RiboGalaxy: A Galaxy-based Web Platform for Ribosome Profiling Data Processing - 2023 Update.

Ribosome profiling (Ribo-Seq) captures a "snapshot" of ribosomes' locations at the entire transcriptome of a cell at sub-codon resolution providing insights into gene expression and enabling the discovery of novel translated regions. RiboGalaxy (https://ribogalaxy.genomicsdatascience.ie/), a Galaxy-based platform for processing Ribo-Seq data is a RiboSeq.Org (https://riboseq.org/) resource. RiboSeq.Org is an online gateway to a set of integrated tools for the processing and analysis of Ribo-Seq data. In this RiboGalaxy update we introduce changes to both the tools available on RiboGalaxy and to how the resource is managed on the backend. For example, in order to improve interoperability between Riboseq.Org resources, we added tools that link RiboGalaxy outputs with Trips-Viz and GWIPS-viz browsers for downstream analysis and visualisation. RiboGalaxy's backend now utilises Ansible configuration management which enhances its stability and jobs are executed within Singularity containers and are managed by Slurm, strengthening reproducibility and performance respectively.

Trips-Viz: an environment for the analysis of public and user-generated ribosome profiling data.

Trips-Viz (https://trips.ucc.ie/) is an interactive platform for the analysis and visualization of ribosome profiling (Ribo-Seq) and shotgun RNA sequencing (RNA-seq) data. This includes publicly available and user generated data, hence Trips-Viz can be classified as a database and as a server. As a database it provides access to many processed Ribo-Seq and RNA-seq data aligned to reference transcriptomes which has been expanded considerably since its inception. Here, we focus on the server functionality of Trips-viz which also has been greatly improved. Trips-viz now enables visualisation of proteomics data from a large number of processed mass spectrometry datasets. It can be used to support translation inferred from Ribo-Seq data. Users are now able to upload a custom reference transcriptome as well as data types other than Ribo-Seq/RNA-Seq. Incorporating custom data has been streamlined with RiboGalaxy (https://ribogalaxy.ucc.ie/) integration. The other new functionality is the rapid detection of translated open reading frames (ORFs) through a simple easy to use interface. The analysis of differential expression has been also improved via integration of DESeq2 and Anota2seq in addition to a number of other improvements of existing Trips-viz features.

Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF.

While near-cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here, we describe a rare case of highly efficient near-cognate initiation. A CUG triplet located in the 5' leader of POLG messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5' of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5' leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

Computational methods for ribosome profiling data analysis.

Since the introduction of the ribosome profiling technique in 2009 its popularity has greatly increased. It is widely used for the comprehensive assessment of gene expression and for studying the mechanisms of regulation at the translational level. As the number of ribosome profiling datasets being produced continues to grow, so too does the need for reliable software that can provide answers to the biological questions it can address. This review describes the computational methods and tools that have been developed to analyze ribosome profiling data at the different stages of the process. It starts with initial routine processing of raw data and follows with more specific tasks such as the identification of translated open reading frames, differential gene expression analysis, or evaluation of local or global codon decoding rates. The review pinpoints challenges associated with each step and explains the ways in which they are currently addressed. In addition it provides a comprehensive, albeit incomplete, list of publicly available software applicable to each step, which may be a beneficial starting point to those unexposed to ribosome profiling analysis. The outline of current challenges in ribosome profiling data analysis may inspire computational biologists to search for novel, potentially superior, solutions that will improve and expand the bioinformatician's toolbox for ribosome profiling data analysis. This article is characterized under: Translation > Ribosome Structure/Function RNA Evolution and Genomics > Computational Analyses of RNA Translation > Translation Mechanisms Translation > Translation Regulation.

Hepatitis C Virus Downregulates Core Subunits of Oxidative Phosphorylation, Reminiscent of the Warburg Effect in Cancer Cells.

