scholarly journals RiboToolkit: an integrated platform for analysis and annotation of ribosome profiling data to decode mRNA translation at codon resolution

2020 ◽  
Vol 48 (W1) ◽  
pp. W218-W229 ◽  
Author(s):  
Qi Liu ◽  
Tanya Shvarts ◽  
Piotr Sliz ◽  
Richard I Gregory
Keyword(s):  
Mrna Translation ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Sequencing Data ◽  
Experimental Conditions ◽  
Web Based ◽  
Rna Translation ◽  
Web Interfaces

Abstract Ribosome profiling (Ribo-seq) is a powerful technology for globally monitoring RNA translation; ranging from codon occupancy profiling, identification of actively translated open reading frames (ORFs), to the quantification of translational efficiency under various physiological or experimental conditions. However, analyzing and decoding translation information from Ribo-seq data is not trivial. Although there are many existing tools to analyze Ribo-seq data, most of these tools are designed for specific or limited functionalities and an easy-to-use integrated tool to analyze Ribo-seq data is lacking. Fortunately, the small size (26–34 nt) of ribosome protected fragments (RPFs) in Ribo-seq and the relatively small amount of sequencing data greatly facilitates the development of such a web platform, which is easy to manipulate for users with or without bioinformatic expertise. Thus, we developed RiboToolkit (http://rnabioinfor.tch.harvard.edu/RiboToolkit), a convenient, freely available, web-based service to centralize Ribo-seq data analyses, including data cleaning and quality evaluation, expression analysis based on RPFs, codon occupancy, translation efficiency analysis, differential translation analysis, functional annotation, translation metagene analysis, and identification of actively translated ORFs. Besides, easy-to-use web interfaces were developed to facilitate data analysis and intuitively visualize results. Thus, RiboToolkit will greatly facilitate the study of mRNA translation based on ribosome profiling.

scholarly journals Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kyle A. Cottrell ◽  
Ryan C. Chiou ◽  
Jason D. Weber
Keyword(s):  
Protein Synthesis ◽  
Tumor Cells ◽  
Ribosomal Proteins ◽  
Human Cancer ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Gene Encoding ◽  
Terminal Oligopyrimidine ◽  
Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

scholarly journals Ribo-ODDR: Oligo design pipeline for experiment-specific rRNA depletion in ribo-seq

2021 ◽  
Author(s):  
Ferhat Alkan ◽  
Joana Silva ◽  
Eric Pintó Barberà ◽  
William J Faller
Keyword(s):  
Ribosome Profiling ◽  
Supplementary Information ◽  
Experimental Conditions ◽  
Computational Framework ◽  
Rna Translation ◽  
Rrna Depletion ◽  
Selection For ◽  
Nucleotide Resolution ◽  
User Friendly ◽  
Oligo Design

Abstract Motivation Ribosome Profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of rRNA fragments. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, they may perform suboptimally in Ribo-seq. In order to overcome this, it is possible to use custom biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. Results Here, we first show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a “one-size-fits-all” approach may be inefficient. Therefore we developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We experimentally show that Ribo-ODDR designed oligos outperform commercially available kits and lead to a significant increase in rRNA depletion in Ribo-seq. Availability Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals ARF suppresses 5’-terminal oligopyrimidine mRNA translation

2020 ◽  
Author(s):  
Kyle A. Cottrell ◽  
Ryan C. Chiou ◽  
Jason D. Weber
Keyword(s):  
Protein Synthesis ◽  
Tumor Cells ◽  
Ribosomal Proteins ◽  
Human Cancer ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Gene Encoding ◽  
Terminal Oligopyrimidine ◽  
Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How many of the mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5’-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 caused a similar increase in 5’-TOP mRNA translation. The 5’-TOP regulators mTORC1, eIF4G1 and LARP1 are dysregulated in ARF and p53 null cells.

