Biology subjects

Kaufman, I. D.

Publications and source records attributed to Kaufman, I. D..

3 recordsLinked to original sources

bioRxiv2026

Overlapping upstream ORFs repress translation and expand proteome diversity in Arabidopsis

Upstream open reading frames (uORFs) are widespread cis-regulatory elements that modulate translation initiation of downstream main ORFs (mORFs). Among them, overlapping uORFs (ouORFs) that overlap with mORFs are predicted to exert the strongest translational repression, yet they remain largely unexplored because of the difficulty of their identification. Here, we developed complementary computational approaches to systematically identify translated ouORFs from super-resolution ribosome profiling data in Arabidopsis. We identified 965 translated ouORFs alongside 7,180 canonical non-overlapping uORFs (nuORFs). We found that ouORFs exert substantially stronger translational repression than nuORFs, and that this repression depends primarily on Kozak context rather than uORF length. In addition, genes containing ouORFs or nuORFs have weaker mORF Kozak contexts than genes without uORFs, which may further reduce mORF translation. Moreover, ouORF translation promotes initiation downstream of the annotated mORF start codon, generating N-terminally truncated protein isoforms with altered domain composition and subcellular localization. Using ATPS2 as an example, we demonstrate that ouORF translation regulates alternative translation initiation to control the balance between chloroplast and cytosolic protein isoforms. Together, our findings establish ouORFs as a versatile class of translational regulatory elements that coordinate both protein abundance and protein diversity, providing the first genome-wide characterization of translated ouORFs in plants.

molecular biology↗

bioRxiv2025

GC3 codons enhance protein production in diverse GC- and AT-rich plant species

Engineering translation holds great promise for maximizing protein yields in agriculture and biotechnology, but the diversity of plant genomes hinders predictable engineering. To identify mRNA features that broadly improve translation, we conducted a comparative translatome analysis across model plants. We found that codons with G or C at the third position (GC3) are consistently associated with higher translation efficiency. Experimental results confirmed that elevating GC3 increases both protein output and mRNA abundance, in both GC- and AT-rich species. Comparative analyses across 80 plant species, spanning a wide range of GC3 levels, show that GC3 content is positively correlated with translation efficiency. Additionally, high GC3-codon usage is conserved among endogenous high-abundance proteins, such as Rubisco small subunits and ribosomal proteins. Finally, tRNA availability likely explains why GC3 codons broadly enhance translation. Together, our results provide a simple guideline for codon optimization: increasing GC3 can enhance protein production across diverse plants.

molecular biology↗

bioRxiv2025

ggRibo: a ggplot-based single-gene viewer for visualizing Ribo-seq and related omics datasets

Seeing is believing. Visualizing Ribo-seq and other sequencing data within genes of interest is a powerful approach to studying gene expression, but its application is limited by a lack of robust tools. Here, we introduce ggRibo, a user-friendly R package for visualizing individual gene expression, integrating Ribo-seq, RNA-seq, and other genome-wide datasets with flexible scaling options. ggRibo visualizes 3-nucleotide periodicity, a hallmark of translating ribosomes, within a gene-structure context, including introns and untranslated regions, enabling the study of novel ORFs, translation of different isoforms, and mechanisms of translational regulation. ggRibo can plot multiple Ribo-seq/RNA-seq datasets from different conditions for comparison. It also contains functions for plotting single-transcript view, reading-frame decomposition, and RNA-seq coverage alone. Importantly, ggRibo supports the visualization of other omics datasets that could also be presented with single-nucleotide resolution, such as RNA degradome, transcription start sites, translation initiation sites, and epitranscriptomic modifications. We demonstrate its utility with examples of upstream ORFs, downstream ORFs, nested ORFs, and differential isoform translation in humans, Arabidopsis, tomato, and rice. We also provide examples of multi-omic comparisons that reveal insights that connect the transcriptome, translatome, and degradome. In summary, ggRibo is an advanced single-gene viewer that offers a valuable resource for studying gene expression regulation through its intuitive and flexible platform.

bioinformatics↗