIF3 subunits. Depletion of eIF4G narrows the range of mRNA translational efficiencies genome wide Although a significant level of translation continues fol lowing the extensive depletion of eIF4G in the degron mutant, it was possible that translation of some mRNAs would be greatly diminished while trans lation of others would continue relatively unaffected or even increase.
To address this possibility, we determined the effect of depleting eIF4G on the translational effi ciencies of mRNAs genome wide. To this end, we con ducted microarray analysis on RNA isolated from the heaviest polysomes, containing 4 or more elongating 80S ribosomes per mRNA, Inhibitors,Modulators,Libraries and also total RNA from WCEs, from both degron mutant and WT cells cultured for 8 h under non per missive conditions. Translational efficiencies were calculated for each gene as the ratio of hybridization intensities on microarrays probed with cDNAs produced from HP versus total RNA samples.
It should Inhibitors,Modulators,Libraries be noted that equal amounts of cDNA are used to probe each microarray and the intensities are scaled so that each array has approximately the same average value. This normalization will diminish the effect of reduced poly some abundance in the eIF4G mutant versus WT cells. The total amount of mRNA could also decline in the mutant owing to reduced transcription or increased mRNA turnover accompanying diminished translation, which would offset the effect of decreased polysome abundance on the calculated translational efficiencies. Hence, comparing TE values can indicate absolute dif ferences in translational efficiency between two genes in the same Inhibitors,Modulators,Libraries strain, but it reveals only relative differences in efficiency for a given gene between two strains.
As a quality control for the polysomal fractionation and mRNA extraction procedures, we first analyzed the distribution of several mRNAs among heavy poly somes, light polysomes, and 80S monosomes Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries using real time RT PCR to quantify mRNA concentrations. The distributions of RPL41A and RPL41B mRNAs were examined because their coding sequences, of only 78 nt, are large enough to accommodate only two translating 80S ribosomes, and at the average ribosome density for yeast mRNAs they should gener ally contain only one translating 80S ribosome at a selleckchem LY2835219 time, hence, the majority of these two mRNAs should occur in the 80S monosome fraction.