scholarly journals RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia

2021 ◽  
Vol 5 (13) ◽  
pp. 2788-2792
Author(s):  
Stavroula Ntoufa ◽  
Marina Gerousi ◽  
Stamatia Laidou ◽  
Fotis Psomopoulos ◽  
Georgios Tsiolas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Lymphocytic Leukemia ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Wild Type ◽  
Recurrent Mutations

Abstract Recent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

scholarly journals Generally applicable transcriptome-wide analysis of translational efficiency using anota2seq

2017 ◽  
Author(s):  
Christian Oertlin ◽  
Julie Lorent ◽  
Valentina Gandin ◽  
Carl Murie ◽  
Laia Masvidal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Analytical Methods ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Mtor Inhibition

ABSTRACTmRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated results in various disorders. Optimal and universally applicable analytical methods to study transcriptome-wide changes in translational efficiency are therefore critical for understanding the complex role of translation regulation under physiological and pathological conditions. Techniques used to interrogate translatomes, including polysome- and ribosome-profiling, require adjustment for changes in total mRNA levels to capture bona fide alterations in translational efficiency. Herein, we present the anota2seq algorithm for such analysis using data from ribosome- or polysome-profiling quantified by DNA-microarrays or RNA sequencing, which outperforms current methods for identification of changes in translational efficiency. In contrast to available analytical methods, anota2seq also allows capture of an underappreciated mode for regulation of gene expression whereby translation acts as a buffering mechanism which maintains constant protein levels despite fluctuations in mRNA levels (“translational buffering”). Application of anota2seq shows that insulin affects gene expression at multiple levels, in a largely mTOR-dependent manner. Moreover, insulin induces levels of a subset of mRNAs independently of mTOR that undergo translational buffering upon mTOR inhibition. Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes and enables studies of translational buffering which represents an unexplored mechanism for regulating of gene expression.

Assessment of translational importance of mammalian mRNA sequence features based on Ribo-Seq and mRNA-Seq data

2016 ◽  
Vol 14 (02) ◽  
pp. 1641006 ◽  
Author(s):  
Oxana A. Volkova ◽  
Yury V. Kondrakhin ◽  
Ivan S. Yevshin ◽  
Tagir F. Valeev ◽  
Ruslan N. Sharipov
Keyword(s):  
Statistical Analysis ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Mrna Sequence ◽  
Protein Coding ◽  
Sequence Features ◽  
Additional Information ◽  
Significant Relationships ◽  
Non Coding Rnas

Ribosome profiling technology (Ribo-Seq) allowed to highlight more details of mRNA translation in cell and get additional information on importance of mRNA sequence features for this process. Application of translation inhibitors like harringtonine and cycloheximide along with mRNA-Seq technique helped to assess such important characteristic as translation efficiency. We assessed the translational importance of features of mRNA sequences with the help of statistical analysis of Ribo-Seq and mRNA-Seq data. Translationally important features known from literature as well as proposed by the authors were used in analysis. Such comparisons as protein coding versus non-coding RNAs and high- versus low-translated mRNAs were performed. We revealed a set of features that allowed to discriminate the compared categories of RNA. Significant relationships between mRNA features and efficiency of translation were also established.

scholarly journals Genome-Wide Identification and Characterization of Small Peptides in Maize

2021 ◽  
Vol 12 ◽  
Author(s):  
Yan Liang ◽  
Wanchao Zhu ◽  
Sijia Chen ◽  
Jia Qian ◽  
Lin Li
Keyword(s):  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Translational Efficiency ◽  
Small Peptides ◽  
Bioinformatics Pipeline ◽  
Protein Coding ◽  
Genome Wide ◽  
A Genome ◽  
Intergenic Regions ◽  
New Perspective

