scholarly journals Inferring translational heterogeneity from ribosome profiling data

2019 ◽  
Author(s):  
Pedro do Couto Bordignon ◽  
Sebastian Pechmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Biosynthesis ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Messenger Rnas ◽  
Translation Elongation ◽  
Cellular Homeostasis ◽  
Ribosome Density ◽  
Experimental Variability ◽  
Underlying Mechanisms

Translation of messenger RNAs into proteins by the ribosome is the most important step of protein biosynthesis. Accordingly, translation is tightly controlled and heavily regulated to maintain cellular homeostasis. Ribosome profiling (Ribo-seq) has revolutionized the study of translation by revealing many of its underlying mechanisms. However, equally many aspects of translation remain mysterious, in part also due to persisting challenges in the interpretation of data obtained from Ribo-seq experiments. Here, we show that some of the variability observed in Ribo-seq data has biological origins and reflects programmed heterogeneity of translation. To systematically identify sequences that are differentially translated (DT) across mRNAs beyond what can be attributed to experimental variability, we performed a comparative analysis of Ribo-seq data from Saccharomyces cerevisiae and derived a consensus ribosome density profile that reflects consistent signals in individual experiments. Remarkably, the thus identified DT sequences link to mechanisms known to regulate translation elongation and are enriched in genes important for protein and organelle biosynthesis. Our results thus highlight examples of translational heterogeneity that are encoded in the genomic sequences and tuned to optimizing cellular homeostasis. More generally, our work highlights the power of Ribo-seq to understand the complexities of translation regulation.

scholarly journals Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

2015 ◽  
Author(s):  
Jeffrey A Hussmann ◽  
Stephanie Patchett ◽  
Arlen Johnson ◽  
Sara Sawyer ◽  
William H Press
Keyword(s):  
Negative Correlation ◽  
Ribosome Profiling ◽  
Messenger Rnas ◽  
Translation Rate ◽  
Ribosome Density ◽  
Weak Negative Correlation ◽  
Specific Elongation ◽  
Positive Correlations ◽  
Cognate Trna

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

scholarly journals Rationalizing Translation Elongation by Reinforcement Learning

2018 ◽  
Author(s):  
Hailin Hu ◽  
Xianggen Liu ◽  
An Xiao ◽  
Sen Song ◽  
Jianyang Zeng
Keyword(s):  
Reinforcement Learning ◽  
Translational Regulation ◽  
State Of The Art ◽  
Policy Network ◽  
Translation Elongation ◽  
Ribosome Density ◽  
Protein Biogenesis ◽  
Potential Applications ◽  
Underlying Mechanisms

AbstractTranslation elongation plays a crucial role in multiple aspects of protein biogenesis. In this study, we develop a novel deep reinforcement learning based framework, named RiboRL, to model the distributions of ribosomes on transcripts. In particular, RiboRL employs a policy network (PolicyNet) to perform a context-dependent feature selection to facilitate the prediction of ribosome density. Extensive tests demonstrate that RiboRL can outperform other state-of-the-art methods in predicting ribosome densities. We also show that the reinforcement learning based strategy can generate more informative features for the prediction task when compared to other commonly used attribution methods in deep learning. Moreover, the in-depth analyses and a case study also indicate the potential applications of the RiboRL framework in generating meaningful biological insights regarding translation elongation dynamics. These results have established RiboRL as a useful computational tool to facilitate the studies of the underlying mechanisms of translational regulation.

scholarly journals Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Villu Kasari ◽  
Tõnu Margus ◽  
Gemma C. Atkinson ◽  
Marcus J.O. Johansson ◽  
Vasili Hauryliuk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Stop Codon ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Additional Function ◽  
Ribosome Density ◽  
Eukaryotic Elongation Factor ◽  
Eukaryotic Organisms

AbstractIn addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5’ part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

scholarly journals Full-length ribosome density prediction by a multi-input and multi-output model

2021 ◽  
Vol 17 (3) ◽  
pp. e1008842
Author(s):  
Tingzhong Tian ◽  
Shuya Li ◽  
Peng Lang ◽  
Dan Zhao ◽  
Jianyang Zeng
Keyword(s):  
Ribosome Profiling ◽  
Full Length ◽  
Translation Efficiency ◽  
Long Distance ◽  
Translation Elongation ◽  
Coding Sequence ◽  
Density Distributions ◽  
Density Prediction ◽  
Ribosome Density ◽  
Elongation Process

