Biology subjects

Hsu, P. Y.

Publications and source records attributed to Hsu, P. Y..

11 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↗

bioRxiv2024

Dispersed Transcription Start Sites Modulate uORF-Mediated Regulation in Arabidopsis

Upstream open reading frames (uORFs) are predicted in over half of the genes in many eukaryotes and often reduce translation and mRNA stability. This raises a paradox: why do uORFs remain so prevalent in eukaryotic genomes despite their repressive effects on gene expression? Using Arabidopsis as a model, we show that transcription start site (TSS) selection controls the inclusion and repressive impact of uORFs within the steady-state RNA pool. We find that TSS selection influences uORF inclusion in >90% of uORF-containing genes and selectively excludes uORFs from many transcripts. Although bioinformatic analyses predict uORFs in 55% of Arabidopsis genes, fewer than 10% of expressed transcripts contain them. This TSS filtering is nonrandom: genes whose uORFs are excluded are enriched for housekeeping functions, whereas genes retaining uORFs are associated with regulatory roles. We show that failing to account for TSS heterogeneity can mask length-dependent uORF repression and lead to misinterpretation of uORF activities. Importantly, uORFs mostly excluded from native mRNAs can still repress translation when artificially placed upstream of a reporter, demonstrating that apparent uORF repressiveness may not reflect their activity in native transcripts. Our findings establish TSS selection as a major determinant of uORF-mediated translational control. Because TSS heterogeneity is widespread among plant and animal genes, we warn that ignoring TSS heterogeneity can confound analyses of regulatory elements in mRNA 5' leaders. We therefore recommend accounting for TSS usage when studying uORFs and other mRNA regulatory elements; otherwise, conclusions may reflect regulatory potential rather than regulatory activity in native transcripts.

plant biology↗

bioRxiv2023

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.

genomics↗

bioRxiv2022

A custom library construction method for super-resolution ribosome profiling in Arabidopsis

Ribosome profiling (aka Ribo-seq) is the deep sequencing of ribosome footprints (RFs). It maps and quantifies ribosome occupancy on mRNA, which enables the identification of coding regions and the accurate quantification of translation efficiency. We previously optimized the Ribo-seq method in Arabidopsis and tomato (Hsu et al., 2016; Wu et al., 2019; Wu and Hsu, 2022) to obtain precise RFs with strong 3-nucleotide periodicity, a feature displayed by actively translating ribosomes and a benchmark of high-quality Ribo-seq (Brar and Weissman, 2015). This strong periodicity allowed us to confidently define numerous unannotated translation events across plants (Hsu et al., 2016; Wu et al., 2019; Wu and Hsu, 2022). Recently, several key commercial reagents used in our methods were discontinued; thus, there is an urgent need to develop a new protocol. Here, we report an updated protocol that adapts two custom library construction methods (McGlincy and Ingolia, 2017; Li et al., 2021) for plants. We applied this new protocol to Arabidopsis seedlings and obtained high-quality data. We describe our step-by-step method and discuss crucial considerations for Ribo-seq experiments. We also provide a bioinformatic pipeline to perform essential quality control analyses on Ribo-seq data. Our approach should be readily applicable to other plant species with minimal modifications.

genomics↗

bioRxiv2021

Identification and Functional Annotation of Long Intergenic Non-coding RNAs in the Brassicaceae

Long intergenic noncoding RNAs (lincRNAs) are a large yet enigmatic class of eukaryotic transcripts with critical biological functions. Despite the wealth of RNA-seq data available, lincRNA identification lags in the plant lineage. In addition, there is a need for a harmonized identification and annotation effort to enable cross-species functional and genomic comparisons. In this study we processed >24 Tbp of RNA-seq data from >16,000 experiments to identify ~130,000 lincRNAs in four Brassicaceae: Arabidopsis thaliana, Camelina sativa, Brassica rapa, and Eutrema salsugineum. We used Nanopore RNA-seq, transcriptome-wide structural information, peptide data, and epigenomic data to characterize these lincRNAs and identify functional motifs. We then used comparative genomic and transcriptomic approaches to highlight lincRNAs in our dataset with sequence or transcriptional evolutionary conservation, including lincRNAs transcribed adjacent to orthologous genes that display little sequence similarity and likely function as transcriptional regulators. Finally, we used guilt-by-association techniques to further classify these lincRNAs according to putative function. LincRNAs with Brassicaceae-conserved putative miRNA binding motifs, short ORFs, and whose expression is modulated by abiotic stress are a few of the annotations that will prioritize and guide future functional analyses.

genomics↗

bioRxiv2021

Actively translated uORFs reduce translation and mRNA stability independent of NMD in Arabidopsis