Hepatitis C Virus (HCV) mainly infects liver hepatocytes and replicates its single-stranded plus strand RNA genome exclusively in the cytoplasm. Viral proteins and RNA interfere with the host cell immune response, allowing the virus to continue replication. Therefore, in about 70% of cases, the viral infection cannot be cleared by the immune system, but a chronic infection is established, often resulting in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Induction of cancer in the host cells can be regarded to provide further advantages for ongoing virus replication. One adaptation in cancer cells is the enhancement of cellular carbohydrate flux in glycolysis with a reduction of the activity of the citric acid cycle and aerobic oxidative phosphorylation. To this end, HCV downregulates the expression of mitochondrial oxidative phosphorylation complex core subunits quite early after infection. This so-called aerobic glycolysis is known as the "Warburg Effect" and serves to provide more anabolic metabolites upstream of the citric acid cycle, such as amino acids, pentoses and NADPH for cancer cell growth. In addition, HCV deregulates signaling pathways like those of TNF-ß and MAPK by direct and indirect mechanisms, which can lead to fibrosis and HCC.

Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling.

BACKGROUND: Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known. METHODS: We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days. RESULTS: Established viral replication does not cause global changes in host gene expression-only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (PPP1R15A/GADD34, DDIT3/CHOP, and TRIB3) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes. CONCLUSION: After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming ("Warburg effect") even in the hepatocellular carcinoma cells used here.

Trips-Viz: a transcriptome browser for exploring Ribo-Seq data.

Ribosome profiling (Ribo-Seq) is a technique that allows for the isolation and sequencing of mRNA fragments protected from nuclease digestion by actively translating ribosomes. Mapping these ribosome footprints to a genome or transcriptome generates quantitative information on translated regions. To provide access to publicly available ribosome profiling data in the context of transcriptomes we developed Trips-Viz (transcriptome-wide information on protein synthesis-visualized). Trips-Viz provides a large range of graphical tools for exploring global properties of translatomes and of individual transcripts. It enables analysis of aligned footprints to evaluate datasets quality, differential gene expression detection, visual identification of upstream ORFs and alternative proteoforms. Trips-Viz is available at https://trips.ucc.ie.

PausePred and Rfeet: webtools for inferring ribosome pauses and visualizing footprint density from ribosome profiling data.

The process of translation is characterized by irregularities in the local decoding rates of specific mRNA codons. This includes the occurrences of long pauses that can take place when ribosomes decode certain peptide sequences, encounter strong RNA secondary structures, or decode "hungry" codons. Examples are known where such pausing or stalling is used for regulating protein synthesis. This can be achieved at the level of translation via direct alteration of ribosome progression through mRNA or by altering mRNA stability via NoGo decay. Ribosome pausing has also been implicated in the cotranslational folding of proteins. Ribosome profiling data often are used for inferring the locations of ribosome pauses. However, no dedicated online software is available for this purpose. Here we present PausePred (https://pausepred.ucc.ie/), which can be used to infer ribosome pauses from ribosome profiling (Ribo-seq) data. Peaks of ribosome footprint density are scored based on their magnitude relative to the background density within the surrounding area. The scoring allows the comparison of peaks across the transcriptome or genome. In addition to the score, PausePred reports the coordinates of the pause, the footprint density at the pause site, and the surrounding nucleotide sequence. The pauses can be visualized in the context of Ribo-seq and RNA-seq density plots generated for specific transcripts or genomic regions with the Rfeet tool. PausePred does not require input on the location of protein coding ORFs (although gene annotations can be optionally supplied). As a result, it can be used universally and its output does not depend on ever evolving annotations.

TASEP modelling provides a parsimonious explanation for the ability of a single uORF to derepress translation during the integrated stress response.

Translation initiation is the rate-limiting step of protein synthesis that is downregulated during the Integrated Stress Response (ISR). Previously, we demonstrated that most human mRNAs that are resistant to this inhibition possess translated upstream open reading frames (uORFs), and that in some cases a single uORF is sufficient for the resistance. Here we developed a computational model of Initiation Complexes Interference with Elongating Ribosomes (ICIER) to gain insight into the mechanism. We explored the relationship between the flux of scanning ribosomes upstream and downstream of a single uORF depending on uORF features. Paradoxically, our analysis predicts that reducing ribosome flux upstream of certain uORFs increases initiation downstream. The model supports the derepression of downstream translation as a general mechanism of uORF-mediated stress resistance. It predicts that stress resistance can be achieved with long slowly decoded uORFs that do not favor translation reinitiation and that start with initiators of low leakiness.

The GWIPS-viz Browser.

