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

   https://doi.org/10.1186/s13007-022-00947-2  

   Wu, Hsin-Yen Larry ; Hsu, Polly Yingshan ( October 2022 , Plant Methods)

   Ribosome profiling, also known as Ribo-seq, is a powerful technique to study genome-wide mRNA translation. It reveals the precise positions and quantification of ribosomes on mRNAs through deep sequencing of ribosome footprints. We previously optimized the resolution of this technique in plants. However, several key reagents in our original method have been discontinued, and thus, there is an urgent need to establish an alternative protocol.

   Here we describe a step-by-step protocol that combines our optimized ribosome footprinting in plants with available custom library construction methods established in yeast and bacteria. We tested this protocol in 7-day-old Arabidopsis seedlings and evaluated the quality of the sequencing data regarding ribosome footprint length, mapped genomic features, and the periodic properties corresponding to actively translating ribosomes through open resource bioinformatic tools. We successfully generated high-quality Ribo-seq data comparable with our original method.

   We established a custom library construction method for super-resolution Ribo-seq in Arabidopsis. The experimental protocol and bioinformatic pipeline should be readily applicable to other plant tissues and species.

    

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2. RiboDiPA: a novel tool for differential pattern analysis in Ribo-seq data

   https://doi.org/10.1093/nar/gkaa1049  

   Li, Keren ; Hope, C Matthew ; Wang, Xiaozhong A ; Wang, Ji-Ping ( November 2020 , Nucleic Acids Research)

   null (Ed.)

   Abstract Ribosome profiling, also known as Ribo-seq, has become a popular approach to investigate regulatory mechanisms of translation in a wide variety of biological contexts. Ribo-seq not only provides a measurement of translation efficiency based on the relative abundance of ribosomes bound to transcripts, but also has the capacity to reveal dynamic and local regulation at different stages of translation based on positional information of footprints across individual transcripts. While many computational tools exist for the analysis of Ribo-seq data, no method is currently available for rigorous testing of the pattern differences in ribosome footprints. In this work, we develop a novel approach together with an R package, RiboDiPA, for Differential Pattern Analysis of Ribo-seq data. RiboDiPA allows for quick identification of genes with statistically significant differences in ribosome occupancy patterns for model organisms ranging from yeast to mammals. We show that differential pattern analysis reveals information that is distinct and complimentary to existing methods that focus on translational efficiency analysis. Using both simulated Ribo-seq footprint data and three benchmark data sets, we illustrate that RiboDiPA can uncover meaningful pattern differences across multiple biological conditions on a global scale, and pinpoint characteristic ribosome occupancy patterns at single codon resolution. 

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3. 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)

   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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4. RiboPlotR: a visualization tool for periodic Ribo-seq reads

   https://doi.org/10.1186/s13007-021-00824-4  

   Wu, Hsin-Yen Larry ; Hsu, Polly Yingshan ( December 2021 , Plant Methods)

   Abstract Background Ribo-seq has revolutionized the study of genome-wide mRNA translation. High-quality Ribo-seq data display strong 3-nucleotide (nt) periodicity, which corresponds to translating ribosomes deciphering three nts at a time. While 3-nt periodicity has been widely used to study novel translation events such as upstream ORFs in 5′ untranslated regions and small ORFs in presumed non-coding RNAs, tools that allow the visualization of these events remain underdeveloped. Results RiboPlotR is a visualization package written in R that presents both RNA-seq coverage and Ribo-seq reads in genomic coordinates for all annotated transcript isoforms of a gene. Specifically, for individual isoform models, RiboPlotR plots Ribo-seq data in the context of gene structures, including 5′ and 3′ untranslated regions and introns, and it presents the reads for all three reading frames in three different colors. The inclusion of gene structures and color-coding the reading frames facilitate observing new translation events and identifying potential regulatory mechanisms. Conclusions RiboPlotR is freely available ( https://github.com/hsinyenwu/RiboPlotR and https://sourceforge.net/projects/riboplotr/ ) and allows the visualization of translated features identified in Ribo-seq data. 

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5. Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control

   https://doi.org/10.1093/nar/gkae285  

   Chen, Katharine Y. ; Park, Heungwon ; Subramaniam, Arvind Rasi ( April 2024 , Nucleic Acids Research)

   Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

    

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