biostudies-literatureMeurs RSwiss National Science Foundation854-876https://www.ebi.ac.uk/biostudies/studies/S-EPMC11790910biostudies-literatureUnknown44(3)Ribosomes scanning from the mRNA 5' cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where 40S subunits resume scanning and initiate another translation event downstream. The noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors or uORF features influencing re-initiation is limited. Here, we establish a cell-free re-initiation assay using HeLa lysates to address this question. Comparing in vivo and in vitro re-initiation on uORF-containing reporters, we validate MCTS1-DENR-dependent re-initiation in vitro. Using this system and ribosome profiling in cells, we found that knockdown of the MCTS1-DENR homThe EMBO journalMCTS2 and distinct eIF2D roles in uORF-dependent translation regulation revealed by in vitro re-initiation assays.PMC11790910212423205601Ban NDe Matos MTeleman AAGuex NWeber TGatfield DBothe AMeurs RfalseMCTS2 and distinct eIF2D roles in uORF-dependent translation regulation revealed by in vitro re-initiation assays.Ribosomes scanning from the mRNA 5' cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where 40S subunits resume scanning and initiate another translation event downstream. The noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors or uORF features influencing re-initiation is limited. Here, we establish a cell-free re-initiation assay using HeLa lysates to address this question. Comparing in vivo and in vitro re-initiation on uORF-containing reporters, we validate MCTS1-DENR-dependent re-initiation in vitro. Using this system and ribosome profiling in cells, we found that knockdown of the MCTS1-DENR hom2025-01-01T00:00:00Z2025 Feb2026-06-01T10:37:36.161Z2025-04-04T01:02:30.307ZS-EPMC117909103974812010.1038/s44318-024-00347-3