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. Overall design: 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 eIF2a 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.