Sub-codon Resolution Research Articles

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

Comparative Analysis of Gene Expression in Virulent and Attenuated Strains of Infectious Bronchitis Virus at Subcodon Resolution.

Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.

Global identification of functional microRNA-mRNA interactions in Drosophila

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. So far, no comprehensive experimental annotation of functional miRNA target sites exists in Drosophila. Here, we generated a transcriptome-wide in vivo map of miRNA-mRNA interactions in Drosophila melanogaster, making use of single nucleotide resolution in Argonaute1 (AGO1) crosslinking and immunoprecipitation (CLIP) data. Absolute quantification of cellular miRNA levels presents the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two CLIP approaches, we identify a similar predictive potential to unambiguously assign thousands of miRNA-mRNA pairs from AGO1 interaction data at unprecedented depth, achieving higher signal-to-noise ratios than with computational methods alone. Quantitative RNA-seq and sub-codon resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites and their quantitative functional impact in Drosophila.

Survey of the translation shifts in hepatocellular carcinoma with ribosome profiling.

Despite the critical position of translation in the multilevel gene expression regulation program, high-resolution and genome-wide view of the landscape of RNA translation in solid tumors is still limited.Methods: With a ribosome profiling procedure optimized for solid tissue samples, we profiled the translatomes of liver tumors and their adjacent noncancerous normal liver tissues from 10 patients with hepatocellular carcinoma (HCC). A set of bioinformatics tools was then applied to these data for the mining of novel insights into the translation shifts in HCC.Results: This is the first translatome data resource for dissecting dysregulated translation in HCC at the sub-codon resolution. Based on our data, quantitative comparisons of mRNA translation rates yielded the genes and processes that were subjected to patient specific or universal dysregulations of translation efficiencies in tumors. For example, multiple proteins involved in extracellular matrix organization exhibited significant translational upregulation in tumors. We then experimentally validated the tumor-promoting functions of two such genes as examples: AGRN and VWA1. In addition, the data was also used for de novo annotation of the translatomes in tumors and normal tissues, including multiple types of novel non-canonical small ORFs, which would be a resource for further functional studies.Conclusions: The present study generates the first survey of the HCC translatome with ribosome profiling, which is an insightful data resource for dissecting the translatome shift in liver cancer, at sub-codon resolution.

Shoelaces: an interactive tool for ribosome profiling processing and visualization

BackgroundThe emergence of ribosome profiling to map actively translating ribosomes has laid the foundation for a diverse range of studies on translational regulation. The data obtained with different variations of this assay is typically manually processed, which has created a need for tools that would streamline and standardize processing steps.ResultsWe present Shoelaces, a toolkit for ribosome profiling experiments automating read selection and filtering to obtain genuine translating footprints. Based on periodicity, favoring enrichment over the coding regions, it determines the read lengths corresponding to bona fide ribosome protected fragments. The specific codon under translation (P-site) is determined by automatic offset calculations resulting in sub-codon resolution. Shoelaces provides both a user-friendly graphical interface for interactive visualisation in a genome browser-like fashion and a command line interface for integration into automated pipelines. We process 79 libraries and show that studies typically discard excessive amounts of quality data in their manual analysis pipelines.ConclusionsShoelaces streamlines ribosome profiling analysis offering automation of the processing, a range of interactive visualization features and export of the data into standard formats. Shoelaces stores all processing steps performed in an XML file that can be used by other groups to exactly reproduce the processing of a given study. We therefore anticipate that Shoelaces can aid researchers by automating what is typically performed manually and contribute to the overall reproducibility of studies. The tool is freely distributed as a Python package, with additional instructions, tutorial and demo datasets available at https://bitbucket.org/valenlab/shoelaces.

De novo annotation and characterization of the translatome with ribosome profiling data.

By capturing and sequencing the RNA fragments protected by translating ribosomes, ribosome profiling provides snapshots of translation at subcodon resolution. The growing needs for comprehensive annotation and characterization of the context-dependent translatomes are calling for an efficient and unbiased method to accurately recover the signal of active translation from the ribosome profiling data. Here we present our new method, RiboCode, for such purpose. Being tested with simulated and real ribosome profiling data, and validated with cell type-specific QTI-seq and mass spectrometry data, RiboCode exhibits superior efficiency, sensitivity, and accuracy for de novo annotation of the translatome, which covers various types of ORFs in the previously annotated coding and non-coding regions. As an example, RiboCode was applied to assemble the context-specific translatomes of yeast under normal and stress conditions. Comparisons among these translatomes revealed stress-activated novel upstream and downstream ORFs, some of which are associated with translational dysregulations of the annotated main ORFs under the stress conditions.

Computational resources for ribosome profiling: from database to Web server and software.

Ribosome profiling is emerging as a powerful technique that enables genome-wide investigation of in vivo translation at sub-codon resolution. The increasing application of ribosome profiling in recent years has achieved remarkable progress toward understanding the composition, regulation and mechanism of translation. This benefits from not only the awesome power of ribosome profiling but also an extensive range of computational resources available for ribosome profiling. At present, however, a comprehensive review on these resources is still lacking. Here, we survey the recent computational advances guided by ribosome profiling, with a focus on databases, Web servers and software tools for storing, visualizing and analyzing ribosome profiling data. This review is intended to provide experimental and computational biologists with a reference to make appropriate choices among existing resources for the question at hand.

Accurate Recovery of Ribosome Positions Reveals Slow Translation of Wobble-Pairing Codons in Yeast.

Ribosome profiling quantitatively captures ribosome locations during translation. The resulting profiles of ribosome locations are widely used to study translational speed. However, an accurate estimation of the ribosome location depends on identifying the A-site from ribosome profiling reads, a problem that was previously unsolved. Here, we propose a novel method to estimate the ribosome A-site positions from high-coverage ribosome profiling reads. Our model allows more reads to be used, accurately explains the 3-nt periodicity of ribosome profiling reads from various lengths, and recovers consistent ribosome positions across different lengths. Our recovered ribosome positions are correctly highly skewed toward a single frame within a codon. They retain subcodon resolution and enable detection of off-frame translational events, such as frameshifts. Our method improves the correlation with other estimates of codon decoding time. Furthermore, the refined profiles show that yeast wobble-pairing codons are translated slower than their synonymous Watson–Crick-pairing codons. These results provide evidence that protein synthetic rate can be tuned by codon usage bias.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Comparative ribosome profiling reveals extensive translational complexity in differentTrypanosoma bruceilife cycle stages

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5′ UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.

Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.