Translation initiation is a key step determining protein synthesis. Studies have uncovered a number of alternative translation initiation sites (TISs) in mammalian mRNAs and showed their roles in reshaping the proteome. However, the extent to which alternative TISs affect gene expression across plants remains largely unclear. Here, by profiling initiating ribosome positions, we globally identified in vivo TISs in tomato and Arabidopsis and found thousands of genes with more than one TIS. Of the identified TISs, >19% and >20% were located at unannotated AUG and non-AUG sites, respectively. CUG and ACG were the most frequently observed codons at non-AUG TISs, a phenomenon also found in mammals. In addition, although alternative TISs were usually found in both orthologous genes, the TIS sequences were not conserved, suggesting the conservation of alternative initiation mechanisms but flexibility in using TISs. Unlike upstream AUG TISs, the presence of upstream non-AUG TISs was not correlated with the translational repression of main open reading frames, a pattern observed across plants. Also, the generation of proteins with diverse N-terminal regions through the use of alternative TISs contributes to differential subcellular localization, as mutating alternative TISs resulted in the loss of organelle localization. Our findings uncovered the hidden coding potential of plant genomes and, importantly, the constraint and flexibility of translational initiation mechanisms in the regulation of gene expression across plant species.

中文翻译：

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替代 AUG 和非 AUG 翻译引发剂的流行及其对植物的调节作用

翻译起始是决定蛋白质合成的关键步骤。研究发现了哺乳动物 mRNA 中的许多替代翻译起始位点 (TIS)，并显示了它们在重塑蛋白质组中的作用。然而，替代 TIS 影响跨植物基因表达的程度在很大程度上仍不清楚。在这里，通过分析启动核糖体的位置，我们在番茄和拟南芥中全球鉴定了体内 TIS并发现了具有不止一种 TIS 的数千个基因。在确定的 TIS 中，>19% 和 >20% 分别位于未注释的 AUG 和非 AUG 位点。CUG 和 ACG 是非 AUG TIS 中最常观察到的密码子，在哺乳动物中也发现了这种现象。此外，虽然通常在两个直系同源基因中都发现了替代的 TIS，但 TIS 序列并不保守，这表明替代起始机制的保守性和使用 TIS 的灵活性。与上游 AUG TIS 不同，上游非 AUG TIS 的存在与主要开放阅读框的翻译抑制无关，这是一种在植物中观察到的模式。此外，通过使用替代 TIS 产生具有不同 N 端区域的蛋白质有助于差异亚细胞定位，因为突变的替代 TIS 导致细胞器定位的丧失。我们的研究结果揭示了植物基因组隐藏的编码潜力，更重要的是，揭示了植物物种间基因表达调控中翻译起始机制的约束和灵活性。