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<p>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 <i>Mcts1</i> retrogene, as a DENR partner promoting re-initiation in vitro, providing a plausible explanation for clinical differences associated with <i>DENR</i> vs. <i>MCTS1</i> mutations in humans.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年1月2日

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  • Romane Meurs,
  • Mara De Matos,
  • Adrian Bothe,
  • Nicolas Guex,
  • Tobias Weber,
  • Aurelio A Teleman,
  • Nenad Ban,
  • David Gatfield

摘要

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.