Reprogramming of translation in yeast cells impaired for ribosome recycling favors short, efficiently translated mRNAs

Yeast strains lacking orthologs of mammalian eIF2D (Tma64), and either MCT-1 (Tma20) or DENR (Tma22) are broadly impaired for 40S recycling; however, it was unknown whether this defect leads to reduced 43S PIC levels with an impact on translation of particular mRNAs. To test this, we by combined ribosome footprint profiling with RNA-seq analysis of mRNA abundance. This study includes a total of 36 samples comprised of 18 RNA-seq samples (mRNA) and 18 ribosome footprint profiling samples (ribo). Experiment 1 includes 12 samples, comprised of 6 RNA-seq samples and 6 ribosome footprint profiling samples, derived from 2 biological replicates each of the BY4741 (WT1) and yeast tma64∆/tma20∆ double mutant H4520 (tma∆∆) which were grown at 30ºC in synthetic complete (SC) medium to log-phase (A600 = 0.5–0.6) and BY4741 grown in SC media lacking isoleucine and valine (SC-Ile/Val) to log-phase (A600 = 0.5–0.6) and treated with the antimetabolite Sulfometuron methyl (SM) at 1 µg/mL for 25 min to induce eIF2α phosphorylation. Experiment 2 examines the effect of the yeast homolog of eIF2D (Tma64) to promote translation initiation on the subset of mRNAs whose translation efficiencies (TEs) are reduced in the tma∆∆ mutant by complementing the function of eIF2 in delivering Met-tRNAi to the 40S subunit. This experiment includes 8 samples, 4 RNA-seq samples and 4 ribosome footprint profiling samples, derived from 2 biological replicates each of WT2 and the tma64∆ mutant 4051, cultured in SC medium at 30ºC. Experiment 3 examines effect of depleting 40S subunit protein on translation efficiencies of strong and weak mRNA and includes 8 samples, 4 RNA-seq samples and 4 ribosome footprint profiling samples, derived from 2 biological replicates each of WT3 strain H2994 and the rps26∆∆ mutant JVY09 were cultured in SC medium (SC medium with 2% galactose+2% raffinose) at 30ºC to log-phase were shifted from galactose- to glucose-containing media for 3h. Experiment 4 examines effect of the transcriptional changes evoked by Gcn4 induction responsible for the translational reprogramming produced by SM treatment of WT cells and includes 8 samples, 4 RNA-seq samples and 4 ribosome footprint profiling samples, derived from 2 biological replicates each of the gcn4∆ mutant 249, cultured in SC with or without SM treatment.