MCTS2 and distinct eIF2D roles in uORF-dependent translation regulation revealed by in vitro re-initiation assays

EMBO J. 2025 Feb;44(3):854-876. doi: 10.1038/s44318-024-00347-3. Epub 2025 Jan 2.

Abstract

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 homolog eIF2D causes widespread gene deregulation unrelated to uORF translation, and thus distinct to MCTS1-DENR-dependent re-initiation regulation. Additionally, we identified MCTS2, encoded by an Mcts1 retrogene, as a DENR partner promoting re-initiation in vitro, providing a plausible explanation for clinical differences associated with DENR vs. MCTS1 mutations in humans.

Keywords: DENR-MCTS1; In Vitro Translation; Re-Initiation; eIF2D; uORF.

MeSH terms

  • Cell Cycle Proteins
  • Cell-Free System
  • Eukaryotic Initiation Factor-2
  • Eukaryotic Initiation Factors
  • Gene Expression Regulation
  • HeLa Cells
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Oncogene Proteins
  • Open Reading Frames*
  • Peptide Chain Initiation, Translational
  • Protein Biosynthesis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Ribosomes / genetics
  • Ribosomes / metabolism

Substances

  • MCTS1 protein, human
  • eIF2D protein, human
  • Membrane Proteins
  • Neoplasm Proteins
  • DENR protein, human
  • RNA, Messenger
  • Oncogene Proteins
  • Eukaryotic Initiation Factor-2
  • Cell Cycle Proteins
  • Eukaryotic Initiation Factors