Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Department of Plant Pathology, Entomology, and Microbiology, Iowa State University, Ames, IA 50011, USA.
Plant Cell. 2024 Feb 26;36(3):510-539. doi: 10.1093/plcell/koad290.
A crucial step in functional genomics is identifying actively translated ORFs and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed noncoding RNAs. Proteomic data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of trans-acting small interfering RNAs (TAS1-4) and microRNAs (pri-MIR163 and pri-MIR169) and periodic ribosome stalling supporting cotranslational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2 to 10 amino acids) and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.
功能基因组学的一个关键步骤是鉴定活跃翻译的 ORF 并将其与生物学功能联系起来。挑战在于鉴定短 ORF,因为它们的鉴定受数据质量和深度的影响很大。在这里,我们改进了拟南芥(Arabidopsis thaliana)超分辨核糖体测序的覆盖范围,揭示了核、叶绿体和线粒体基因的未表征翻译事件。在转录组组装的辅助下,我们鉴定了 7751 个非常规翻译事件,包括注释蛋白编码基因上的 6996 个上游 ORF(uORF)和 209 个下游 ORF,以及 546 个假定非编码 RNA 中的 ORF。蛋白质组学数据证实了这些未注释翻译事件中的一些产生了稳定的蛋白质。我们提供了证据证明,反式作用小干扰 RNA(TAS1-4 和 microRNA(pri-MIR163 和 pri-MIR169)的初级转录物以及支持共翻译衰减的周期性核糖体停顿都有活跃的翻译。此外,我们开发了一种鉴定极短 uORF 的方法,包括 370 个最小 uORF(AUG-stop)、2921 个微小 uORF(2 到 10 个氨基酸)和 681 个相互重叠的 uORF。值得注意的是,这些短 uORF 表现出与长 uORF 一样强烈的翻译抑制作用。我们还系统地发现了 594 个受可变剪接调控的 uORF,表明存在广泛的同工型特异性翻译调控。最后,这些普遍存在的 uORF 与许多重要的途径有关。总之,我们改进的拟南芥翻译图谱为研究基因表达调控提供了有价值的资源。