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1. Variation in upstream open reading frames contributes to allelic diversity in maize protein abundance

   https://doi.org/10.1073/pnas.2112516119  

   Gage, Joseph L.; Mali, Sujina; McLoughlin, Fionn; Khaipho-Burch, Merritt; Monier, Brandon; Bailey-Serres, Julia; Vierstra, Richard D.; Buckler, Edward S. (April 2022, Proceedings of the National Academy of Sciences)

   The 5 ′ untranslated region (UTR) sequence of eukaryotic mRNAs may contain upstream open reading frames (uORFs), which can regulate translation of the main ORF (mORF). The current model of translational regulation by uORFs posits that when a ribosome scans a mRNA and encounters an uORF, translation of that uORF can prevent ribosomes from reaching the mORF and cause decreased mORF translation. In this study, we first observed that rare variants in the 5 ′ UTR dysregulate maize ( Zea mays L. ) protein abundance. Upon further investigation, we found that rare variants near the start codon of uORFs can repress or derepress mORF translation, causing allelic changes in protein abundance. This finding holds for common variants as well, and common variants that modify uORF start codons also contribute disproportionately to metabolic and whole-plant phenotypes, suggesting that translational regulation by uORFs serves an adaptive function. These results provide evidence for the mechanisms by which natural sequence variation modulates gene expression, and ultimately, phenotype. 

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2. Ribosome profiling reveals distinct translational programs underlying Arabidopsis seed dormancy and germination

   https://doi.org/10.64898/2026.01.08.696037  

   Gomez_Roldan, Maria Victoria; Layat, Elodie; Bailey-Serres, Julia; Bazin, Jérémie; Bailly, Christophe (January 2026, bioRxiv)

   Abstract Seed dormancy and germination represent a critical developmental transition that determines plant fitness, yet the contribution of translational regulation to this process remains poorly understood. Here, we used genome-wide ribosome profiling (Ribo-seq) combined with RNA sequencing (RNA-seq) to investigate how translational control shapes the transition from dormancy to germination inArabidopsis thalianaseeds. We analyzed dry dormant seeds, stratified non-dormant seeds, and seeds during early imbibition, enabling simultaneous assessment of transcript abundance and ribosome occupancy. Our analyses reveal that dry seeds harbor an unexpectedly organized translational machinery, with ribosomes pre-positioned at start codons and within coding regions of thousands of stored mRNAs, indicating a poised translational state. Dormancy release and early imbibition triggered extensive gene-specific changes in translational efficiency that were largely uncoupled from transcript abundance, highlighting selective translation as a key regulatory layer. Genes involved in ribosome biogenesis, protein folding, and hormone signaling were preferentially translated during dormancy maintenance, whereas germination-promoting factors showed increased ribosome occupancy following stratification. Global ribosome profiling further uncovered dynamic ribosome pausing at stop codons and pronounced modulation of translation initiation during imbibition.We also identified widespread translation of upstream open reading frames (uORFs) and demonstrated that uORF-mediated repression constitutes a major translational checkpoint during seed imbibition. Functional assays confirmed that uORFs fromMARD1andPAO4repress downstream translationin vivo. Together, our results establish translational regulation as a central mechanism governing seed dormancy and germination, revealing how ribosome positioning and uORF activity fine-tune protein synthesis to control developmental transitions in response to environmental cues. 

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3. Translational buffering by ribosome stalling in upstream open reading frames

   https://doi.org/10.1371/journal.pgen.1010460  

   Bottorff, Ty A.; Park, Heungwon; Geballe, Adam P.; Subramaniam, Arvind Rasi (October 2022, PLOS Genetics)

   Barsh, Gregory S. (Ed.)

   Upstream open reading frames (uORFs) are present in over half of all human mRNAs. uORFs can potently regulate the translation of downstream open reading frames through several mechanisms: siphoning away scanning ribosomes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. Here, we performed systematic measurements on the uORF-containing 5′ UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF protein expression when ribosome loading onto the mRNA is reduced. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. We identify several human uORFs that repress main ORF protein expression via a similar terminal diproline motif. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions. 

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4. Modeling the structure and DAP5-binding site of the FGF-9 5′-UTR RNA utilized in cap-independent translation

   https://doi.org/10.1261/rna.080013.124  

   Whittaker, Amanda; Goss, Dixie J (August 2024, RNA)

   Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5′ untranslated regions (5′ UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem–loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5′ UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5′ UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5′ UTRs, some of which require an unpaired, accessible 5′ end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5′-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5–RNA interactions. Modeling of FGF-9 5′-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies. 

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5. Improved Super-Resolution Ribosome Profiling Reveals Prevalent Translation of Upstream ORFs and Small ORFs in Arabidopsis

   https://doi.org/10.1093/plcell/koad290  

   Wu, Hsin-Yen Larry; Ai, Qiaoyun; Teixeira, Rita Teresa; Nguyen, Phong H; Song, Gaoyuan; Montes, Christian; Elmore, J Mitch; Walley, Justin W; Hsu, Polly Yingshan (November 2023, The Plant Cell)

   Abstract A crucial step in functional genomics is identifying actively translated open reading frames (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 non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from 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 uORFs (AUG-stop), and 2,921 tiny uORFs (2–10 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. 

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