Improved Super-Resolution Ribosome Profiling Revealed Prevalent Translation of Upstream ORFs and Small ORFs in Arabidopsis

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.