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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)

   Abstract BackgroundRibosome 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. ResultsHere 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. ConclusionsWe 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. 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)

   Abstract 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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4. Ribosome-induced mRNA pseudoknot interactions visualized by DMS MaP-Seq

   https://doi.org/10.1101/2024.12.07.627321  

   Kellenberger, Preston A; Song, Zhenwei; Heng, Xiao; Cornish, Peter V (December 2024, bioRxiv)

   Abstract Examining the dynamicity of RNA structure has deepened our understanding of its vast biological functions. Perhaps the protein complex that encounters the most diverse landscape of RNA structure is the ribosome. In translation, the ribosome must linearize countless mRNA conformations for proper protein production. Some RNA structures, however, reliably make up sequences which hinder the ability of the ribosome to maintain its reading frame. The most well-studied of these structures is the RNA pseudoknot. Here, we present an approach utilizing dimethyl sulfate probing with mutational profiling and sequencing (DMS MaP-Seq) to precisely examine RNA unwinding. We employ the method to understand the unfolding of the Sugarcane Yellow Leaf Virus pseudoknot (ScYLV_PK). Notably, we find that the helical junction is stabilized in the presence of the ribosome and is contingent upon hydrogen bonding at the 27^thresidue of ScYLV_PK. Additionally, it is demonstrated that the ribosome destabilizes wildtype ScYLV_PKin a manner independent of A/P-site occupancy. Together, these results establish DMS MaP-Seq as a sensitive tool for detecting ribosome-induced RNA conformational changes and reveal specific structural motifs that govern pseudoknot stability during translation. 

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5. riboviz 2: a flexible and robust ribosome profiling data analysis and visualization workflow

   https://doi.org/10.1093/bioinformatics/btac093  

   Cope, Alexander L; Anderson, Felicity; Favate, John; Jackson, Michael; Mok, Amanda; Kurowska, Anna; Liu, Junchen; MacKenzie, Emma; Shivakumar, Vikram; Tilton, Peter; et al (February 2022, Bioinformatics)

   Valencia, Alfonso (Ed.)

   Abstract Motivation Ribosome profiling, or Ribo-seq, is the state-of-the-art method for quantifying protein synthesis in living cells. Computational analysis of Ribo-seq data remains challenging due to the complexity of the procedure, as well as variations introduced for specific organisms or specialized analyses. Results We present riboviz 2, an updated riboviz package, for the comprehensive transcript-centric analysis and visualization of Ribo-seq data. riboviz 2 includes an analysis workflow built on the Nextflow workflow management system for end-to-end processing of Ribo-seq data. riboviz 2 has been extensively tested on diverse species and library preparation strategies, including multiplexed samples. riboviz 2 is flexible and uses open, documented file formats, allowing users to integrate new analyses with the pipeline. Availability and implementation riboviz 2 is freely available at github.com/riboviz/riboviz. 

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