Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892.
HHMI, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 2022 Mar 1;119(9). doi: 10.1073/pnas.2117226119.
Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox () gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNAs (mRNAs) from mouse somites revealed that the 5' leaders of mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements. We show that the conserved uORFs inhibit reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.
真核生物翻译起始位点的选择受到侧翼 AUG 密码子的上下文核苷酸和真核翻译起始因子 eIF1 和 eIF5 的水平的影响。在对哺乳动物基因的搜索中，我们鉴定了 5 个同源盒（）基因，它们的 AUG 密码子在保守的次优上下文起始，以及 13 个含有进化上保守的上游开放阅读框（uORF）的基因，这些基因的 AUG 密码子在较差的序列背景下起始。对已发表的基因表达帽分析测序 (CAGE-seq) 数据和生成的来自小鼠体节的信使 RNA (mRNA) 的 CAGE-seq 数据的分析表明，感兴趣的 mRNA 的 5' 先导含有保守的 uORF，通常比报道的短得多，并且缺乏先前提出的内部核糖体进入位点元件。我们表明，保守的 uORF 抑制报告基因的表达，并且通过过表达 eIF1 或 eIF5 改变起始密码子选择的严格性可以调节报告基因的表达。我们还表明，通过耗尽大亚基核糖体蛋白或用亚致死浓度的嘌呤霉素处理细胞来修饰核糖体稳态会导致起始密码子选择的严格性降低。因此，通过改变起始密码子选择的严格性，改变全局翻译可以赋予基因特异性效应。
