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We present a deep learning based framework, called ROSE, to accurately predict ribosome stalling events in translation elongation from coding sequences based on high-throughput ribosome profiling data. Our validation results demonstrate the superior performance of ROSE over conventional prediction models. ROSE provides an effective index to estimate the likelihood of translational pausing at codon resolution and understand diverse putative regulatory factors of ribosome stalling. Also, the ribosome stalling landscape computed by ROSE can recover the functional interplay between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particle (SRP) and protein secondary structure formation.
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Integrative detection of genome-wide translation using iRibo
Ribosome profiling is a sequencing technique that provides a global picture of translation across a genome. Here, we present iRibo, a software program for integrating any number of ribosome profiling samples to obtain sensitive inference of annotated or unannotated translated open reading frames. We describe the process of using iRibo to generate a species’ translatome from a set of ribosome profiling samples using S. cerevisiae as an example.
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- Award ID(s):
- 2144349
- PAR ID:
- 10548921
- Publisher / Repository:
- CellPress
- Date Published:
- Journal Name:
- STAR Protocols
- Volume:
- 5
- Issue:
- 1
- ISSN:
- 2666-1667
- Page Range / eLocation ID:
- 102826
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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Ellermeier, Craig D (Ed.)ABSTRACT Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized; however, little is known about the impacts of oxidative stress at the translation level. Using the model archaeonHaloferax volcanii, we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded by these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins inH. volcanii, revealed additional regulatory layers and underscored the multifaceted architecture of stress-responsive regulatory networks.IMPORTANCEArchaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. UsingHaloferax volcaniias a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation.more » « less
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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.more » « less
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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.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.xpro.2023.102826
