Rli1/ABCE1 recycles terminating ribosomes and controls translation reinitiation in 3′UTRs in vivo

Summary To study the function of Rli1/ABCE1 in vivo, we used ribosome profiling and biochemistry to characterize its contribution to ribosome recycling. When Rli1 levels were diminished, 80S ribosomes accumulated both at stop codons and in the adjoining 3′UTRs of most mRNAs. Frequently, these ribosomes reinitiated translation without the need for a canonical start codon, as small peptide products predicted by 3′UTR ribosome occupancy in all three reading frames were confirmed by western analysis and mass spectrometry. Eliminating the ribosome-rescue factor Dom34 dramatically increased 3′UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and controls ribosome homeostasis. 3′UTR translation occurs in wild-type cells as well, and observations of elevated 3′UTR ribosomes during stress suggest that modulating recycling and reinitiation is involved in responding to environmental changes.