GWIPS-viz is a publicly available browser that provides Genome Wide Information on Protein Synthesis through the visualization of ribosome profiling data. Ribosome profiling (Ribo-seq) is a high-throughput technique which isolates fragments of messenger RNA that are protected by the ribosome. The alignment of the ribosome-protected fragments or footprint sequences to the corresponding reference genome and their visualization using GWIPS-viz allows for unique insights into the genome loci that are expressed as potentially translated RNA. The GWIPS-viz browser hosts both Ribo-seq data and corresponding mRNA-seq data from publicly available studies across a number of genomes, avoiding the need for computational processing on the user side. Since its initial publication in 2014, over 1885 tracks have been produced across 24 genomes. This unit describes the navigation of the GWIPS-viz genome browser, the uploading of custom tracks, and the downloading of the Ribo-seq/mRNA-seq alignment data. © 2018 by John Wiley & Sons, Inc.

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation.

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage.

GWIPS-viz: 2018 update.

The GWIPS-viz browser (http://gwips.ucc.ie/) is an on-line genome browser which is tailored for exploring ribosome profiling (Ribo-seq) data. Since its publication in 2014, GWIPS-viz provides Ribo-seq data for an additional 14 genomes bringing the current total to 23. The integration of new Ribo-seq data has been automated thereby increasing the number of available tracks to 1792, a 10-fold increase in the last three years. The increase is particularly substantial for data derived from human sources. Following user requests, we added the functionality to download these tracks in bigWig format. We also incorporated new types of data (e.g. TCP-seq) as well as auxiliary tracks from other sources that help with the interpretation of Ribo-seq data. Improvements in the visualization of the data have been carried out particularly for bacterial genomes where the Ribo-seq data are now shown in a strand specific manner. For higher eukaryotic datasets, we provide characteristics of individual datasets using the RUST program which includes the triplet periodicity, sequencing biases and relative inferred A-site dwell times. This information can be used for assessing the quality of Ribo-seq datasets. To improve the power of the signal, we aggregate Ribo-seq data from several studies into Global aggregate tracks for each genome.

Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity.

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitroIn vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

RiboGalaxy: A browser based platform for the alignment, analysis and visualization of ribosome profiling data.

Ribosome profiling (ribo-seq) is a technique that uses high-throughput sequencing to reveal the exact locations and densities of translating ribosomes at the entire transcriptome level. The technique has become very popular since its inception in 2009. Yet experimentalists who generate ribo-seq data often have to rely on bioinformaticians to process and analyze their data. We present RiboGalaxy ( http://ribogalaxy.ucc.ie ), a freely available Galaxy-based web server for processing and analyzing ribosome profiling data with the visualization functionality provided by GWIPS-viz ( http://gwips.ucc.ie ). RiboGalaxy offers researchers a suite of tools specifically tailored for processing ribo-seq and corresponding mRNA-seq data. Researchers can take advantage of the published workflows which reduce the multi-step alignment process to a minimum of inputs from the user. Users can then explore their own aligned data as custom tracks in GWIPS-viz and compare their ribosome profiles to existing ribo-seq tracks from published studies. In addition, users can assess the quality of their ribo-seq data, determine the strength of the triplet periodicity signal, generate meta-gene ribosome profiles as well as analyze the relative impact of mRNA sequence features on local read density. RiboGalaxy is accompanied by extensive documentation and tips for helping users. In addition we provide a forum ( http://gwips.ucc.ie/Forum ) where we encourage users to post their questions and feedback to improve the overall RiboGalaxy service.

GWIPS-viz as a tool for exploring ribosome profiling evidence supporting the synthesis of alternative proteoforms.

