Paget-Bailly Philippe, Helpiquet Alexandre, Decourcelle Mathilde, Bories Roxane, Bravo Ignacio G
Laboratory MIVEGEC (Univ. Montpellier, CNRS, IRD), French National Center for Scientific Research (CNRS), Montpellier, France.
Functional Proteomics Platform, BioCampus Montpellier (University of Montpellier, CNRS, INSERM), Montpellier, France.
Protein Sci. 2025 Feb;34(2):e70036. doi: 10.1002/pro.70036.
Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.
生物化学教科书将真核生物信使核糖核酸（mRNA）描述为单顺反子。然而，越来越多的证据表明，在“主要”开放阅读框（ORF）之前广泛存在上游开放阅读框并可进行翻译。感染真核生物的DNA和RNA病毒通常会产生多顺反子mRNA，并且病毒已经进化出多种操纵宿主翻译机制的方式。在此，我们引入一种实验模型来研究人类细胞中病毒样双顺反子mRNA的基因表达调控。该模型由一个短的上游ORF和一个编码荧光蛋白的下游报告ORF组成。我们设计了上游ORF的同义变体，以探索广阔的参数空间，包括密码子使用偏好、mRNA折叠特征和剪接倾向。我们发现，人类翻译机制可以从双顺反子mRNA翻译下游ORF，尽管报告蛋白水平比上游ORF低数千倍。此外，仅在延伸过程中对上游ORF进行同义编码会显著影响其自身的翻译效率，揭示隐蔽的剪接信号，并调节下游ORF翻译的概率。我们的结果与一种促进人类细胞中双顺反子mRNA下游ORF翻译的漏扫描机制一致，为上游ORF在翻译调控中的作用提供了新的见解。