Small peptides (sPeptides), <100 amino acids (aa) long, are encoded by small open reading frames (sORFs) often found in the 5′ and 3′ untranslated regions (or other parts) of mRNAs, in long non-coding RNAs, or transcripts from introns and intergenic regions; various sPeptides play important roles in multiple biological processes. In this study, we conducted a comprehensive study of maize (Zea mays) sPeptides using mRNA sequencing, ribosome profiling (Ribo-seq), and mass spectrometry (MS) on six tissues (each with at least two replicates). To identify maize sORFs and sPeptides from these data, we set up a robust bioinformatics pipeline and performed a genome-wide scan. This scan uncovered 9,388 sORFs encoding peptides of 2–100 aa. These sORFs showed distinct genomic features, such as different Kozak region sequences, higher specificity of translation, and high translational efficiency, compared with the canonical protein-coding genes. Furthermore, the MS data verified 2,695 sPeptides. These sPeptides perfectly discriminated all the tissues and were highly associated with their parental genes. Interestingly, the parental genes of sPeptides were significantly enriched in multiple functional gene ontology terms related to abiotic stress and development, suggesting the potential roles of sPeptides in the regulation of their parental genes. Overall, this study lays out the guidelines for genome-wide scans of sORFs and sPeptides in plants by integrating Ribo-seq and MS data and provides a more comprehensive resource of functional sPeptides in maize and gives a new perspective on the complex biological systems of plants.

scholarly journals Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors

2017 ◽  
Author(s):  
Nahuel A. Paolini ◽  
Kat S. Moore ◽  
Franca M. di Summa ◽  
Ivo F.A.C. Fokkema ◽  
Peter A.C. ‘t Hoen ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translation Initiation Factor ◽  
List Type ◽  
Translation Efficiency ◽  
Proteotoxic Stress ◽  
Advantages And Disadvantages ◽  
Ribosome Density ◽  
Eif2 Phosphorylation

AbstractThe regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how eIF2 phosphorylation affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict eIF2-dependent translation efficiency, but uORF identity differs. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.- eif2 dependent translation in erythroblasts during proteotoxic stress determined by ribosome footprinting- identification of transcription factors upregulated in response to eIF2 phosphorylation- Advantages and disadvantages of translation initiation site determination using harringtonine- distinct uORF pattern in transcripts with enhanced, or more than average reduced translation upon proteotoxic stress

scholarly journals Ssd1 and Gcn2 suppress global translation efficiency in replicatively aged yeast while their activation extends lifespan

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Zheng Hu ◽  
Bo Xia ◽  
Spike DL Postnikoff ◽  
Zih-Jie Shen ◽  
Alin S Tomoiaga ◽  
...  
Keyword(s):  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
P Bodies ◽  
Downstream Target ◽  
Autophagy Induction ◽  
Extended Lifespan ◽  
Stress Kinase ◽  
Global Translation

Translational efficiency correlates with longevity, yet its role in lifespan determination remains unclear. Using ribosome profiling, translation efficiency is globally reduced during replicative aging in budding yeast by at least two mechanisms: Firstly, Ssd1 is induced during aging, sequestering mRNAs to P-bodies. Furthermore, Ssd1 overexpression in young cells reduced translation and extended lifespan, while loss of Ssd1 reduced the translational deficit of old cells and shortened lifespan. Secondly, phosphorylation of eIF2α, mediated by the stress kinase Gcn2, was elevated in old cells, contributing to the global reduction in translation without detectable induction of the downstream Gcn4 transcriptional activator. tRNA overexpression activated Gcn2 in young cells and extended lifespan in a manner dependent on Gcn4. Moreover, overexpression of Gcn4 sufficed to extend lifespan in an autophagy-dependent manner in the absence of changes in global translation, indicating that Gcn4-mediated autophagy induction is the ultimate downstream target of activated Gcn2, to extend lifespan.

scholarly journals ORFik: a comprehensive R toolkit for the analysis of translation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Håkon Tjeldnes ◽  
Kornel Labun ◽  
Yamila Torres Cleuren ◽  
Katarzyna Chyżyńska ◽  
Michał Świrski ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Sequencing Data ◽  
Protein Coding ◽  
High Throughput Sequencing Data ◽  
Upstream Open Reading Frames ◽  
Many Core ◽  
User Friendly

Abstract Background With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. Results Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5′UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5′ UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. Conclusion In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment. Availability http://bioconductor.org/packages/ORFik.

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