scholarly journals RiboDiff: Detecting Changes of Translation Efficiency from Ribosome Footprints

2015 ◽  
Author(s):  
Yi Zhong ◽  
Theofanis Karaletsos ◽  
Philipp Drewe ◽  
Vipin Thankam T Sreedharan ◽  
David Kuo ◽  
...  
Keyword(s):  
Linear Models ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Analysis Tool ◽  
Translation Efficiency ◽  
Case Control Studies ◽  
Statistical Framework ◽  
Experimental Treatments ◽  
Over Dispersion

Motivation: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. Results: We present a statistical framework and analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy. Availability: Source code and documentation are available at http://github.com/ratschlab/ribodiff. Supplementary Material can be found at http://bioweb.me/ribo.

scholarly journals Ribosome Profiling and RNA Sequencing Reveal Genome-Wide Cellular Translation and Transcription Regulation Under Osmotic Stress in Lactobacillus rhamnosus ATCC 53103

2021 ◽  
Vol 12 ◽  
Author(s):  
Xuejing Fan ◽  
Tianyu Bao ◽  
Huaxi Yi ◽  
Zongcai Zhang ◽  
Kenan Zhang ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Fatty Acid Biosynthesis ◽  
Lactobacillus Rhamnosus ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Phosphotransferase System ◽  
Reading Frame ◽  
Cellular Translation ◽  
Mannose Metabolism

To determine whether osmotic pressure affects the translation efficiency of Lactobacillus rhamnosus, the ribosome profiling assay was performed to analyze the changes in translation efficiency in L. rhamnosus ATCC 53103. Under osmotic stress, differentially expressed genes (DEGs) involved in fatty acid biosynthesis and metabolism, ribosome, and purine metabolism pathways were co-regulated with consistent expression direction at translation and transcription levels. DEGs involved in the biosynthesis of phenylalanine, tyrosine, and tryptophan, and the phosphotransferase system pathways also were co-regulated at translation and transcription levels, while they showed opposite expression direction at two levels. Moreover, DEGs involved in the two-component system, amino acid metabolism, and pyruvate metabolism pathways were only regulated at the transcription level. And DEGs involved in fructose and mannose metabolism were only regulated at the translation level. The translation efficiency of DEGs involved in the biosynthesis of amino acids was downregulated while in quorum sensing and PTS pathways was upregulated. In addition, the ribosome footprints accumulated in open reading frame regions resulted in impaired translation initiation and elongation under osmotic stress. In summary, L. rhamnosus ATCC 53103 could respond to osmotic stress by translation regulation and control the balance between survival and growth of cells by transcription and translation.

scholarly journals Scikit-ribo: Accurate estimation and robust modeling of translation dynamics at codon resolution

2017 ◽  
Author(s):  
Han Fang ◽  
Yi-Fei Huang ◽  
Aditya Radhakrishnan ◽  
Adam Siepel ◽  
Gholson J. Lyon ◽  
...  
Keyword(s):  
Open Source Software ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Accurate Estimation ◽  
Translation Efficiency ◽  
Sampling Errors ◽  
Genome Wide ◽  
Rnaseq Data ◽  
A Site ◽  
Level Analysis

AbstractRibosome profiling (Riboseq) is a powerful technique for measuring protein translation, however, sampling errors and biological biases are prevalent and poorly understand. Addressing these issues, we present Scikit-ribo (https://github.com/hanfang/scikit-ribo), the first open-source software for accurate genome-wide A-site prediction and translation efficiency (TE) estimation from Riboseq and RNAseq data. Scikit-ribo accurately identifies A-site locations and reproduces codon elongation rates using several digestion protocols (r = 0.99). Next we show commonly used RPKM-derived TE estimation is prone to biases, especially for low-abundance genes. Scikit-ribo introduces a codon-level generalized linear model with ridge penalty that correctly estimates TE while accommodating variable codon elongation rates and mRNA secondary structure. This corrects the TE errors for over 2000 genes in S. cerevisiae, which we validate using mass spectrometry of protein abundances (r = 0.81) and allows us to determine the Kozak-like sequence directly from Riboseq. We conclude with an analysis of coverage requirements needed for robust codon-level analysis, and quantify the artifacts that can occur from cycloheximide treatment.

scholarly journals XPRESSyourself: Enhancing, Standardizing, and Automating Ribosome Profiling Computational Analyses Yields Improved Insight into Data

2019 ◽  
Author(s):  
Jordan A. Berg ◽  
Jonathan R. Belyeu ◽  
Jeffrey T. Morgan ◽  
Yeyun Ouyang ◽  
Alex J. Bott ◽  
...  
Keyword(s):  
Best Practices ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Computational Tools ◽  
Current State ◽  
Reference Implementation ◽  
Computational Analyses ◽  
Profiling Analysis ◽  
Insight Into

