Meurs Romane, De Matos Mara, Bothe Adrian, Guex Nicolas, Weber Tobias, Teleman Aurelio A, Ban Nenad, Gatfield David
Center for Integrative Genomics, University of Lausanne, 1015, Lausanne, Switzerland.
Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland.
EMBO J. 2025 Feb;44(3):854-876. doi: 10.1038/s44318-024-00347-3. Epub 2025 Jan 2.
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.
核糖体从mRNA的5'帽向起始密码子扫描时，可能在上游开放阅读框（uORF）处起始，从而减少蛋白质生物合成。在uORF处终止可导致重新起始，即40S亚基重新开始扫描并在下游启动另一个翻译事件。非经典翻译因子MCTS1-DENR参与特定uORF处的重新起始，但关于影响重新起始的其他反式作用因子或uORF特征的了解有限。在这里，我们建立了一种使用HeLa细胞裂解物的无细胞重新起始测定法来解决这个问题。通过比较含uORF报告基因在体内和体外的重新起始情况，我们在体外验证了MCTS1-DENR依赖性重新起始。利用该系统和细胞中的核糖体图谱分析，我们发现MCTS1-DENR同源物eIF2D的敲低会导致与uORF翻译无关的广泛基因失调，因此与MCTS1-DENR依赖性重新起始调节不同。此外，我们鉴定出由Mcts1反转录基因编码的MCTS2作为促进体外重新起始的DENR伙伴，为人类中与DENR和MCTS1突变相关的临床差异提供了一个合理的解释。
