Improved super-resolution ribosome profiling reveals prevalent translation of upstream ORFs and small ORFs in Arabidopsis
Improved super-resolution ribosome profiling reveals prevalent translation of upstream ORFs and small ORFs in Arabidopsis
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DOI：
10.1093/plcell/koad290
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发表时间：
2023-09
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影响因子：
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通讯作者：
H. Wu;Qiaoyun Ai;Rita Teresa Teixeira;Phong H T Nguyen;Gaoyuan Song;Christian Montes;J. M. Elmore;J. Walley;P. Hsu
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作者：
H. Wu;Qiaoyun Ai;Rita Teresa Teixeira;Phong H T Nguyen;Gaoyuan Song;Christian Montes;J. M. Elmore;J. Walley;P. Hsu
功能基因组学的关键一步是确定连接生物功能的主动翻译的开放阅读框架(ORF)。挑战在于识别较短的ORF，因为它们受到数据质量和深度的极大影响。在这里，我们改进了超分辨率Ribo-seq在拟南芥中的覆盖率，揭示了核、叶绿体和线粒体编码基因中的未知翻译事件。我们鉴定了7,751个非常规翻译事件，包括6,996个上游ORF(UORF)和209个下游ORF(注释蛋白编码基因)，以及546个非编码RNA上的ncORF。蛋白质组学数据证实，从一些未注释的翻译事件中产生了稳定的蛋白质。我们提供了tasiRNAs(TAS1-4)和microRNAs(pri-miR163，pri-miR169)初级转录本上活跃翻译的证据，以及支持共翻译衰退的周期性核糖体停滞。此外，我们开发了一种识别极短uORF的方法，包括370个最小uORF(Aug-Stop)，2984个微小uORF(2-10个AA)，以及681个相互重叠的uORF。值得注意的是，与较长的uORF相比，这些较短的uORF表现出较强的翻译抑制。我们还系统地发现了594个受选择性剪接调控的uORF，这表明存在广泛的异构体特异性翻译控制。最后，这些普遍存在的uORF与许多重要的途径有关。综上所述，我们改进的拟南芥翻译图谱为研究基因表达调控提供了宝贵的资源。
A crucial step in functional genomics is identifying actively translated open reading frames (ORFs) that link biological functions. The challenge lies in identifying short ORFs, as they are greatly impacted by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis, revealing uncharacterized translation events in nucleus-, chloroplast-, and mitochondria-encoded genes. We identified 7,751 unconventional translation events, including 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ncORFs on presumed non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of the unannotated translation events. We present evidence of active translation on primary transcripts of tasiRNAs (TAS1-4) and microRNAs (pri-miR163, pri-miR169), and periodic ribosome stalling supporting co-translational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORF (AUG-stop), and 2,984 tiny uORFs (2-10 aa), as well as 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as 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.