AbstractRibosome profiling, an application of nucleic acid sequencing for monitoring ribosome activity, has revolutionized our understanding of protein translation dynamics. This technique has been available for a decade, yet the current state and standardization of publicly available computational tools for these data is bleak. We introduce XPRESSyourself, an analytical toolkit that eliminates barriers and bottlenecks associated with this specialized data type by filling gaps in the computational toolset for both experts and non-experts of ribosome profiling. XPRESSyourself automates and standardizes analysis procedures, decreasing time-to-discovery and increasing reproducibility. This toolkit acts as a reference implementation of current best practices in ribosome profiling analysis. We demonstrate this toolkit’s performance on publicly available ribosome profiling data by rapidly identifying hypothetical mechanisms related to neurodegenerative phenotypes and neuroprotective mechanisms of the small-molecule ISRIB during acute cellular stress. XPRESSyourself brings robust, rapid analysis of ribosome-profiling data to a broad and ever-expanding audience and will lead to more reproducible and accessible measurements of translation regulation. XPRESSyourself software is perpetually open-source under the GPL-3.0 license and is hosted at https://github.com/XPRESSyourself, where users can access additional documentation and report software issues.

scholarly journals Aberrant BCMA Signaling Promotes Tumor Growth by Altering Protein Translation Machinery, a Therapeutic Target for the Treatment of Relapse/Refractory Multiple Myeloma

2021 ◽  
Author(s):  
Yu Rebecca Miao ◽  
Can Cenik ◽  
Dadi Jiang ◽  
Kazue Mizuno ◽  
Grace Caiyun Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Fusion Protein ◽  
Tumor Cells ◽  
Clinical Success ◽  
Transcript Level ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Translation Machinery

B-cell maturation antigen (BCMA) is critical for the viability of Multiple Myeloma (MM) tumor cells and targeting BCMA poses a remarkable opportunity as a potential therapeutic in this cancer. Recent approval of BCMA directed CAR-T and Antibody-Drug-Conjugates (ADCs) have revolutionized MM treatment landscape. Despite such clinical success, treatment resistance and dose limiting toxicity remain as major clinical challenges. Using ribosome profiling, we established a molecular link between BCMA signaling inhibition and protein translation machinery. In addition, BCMA signaling alters the translation efficiency of a transcriptional regulator ATMIN without changing the total mRNA transcript level. Furthermore, ATMIN can transcriptionally regulate IL-6, a critical survival factor for MM. To inhibit the BCMA signaling pathway, we devised both genetic knockdown strategy and pharmacological inhibition by using a soluble BCMA decoy receptor fusion protein (sBCMA-Fc) to trap both of its ligands, APRIL and BAFF. We demonstrated that treatment of MM tumor cells with sBCMA-Fc inhibits tumor progression in numerous in vivo and syngenic PDX tumors models without significant adverse effects. Furthermore, the addition of sBCMA-Fc treatment can restore bortezomib sensitivity in previously bortezomib resistant MM tumors, further adding to its therapeutic value in the treatment of relapse /refractory MM patients. Inhibiting BCMA signaling through neutralization of its ligands APRIL and BAFF with a sBCMA-Fc fusion protein represents a safe and efficacious treatment strategy for the treatment of relapse and refractory MM.

scholarly journals The transcription factor SlHY5 regulates the ripening of tomato fruit at both the transcriptional and translational levels

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Weihao Wang ◽  
Peiwen Wang ◽  
Xiaojing Li ◽  
Yuying Wang ◽  
Shiping Tian ◽  
...  
Keyword(s):  
Fruit Ripening ◽  
Nutritional Quality ◽  
Ribosomal Proteins ◽  
Anthocyanin Biosynthesis ◽  
Tomato Fruit ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Protein Translation ◽  
Transcriptional Level ◽  
Translation Efficiency

AbstractLight plays a critical role in plant growth and development, but the mechanisms through which light regulates fruit ripening and nutritional quality in horticultural crops remain largely unknown. Here, we found that ELONGATED HYPOCOTYL 5 (HY5), a master regulator in the light signaling pathway, is required for normal fruit ripening in tomato (Solanum lycopersicum). Loss of function of tomato HY5 (SlHY5) impairs pigment accumulation and ethylene biosynthesis. Transcriptome profiling identified 2948 differentially expressed genes, which included 1424 downregulated and 1524 upregulated genes, in the Slhy5 mutants. In addition, genes involved in carotenoid and anthocyanin biosynthesis and ethylene signaling were revealed as direct targets of SlHY5 by chromatin immunoprecipitation. Surprisingly, the expression of a large proportion of genes encoding ribosomal proteins was downregulated in the Slhy5 mutants, and this downregulation pattern was accompanied by a decrease in the abundance of ribosomal proteins. Further analysis demonstrated that SlHY5 affected the translation efficiency of numerous ripening-related genes. These data indicate that SlHY5 regulates fruit ripening both at the transcriptional level by targeting specific molecular pathways and at the translational level by affecting the protein translation machinery. Our findings unravel the regulatory mechanisms of SlHY5 in controlling fruit ripening and nutritional quality and uncover the multifaceted regulation of gene expression by transcription factors.

