Cellular, Molecular, and Biochemical Sciences Program, The Ohio State University, Columbus, Ohio 43210, USA.
The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
RNA. 2023 Jun;29(6):735-744. doi: 10.1261/rna.079525.122. Epub 2023 Mar 6.
It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation reinitiation. However, termination in the 5' UTR at the end of uORFs resembles premature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation reinitiation has been proposed as a method for mRNAs to prevent NMD. Here, we test how uORF length influences translation reinitiation and mRNA stability in HeLa cells. Using custom 5' UTRs and uORF sequences, we show that reinitiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life data sets for cumulative predicted uORF length, we conclude that translation reinitiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggest that the decision of whether NMD ensues after translating uORFs occurs before reinitiation in mammalian cells.
据估计，近 50%的哺乳动物转录本至少含有一个上游开放阅读框（uORF），其通常比下游主要开放阅读框小一到两个数量级。大多数 uORF 被认为是抑制性的，因为它们会隔离扫描核糖体，但在某些情况下允许重新起始翻译。然而，在 uORF 末端的 5'UTR 中终止类似于正常由无意义介导的 mRNA 降解（NMD）途径感知到的过早终止。重新起始翻译被提议为 mRNA 防止 NMD 的一种方法。在这里，我们测试 uORF 长度如何影响 HeLa 细胞中的翻译重新起始和 mRNA 稳定性。使用定制的 5'UTR 和 uORF 序列，我们表明可以在异源 mRNA 序列上发生重新起始，有利于较小的 uORF，并且当更多起始因子参与起始时得到支持。在确定 HeLa 细胞中报告 mRNA 的半衰期并为累积预测的 uORF 长度挖掘可用的 mRNA 半衰期数据集之后，我们得出结论，uORFs 之后的翻译重新起始不是 mRNA 防止 NMD 的有效方法。总之，这些数据表明，在哺乳动物细胞中，uORFs 翻译后是否发生 NMD 的决定发生在重新起始之前。