Upstream ORFs (uORFs) are common regulatory elements in the 5 untranslated regions of eukaryotic mRNAs. In addition to repressing main ORF translation, uORF translation in animals also reduces mRNA stability through nonsense-mediated decay (NMD). In contrast, the roles of uORFs in plants are less understood. Here, we identified actively translated uORFs (TuORFs) in Arabidopsis through ribosome profiling and systematically examined their roles in gene expression. Like animal systems, Arabidopsis TuORFs are associated with 38%, 14%, and 43% reductions in translation efficiency, mRNA half-lives, and protein levels, respectively. However, we found TuORF-containing mRNAs have 51% higher transcript levels, and this phenomenon is persistent in diverse tissues and developmental stages across plants. We present multiple lines of evidence that indicate Arabidopsis uORF-containing mRNAs generally exhibit distinct behavior from known NMD targets. First, TuORF-containing mRNAs are not increased in NMD mutants. Second, TuORF-containing mRNAs and known NMD targets have distinct expression patterns, and they are translationally repressed via different mechanisms. Finally, TuORF- containing mRNAs and NMD targets are degraded through separate pathways. Our results suggest that Arabidopsis TuORFs reduce mRNA stability and translation through mechanisms different from NMD and highlight a fundamental difference in gene regulation mediated by TuORFs in plants and animals.

genomics↗

bioRxiv2019

Visualizing the periodic Ribo-seq reads with RiboPlotR

BackgroundRibo-seq has revolutionized the study of mRNA translation in a genome-wide scale. High-quality Ribo-seq data display strong 3-nucleotide (nt) periodicity, which corresponds to translating ribosomes decipher three nucleotides each time. While the 3-nt periodicity has been widely used to study novel translation events and identify small open reading frames on presumed non-coding RNAs, tools which allow the visualization of those events remain underdeveloped.\n\nFindingsRiboPlotR is a visualization package written in R that presents both RNA-seq coverage and Ribo-seq reads for all annotated transcript isoforms in a context of a given gene. In particular, RiboPlotR plots Ribo-seq reads mapped in three reading frames using three colors for one isoform model at a time. Moreover, RiboPlotR shows Ribo-seq reads on upstream ORFs, 5 and 3 untranslated regions and introns, which is critical for observing new translation events and potential regulatory mechanisms.\n\nConclusionsRiboPlotR is freely available (https://github.com/hsinyenwu/RiboPlotR) and allows the visualization of the translating features in Ribo-seq data.

bioinformatics↗

bioRxiv2019

Translational landscape in tomato revealed by transcriptome assembly and ribosome profiling

mRNA translation is a critical step in gene expression, but our understanding of the landscape and control of translation in diverse crops remains lacking. Here, we combined de novo transcriptome assembly and ribosome profiling to study global mRNA translation in tomato roots. Taking advantage of the 3-nucleotide periodicity displayed by translating ribosomes, we identified 354 novel small ORFs (sORFs) translated from previously unannotated transcripts, as well as 1329 upstream ORFs (uORFs) translated within the 5 UTRs of annotated protein-coding genes. Proteomic analysis confirmed that some of these novel uORFs and sORFs generate stable proteins in planta. Compared with the annotated ORFs, the uORFs use more flexible Kozak sequences around translation start sites. Interestingly, uORF-containing genes are enriched for protein phosphorylation/dephosphorylation and signaling transduction pathways, suggesting a regulatory role for uORFs in these processes. We also demonstrated that ribosome profiling is useful to facilitate the annotation of translated ORFs and noncanonical translation initiation sites. In addition to defining the translatome, our results revealed the global control of mRNA translation by uORFs and microRNAs in tomato. In summary, our approach provides a high-throughput method to discover unannotated ORFs, elucidates evolutionarily conserved translational features, and identifies new regulatory mechanisms hidden in a crop genome. SignificanceSeveral studies have shown that altering mRNA translation is a powerful way of improving crop performance. However, due to limited genomic resources and methods, translational regulation remains poorly understood in crops. By leveraging de novo transcriptome assembly and ribosome profiling, we mapped and quantified translating ribosomes across the entire transcriptome in tomato roots. This is the first experiment-based survey to systematically identify actively translated ORFs in a crop. Our results reveal numerous unannotated translation events and uncover new regulatory mechanisms of gene expression in tomato. Our approach not only facilitates our understanding of the tomato translational landscape but also provides a practical strategy to study the translatomes of other species. O_LIThe raw RNA-seq and Ribo-seq data have been deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE124962. C_LIO_LIProteomics raw data files and MaxQuant Search results have been deposited at the MassIVE repository with dataset identifier: MSV000083363. C_LI

plant biology↗

bioRxiv2018

Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling

A major challenge in the field of proteomics is obtaining high quality peptides for comprehensive proteome profiling by liquid chromatography mass spectrometry for many organisms. Here we evaluate and modify a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissues from several developmental timepoints. We find that inclusion of FASP-based on filter digestion improves all protein extraction methods tested. Ultimately, we show that a detergent-free urea-FASP approach enables deep and robust quantification of leaf proteomes. For example, from 4-day-old leaf tissue we profiled up to 11,690 proteins from a single sample replicate. This method should be broadly applicable to researchers working on difficult to process samples from a range of plant and non-plant organisms.\n\nAbbreviations

plant biology↗