ИСТИНА

Ribosome recycling is a vital cellular process that takes place after the ribosome reaches a stop codon and releases the completed peptide. The ATPase ABCE1/Rli1 first removes the 60S subunit, leaving the tRNA-bound 40S subunit associated with the mRNA. Dissociation of the remaining tRNA, mRNA, and 40S subunit has been reconstituted in vitro by two distinct pathways, the first involving the canonical initiation factors eIF1, eIF1A, eIF3 and eIF3j/Hcr1, and the second involving eIF2D/ligatin (yeast Tma64) or the MCT-1/DENR proteins (yeast Tma20/Tma22). Both Tma22 and Tma64 contain SUI1 domains and may function like eIF1, potentially modulating access to the P-site of the 40S subunit during recycling. To address whether these pathways are active in vivo and are redundant, we performed ribosome profiling of 80S ribosomes in tma64∆/tma20∆ and tma64∆/tma22∆ double deletion strains. In contrast to the recycling defects introduced by depletion of Rli1, we observed no stalling of 80S ribosomes at stop codons in the tma double mutants. Instead, ribosomes accumulated 28 nt (one footprint length) behind the stop codon, consistent with the “penultimate” elongating ribosome queueing behind an unrecycled 40S subunit stalled at the stop codon. The tma double deletion strains also showed increased 3’UTR ribosome occupancy relative to WT though at a lower level than in Rli1-depleted cells. Increased ribosome density at 3’ UTR AUG codons and detection of peptides encoded by 3’UTRs in the tma mutant strains strongly imply that 40S ribosomes can initiate translation in the 3’UTR via the canonical pathway when they are not properly recycled. The impaired ribosome recycling also affected translation of uORF containing mRNAs, as revealed by in vitro translation experiments. To investigate whether the Tma proteins and canonical initiation factor eIF1 act redundantly, we profiled TMA+ and tma64∆/tma20∆ strains containing the eIF1 mutation sui1-L96P. Interestingly, sui1-L96P conferred a small increase in 3’UTR ribosome occupancy in the triple mutant with tma64∆/tma20∆ but not in otherwise wild-type cells, suggesting that eIF1 can partially substitute for the Tma proteins in their absence. Our results show that the yeast orthologs of eIF2D, MCT-1, and DENR function in recycling of 40S subunits at stop codons in vivo, and function more prominently than eIF1 in this step of translation. Given known association of high MCT-1 levels with lymphoma and DENR mutations with developmental neurological defects, our findings suggest improper translation reinitiation can trigger disease.