Improved super-resolution ribosome profiling reveals prevalent translation of upstream ORFs and small ORFs in Arabidopsis

Abstract

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.

Dispersed Transcription Start Sites Modulate uORF-Mediated Regulation in Arabidopsis

“…We previously identified translated uORFs (TuORFs) using two approaches, RiboTaper and CiPS, with our high-coverage super-resolution Ribo-seq data in Arabidopsis seedlings (Wu et al 2024). RiboTaper identifies TuORFs based on 3-nucleotide periodicity corresponding to ribosomes translocating 3 nucleotides per codon.…”

“…RiboTaper identifies TuORFs based on 3-nucleotide periodicity corresponding to ribosomes translocating 3 nucleotides per codon. While RiboTaper performs well for longer uORFs, it can overlook shorter uORFs (Wu et al 2024). We developed CiPS to tackle short TuORF identification by accounting for ribosome conformational changes at translation termination (Alkalaeva et al 2006; Brown et al 2015; Matheisl et al 2015), which strongly impact the 3-nucleotide periodicity of short TuORFs (Wu et al 2024).…”

“…While RiboTaper performs well for longer uORFs, it can overlook shorter uORFs (Wu et al 2024). We developed CiPS to tackle short TuORF identification by accounting for ribosome conformational changes at translation termination (Alkalaeva et al 2006; Brown et al 2015; Matheisl et al 2015), which strongly impact the 3-nucleotide periodicity of short TuORFs (Wu et al 2024). Together this comprehensive TuORF catalog and deep translatome data provide an opportunity to examine potential regulation on different groups of TuORFs quantitively.…”

“…Similar normalization strategies have been applied to study uORF-mediated translational repression (Juntawong et al 2014; Chew et al 2016). We first focused on 1252 genes with one longer TuORF (RiboTaper-identified) to avoid cumulative translational repression from multiple TuORFs and the strong stalling effects from short TuORFs (Wu et al 2024), which could bias the quantification of RuM (see Materials and Methods for details).…”

“…One related question to weak uORFs is why thousands of predicted uORFs have not been detected translated, even in deep-coverage Ribo-seq data (i.e., 298 million mapped Ribo-seq reads in the 125 Mb Arabidopsis genome, after excluding rRNAs, tRNAs, snRNAs, and snoRNAs) (Wu et al 2024). Are these undetected uORFs (UuORFs) genuinely disregarded by ribosomes, or could they be excluded from mRNAs through TSS regulation?…”