Translation elongation is regulated by a series of complicated mechanisms in both prokaryotes and eukaryotes. Although recent advance in ribosome profiling techniques has enabled one to capture the genome-wide ribosome footprints along transcripts at codon resolution, the regulatory codes of elongation dynamics are still not fully understood. Most of the existing computational approaches for modeling translation elongation from ribosome profiling data mainly focus on local contextual patterns, while ignoring the continuity of the elongation process and relations between ribosome densities of remote codons. Modeling the translation elongation process in full-length coding sequence (CDS) level has not been studied to the best of our knowledge. In this paper, we developed a deep learning based approach with a multi-input and multi-output framework, named RiboMIMO, for modeling the ribosome density distributions of full-length mRNA CDS regions. Through considering the underlying correlations in translation efficiency among neighboring and remote codons and extracting hidden features from the input full-length coding sequence, RiboMIMO can greatly outperform the state-of-the-art baseline approaches and accurately predict the ribosome density distributions along the whole mRNA CDS regions. In addition, RiboMIMO explores the contributions of individual input codons to the predictions of output ribosome densities, which thus can help reveal important biological factors influencing the translation elongation process. The analyses, based on our interpretable metric named codon impact score, not only identified several patterns consistent with the previously-published literatures, but also for the first time (to the best of our knowledge) revealed that the codons located at a long distance from the ribosomal A site may also have an association on the translation elongation rate. This finding of long-range impact on translation elongation velocity may shed new light on the regulatory mechanisms of protein synthesis. Overall, these results indicated that RiboMIMO can provide a useful tool for studying the regulation of translation elongation in the range of full-length CDS.

scholarly journals Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

2020 ◽  
Author(s):  
Ganapathi Kandasamy ◽  
Ashis Kumar Pradhan ◽  
R Palanimurugan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Rna Degradation ◽  
26S Proteasome ◽  
Cellular Proteins ◽  
Cellular Homeostasis ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

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.

scholarly journals Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Maxim V. Gerashchenko ◽  
Mikhail V. Nesterchuk ◽  
Elena M. Smekalova ◽  
Joao A. Paulo ◽  
Piotr S. Kowalski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Mouse Liver ◽  
Negative Impact ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Elongation Factor 2

Abstract Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

scholarly journals Inferring efficiency of translation initiation and elongation from ribosome profiling

2020 ◽  
Vol 48 (17) ◽  
pp. 9478-9490
Author(s):  
Juraj Szavits-Nossan ◽  
Luca Ciandrini
Keyword(s):  
Protein Synthesis ◽  
Mathematical Modelling ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Recent Advances ◽  
Two Stages

Abstract One of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome’s stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima and traffic is minimized at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach $50\%$. Our work provides new measures of translation initiation and elongation efficiencies, emphasizing the importance of rating these two stages of translation separately.

Comparative analysis of protein-coding and long non-coding transcripts based on RNA sequence features

2018 ◽  
Vol 16 (02) ◽  
pp. 1840013 ◽  
Author(s):  
Oxana A. Volkova ◽  
Yury V. Kondrakhin ◽  
Timur A. Kashapov ◽  
Ruslan N. Sharipov
Keyword(s):  
Position Weight Matrix ◽  
Ribosome Profiling ◽  
Messenger Rnas ◽  
Putative Orfs ◽  
Protein Coding ◽  
Rna Translation ◽  
Cell Life ◽  
Experimental Tool ◽  
Non Coding Rnas ◽  
Small Orfs

RNA plays an important role in the intracellular cell life and in the organism in general. Besides the well-established protein coding RNAs (messenger RNAs, mRNAs), long non-coding RNAs (lncRNAs) have gained the attention of recent researchers. Although lncRNAs have been classified as non-coding, some authors reported the presence of corresponding sequences in ribosome profiling data (Ribo-seq). Ribo-seq technology is a powerful experimental tool utilized to characterize RNA translation in cell with focus on initiation (harringtonine, lactimidomycin) and elongation (cycloheximide). By exploiting translation starts obtained from the Ribo-seq experiment, we developed a novel position weight matrix model for the prediction of translation starts. This model allowed us to achieve 96% accuracy of discrimination between human mRNAs and lncRNAs. When the same model was used for the prediction of putative ORFs in RNAs, we discovered that the majority of lncRNAs contained only small ORFs ([Formula: see text][Formula: see text]nt) in contrast to mRNAs.

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