Comparative survey of the relative impact of mRNA features on local ribosome profiling read density

Ribosome profiling (Ribo-seq), a promising technology for exploring ribosome decoding rates, is characterized by the presence of infrequent high peaks in ribosome footprint density and by long alignment gaps. Here, to reduce the impact of data heterogeneity we introduce a simple normalization method, Ribo-seq Unit Step Transformation (RUST). RUST is robust and outperforms other normalization techniques in the presence of heterogeneous noise. We illustrate how RUST can be used for identifying mRNA sequence features that affect ribosome footprint densities globally. We show that a few parameters extracted with RUST are sufficient for predicting experimental densities with high accuracy. Importantly the application of RUST to 30 publicly available Ribo-seq data sets revealed a substantial variation in sequence determinants of ribosome footprint frequencies, questioning the reliability of Ribo-seq as an accurate representation of local ribosome densities without prior quality control. This emphasizes our incomplete understanding of how protocol parameters affect ribosome footprint densities

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

Insights into the mechanisms of eukaryotic translation gained with ribosome profiling

The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and it thereby provides a â snapshotâ of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation

Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes

Background: Oxygen and glucose metabolism play pivotal roles in many (patho) physiological conditions. In particular, oxygen and glucose deprivation (OGD) during ischemia and stroke results in extensive tissue injury and cell death. Results: Using time-resolved ribosome profiling, we assess gene expression levels in a neural cell line, PC12, during the first hour of OGD. The most substantial alterations are seen to occur within the first 20 minutes of OGD. While transcription of only 100 genes is significantly altered during one hour of OGD, the translation response affects approximately 3,000 genes. This response involves reprogramming of initiation and elongation rates, as well as the stringency of start codon recognition. Genes involved in oxidative phosphorylation are most affected. Detailed analysis of ribosome profiles reveals salient alterations of ribosome densities on individual mRNAs. The mRNA-specific alterations include increased translation of upstream open reading frames, site-specific ribosome pauses, and production of alternative protein isoforms with amino-terminal extensions. Detailed analysis of ribosomal profiles also reveals six mRNAs with translated ORFs occurring downstream of annotated coding regions and two examples of dual coding mRNAs, where two protein products are translated from the same long segment of mRNA, but in two different frames. Conclusions: These findings uncover novel regulatory mechanisms of translational response to OGD in mammalian cells that are different from the classical pathways such as hypoxia inducible factor (HIF) signaling, while also revealing sophisticated organization of protein coding information in certain genes

Rocaglates induce gain-of-function alterations to eIF4A and eIF4F

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5' leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA.P50 GM067041 - NIGMS NIH HHS; R24 GM111625 - NIGMS NIH HHS; R35 GM118173 - NIGMS NIH HHSPublished versio

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

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 process1,2. 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: 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

The Children's Respiratory and Environmental Workgroup (CREW) birth cohort consortium: design, methods, and study population

Background: Single birth cohort studies have been the basis for many discoveries about early life risk factors for childhood asthma but are limited in scope by sample size and characteristics of the local environment and population. The Children’s Respiratory and Environmental Workgroup (CREW) was established to integrate multiple established asthma birth cohorts and to investigate asthma phenotypes and associated causal pathways (endotypes), focusing on how they are influenced by interactions between genetics, lifestyle, and environmental exposures during the prenatal period and early childhood. Methods and results: CREW is funded by the NIH Environmental influences on Child Health Outcomes (ECHO) program, and consists of 12 individual cohorts and three additional scientific centers. The CREW study population is diverse in terms of race, ethnicity, geographical distribution, and year of recruitment. We hypothesize that there are phenotypes in childhood asthma that differ based on clinical characteristics and underlying molecular mechanisms. Furthermore, we propose that asthma endotypes and their defining biomarkers can be identified based on personal and early life environmental risk factors. CREW has three phases: 1) to pool and harmonize existing data from each cohort, 2) to collect new data using standardized procedures, and 3) to enroll new families during the prenatal period to supplement and enrich extant data and enable unified systems approaches for identifying asthma phenotypes and endotypes. Conclusions: The overall goal of CREW program is to develop a better understanding of how early life environmental exposures and host factors interact to promote the development of specific asthma endotypes.HHS/NIH [5UG3OD023282]; Columbia University [P01ES09600, R01 ES008977, P30ES09089, R01 ES013163, R827027]; Tucson Children's Respiratory Study (TCRS) [NHLBI 132523]; Infant Immune Study (IIS) [HL-56177]; Childhood Origins of Asthma Study (COAST) [P01 HL070831, U10 HL064305, R01 HL061879]; Wayne County Health, Environment, Allergy and Asthma Longitudinal Study (WHEALS) [R01 AI050681, R56 AI050681, R01 AI061774, R21 AI059415, K01 AI070606, R21 AI069271, R01 HL113010, R21 ES022321, P01 AI089473, R21 AI080066, R01 AI110450, R01 HD082147]; Fund for Henry Ford Health System; Childhood Allergy Study (CAS) [R01 AI024156, R03 HL067427, R01 AI051598]; Blue Cross Foundation Johnson; Fund for Henry Ford Hospital; Microbes, Allergy, Asthma and Pets (MAAP) [P01 AI089473]; Infant Susceptibility to Pulmonary Infections and Asthma following RSV Exposure (INSPIRE) [NIH/NIAID U19 AI 095227, NIH/NCATS UL1 TR 002243, NIH/NIAID K24 AI 077930, NIH/NHLBI R21 HD 087864, NIH/NHLBI X01 HL 134583]; Wisconsin Infant Study Cohort (WISC) [U19 AI104317, NCATS UL1TR000427]; Upper Midwest Agricultural Safety and Health Center (UMASH) [U54 OH010170]; RTI International, Research Triangle Park, North Carolina, USA; NIH [U24OD023382]; Urban Environment and Childhood Asthma Study (URECA) [NO1-AI-25482, HHSN272200900052C, HHSN272201000052I, NCRR/NIH RR00052, M01RR00533, 1UL1RR025771, M01RR00071, 1UL1RR024156, UL1TR001079, 5UL1RR024992-02, NCATS/NIH UL1TR000040]; Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) [R01 ES11170, R01 ES019890]; Epidemiology of Home Allergens and Asthma Study (EHAAS) [R01 AI035786]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]