scholarly journals Loss of Ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with X-linked intellectual disability

2021 ◽  
Vol 7 (13) ◽  
pp. eabf3072
Author(s):  
Y. Nagayoshi ◽  
T. Chujo ◽  
S. Hirata ◽  
H. Nakatsuka ◽  
C.-W. Chen ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
State Level ◽  
Memory Deficits ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Trna Modification ◽  
Synaptic Organization ◽  
Transfer Rnas ◽  
The Brain

FtsJ RNA 2′-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2′-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient–derived cells. Loss of 2′-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.

scholarly journals m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

2021 ◽  
Vol 22 (6) ◽  
pp. 2941
Author(s):  
Marisa Pereira ◽  
Diana R. Ribeiro ◽  
Miguel M. Pinheiro ◽  
Margarida Ferreira ◽  
Stefanie Kellner ◽  
...  
Keyword(s):  
Stress Response ◽  
Small Rnas ◽  
Mammalian Cells ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Down Regulation ◽  
Gene Expression Control ◽  
The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

scholarly journals RiboA: a web application to identify ribosome A-site locations in ribosome profiling data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Danying Shao ◽  
Nabeel Ahmed ◽  
Nishant Soni ◽  
Edward P. O’Brien
Keyword(s):  
Integer Programming ◽  
Web Application ◽  
Stop Codon ◽  
Ribosome Profiling ◽  
Programming Method ◽  
Analysis Tool ◽  
Site Location ◽  
Link Type ◽  
Wide Range ◽  
A Site

Abstract Background Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations. Results We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms. Conclusions The Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/. The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.

scholarly journals Retrograde Control of Cytosolic Translation Targets Synthesis of Plastid Localized Proteins and Nuclear Responses for Efficient Light Acclimation

2021 ◽  
Author(s):  
Marten Moore ◽  
Aaron Smith ◽  
Corinna Wesemann ◽  
Sonja Schmidtpott ◽  
Melanie Wegener ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Transcript Abundance ◽  
Protein Translation ◽  
Retrograde Signaling ◽  
Just In Time ◽  
Protein Accumulation ◽  
Translation Efficiency ◽  
Multiple Components ◽  
Associated Proteins ◽  
Nuclear Responses

AbstractCanonical retrograde signaling is the transmission of information from organelles to the nucleus. Discrepancies between protein accumulation and transcript abundance in response to oxidative stress were suggestive of protein translation responding to retrograde signaling. Here we uncover multiple components of a translation-dependent retrograde signaling pathway that impact translation efficiency and gene expression, including the kinases, MPK6 and the SnRK1 subunit, AKIN10. Global ribosome foot-printing demonstrated rapid differential loading of 939 of transcripts from polyribosomes within 10 min after transfer from Low to High-light. Translationally regulated transcripts shared motifs in their 5’-UTR that act as binding sites for RBPs such as GAPC. The Stress Associated Proteins 2 and 3 carry such motifs in their UTRs and interact with the calcium sensor Calmodulin-like 49, relocating to the nucleus to co-regulate a translation-dependent transcriptional response. Translation dependent retrograde signaling bifurcates into a direct translational circuit and a translation-reliant nuclear circuit synchronizing translation, nuclear and anterograde response pathways, which may serve as a just in time-provision of needed proteins to the plastids.

scholarly journals Seeking a Role for Translational Control by Alternative Polyadenylation in Saccharomyces cerevisiae

2021 ◽  
Vol 9 (9) ◽  
pp. 1885
Author(s):  
Rachael E. Turner ◽  
Traude H. Beilharz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translational Control ◽  
Model Organism ◽  
Alternative Polyadenylation ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Mrna Isoforms ◽  
Cleavage And Polyadenylation ◽  
Made In

Alternative polyadenylation (APA) represents an important mechanism for regulating isoform-specific translation efficiency, stability, and localisation. Though some progress has been made in understanding its consequences in metazoans, the role of APA in the model organism Saccharomyces cerevisiae remains a relative mystery because, despite abundant studies on the translational state of mRNA, none differentiate mRNA isoforms’ alternative 3′-end. This review discusses the implications of alternative polyadenylation in S. cerevisiae using other organisms to draw inferences. Given the foundational role that research in this yeast has played in the discovery of the mechanisms of cleavage and polyadenylation and in the drivers of APA, it is surprising that such an inference is required. However, because advances in ribosome profiling are insensitive to APA, how it impacts translation is still unclear. To bridge the gap between widespread observed APA and the discovery of any functional consequence, we also provide a review of the experimental techniques used to uncover the functional importance of 3′ UTR isoforms on translation.

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