The boundaries of protein coding sequences are more difficult to define at the 5' end than at the 3' end due to potential multiple translation initiation sites (TISs). Even in the presence of phylogenetic data, the use of sequence information only may not be sufficient for the accurate identification of TISs. Traditional proteomics approaches may also fail because the N-termini of newly synthesized proteins are often processed. Thus ribosome profiling (ribo-seq), producing a snapshot of the ribosome distribution across the entire transcriptome, is an attractive experimental technique for the purpose of TIS location exploration. The GWIPS-viz (Genome Wide Information on Protein Synthesis visualized) browser (http://gwips.ucc.ie) provides free access to the genomic alignments of ribo-seq data and corresponding mRNA-seq data along with relevant annotation tracks. In this brief, we illustrate how GWIPS-viz can be used to explore the ribosome occupancy at the 5' ends of protein coding genes to assess the activity of AUG and non-AUG TISs responsible for the synthesis of proteoforms with alternative or heterogeneous N-termini. The presence of ribo-seq tracks for various organisms allows for cross-species comparison of orthologous genes and the availability of datasets from multiple laboratories permits the assessment of the technical reproducibility of the ribosome densities.

Computational approach for calculating the probability of eukaryotic translation initiation from ribo-seq data that takes into account leaky scanning.

BACKGROUND: Ribosome profiling (ribo-seq) provides experimental data on the density of elongating or initiating ribosomes at the whole transcriptome level that can be potentially used for estimating absolute levels of translation initiation at individual Translation Initiation Sites (TISs). These absolute levels depend on the mutual organisation of TISs within individual mRNAs. For example, according to the leaky scanning model of translation initiation in eukaryotes, a strong TIS downstream of another strong TIS is unlikely to be productive, since only a few scanning ribosomes would be able to reach the downstream TIS. In order to understand the dependence of translation initiation efficiency on the surrounding nucleotide context, it is important to estimate the strength of TISs independently of their mutual organisation, i.e. to estimate with what probability a ribosome would initiate at a particular TIS. RESULTS: We designed a simple computational approach for estimating the probabilities of ribosomes initiating at individual start codons using ribosome profiling data. The method is based on the widely accepted leaky scanning model of translation initiation in eukaryotes which postulates that scanning ribosomes may skip a start codon if the initiation context is unfavourable and continue on scanning. We tested our approach on three independent ribo-seq datasets obtained in mammalian cultured cells. CONCLUSIONS: Our results suggested that the method successfully discriminates between weak and strong TISs and that the majority of numerous non-AUG TISs reported recently are very weak. Therefore the high frequency of non-AUG TISs observed in ribosome profiling experiments is due to their proximity to mRNA 5'-ends rather than their strength. Detectable translation initiation at non-AUG codons downstream of AUG codons is comparatively infrequent. The leaky scanning method will be useful for the characterization of differences in start codon selection between tissues, developmental stages and in response to stress conditions.

GWIPS-viz: development of a ribo-seq genome browser.

We describe the development of GWIPS-viz (http://gwips.ucc.ie), an online genome browser for viewing ribosome profiling data. Ribosome profiling (ribo-seq) is a recently developed technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome-protected messenger RNA (mRNA) fragments, which allows the ribosome density along all mRNA transcripts present in the cell to be quantified. Since its inception, ribo-seq has been carried out in a number of eukaryotic and prokaryotic organisms. Owing to the increasing interest in ribo-seq, there is a pertinent demand for a dedicated ribo-seq genome browser. GWIPS-viz is based on The University of California Santa Cruz (UCSC) Genome Browser. Ribo-seq tracks, coupled with mRNA-seq tracks, are currently available for several genomes: human, mouse, zebrafish, nematode, yeast, bacteria (Escherichia coli K12, Bacillus subtilis), human cytomegalovirus and bacteriophage lambda. Our objective is to continue incorporating published ribo-seq data sets so that the wider community can readily view ribosome profiling information from multiple studies without the need to carry out computational processing.

Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale.

Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes.

Observation of dually decoded regions of the human genome using ribosome profiling data.

The recently developed ribosome profiling technique (Ribo-Seq) allows mapping of the locations of translating ribosomes on mRNAs with subcodon precision. When ribosome protected fragments (RPFs) are aligned to mRNA, a characteristic triplet periodicity pattern is revealed. We utilized the triplet periodicity of RPFs to develop a computational method for detecting transitions between reading frames that occur during programmed ribosomal frameshifting or in dual coding regions where the same nucleotide sequence codes for multiple proteins in different reading frames. Application of this method to ribosome profiling data obtained for human cells allowed us to detect several human genes where the same genomic segment is translated in more than one reading frame (from different transcripts as well as from the same mRNA) and revealed the translation of hitherto unpredicted coding open reading frames.