scholarly journals Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing

2019 ◽  
Author(s):  
Alaaddin Bulak Arpat ◽  
Angélica Liechti ◽  
Mara De Matos ◽  
René Dreos ◽  
Peggy Janich ◽  
...  
Keyword(s):  
Protein Production ◽  
Evolutionary Conservation ◽  
Ribosome Profiling ◽  
Translation Rate ◽  
Translation Elongation ◽  
Peptide Motifs ◽  
Functional Relevance ◽  
Polypeptide Structure ◽  
The Impact ◽  
Pause Site

AbstractTranslation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of non-uniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites, independent of translation rate, is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs and nascent polypeptide structure, is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

scholarly journals Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knock-Out Mice

2018 ◽  
Author(s):  
Sohani Das Sharma ◽  
Jordan B. Metz ◽  
Hongyu Li ◽  
Benjamin D. Hobson ◽  
Nicholas Hornstein ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Translation Initiation ◽  
Rna Binding ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Knock Out ◽  
Knock Out Mice ◽  
Mrna Binding ◽  
The Impact

SummaryFMRP is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in Fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene-specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we used ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24 day-old Fmr1 knock-out mice. We found a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP, and an increase for terminal oligo-pyrimidine (TOP) motif-containing genes canonically controlled by mTOR-4EBP-eIF4E signaling. Amino acid motif- and gene-level analyses both showed a widespread reduction of translational pausing in Fmr1 knock-out mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in Fragile X syndrome.

scholarly journals The impact of ribosomal interference, codon usage, and exit tunnel interactions on translation elongation rate variation

2016 ◽  
Author(s):  
Khanh Dao Duc ◽  
Yun S. Song
Keyword(s):  
Electrostatic Interactions ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Rate Variation ◽  
Biophysical Properties ◽  
Translation Elongation ◽  
Nascent Polypeptide ◽  
Exit Tunnel ◽  
Pass Through ◽  
The Impact

ABSTRACTPrevious studies have shown that translation elongation is regulated by multiple factors, but the observed heterogeneity remains only partially explained. To dissect quantitatively the different determinants of elongation speed, we use probabilistic modeling to estimate initiation and local elongation rates from ribosome profiling data. This model-based approach allows us to quantify the extent of interference between ribosomes on the same transcript. We show that neither interference nor the distribution of slow codons is sufficient to explain the observed heterogeneity. Instead, we find that electrostatic interactions between the ribosomal exit tunnel and specific parts of the nascent polypeptide govern the elongation rate variation as the polypeptide makes its initial pass through the tunnel. Once the N-terminus has escaped the tunnel, the hydropathy of the nascent polypeptide within the ribosome plays a major role in modulating the speed. We show that our results are consistent with the biophysical properties of the tunnel.

scholarly journals Accurate design of translational output by a neural network model of ribosome distribution

2017 ◽  
Author(s):  
Robert J Tunney ◽  
Nicholas J McGlincy ◽  
Monica E Graham ◽  
Nicki Naddaf ◽  
Lior Pachter ◽  
...  
Keyword(s):  
Neural Network ◽  
Fluorescent Protein ◽  
Synonymous Codon ◽  
Full Range ◽  
Rna Stability ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Accurate Design ◽  
The Impact

Synonymous codon choice can have dramatic effects on ribosome speed, RNA stability, and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs, exposing a need for models of coding sequences that capture the full range of empirically observed variation. We present a method, Ixnos, that models this variation in translation elongation using a feedforward neural network to predict the translation elongation rate at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and quantified the impact of large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of possible translation speeds predicted with our model. We found that levels of the fluorescent protein in yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo, and that control of translation elongation alone is sufficient to produce large, quantitative differences in protein output.

scholarly journals Maximizing protein translation rate in the non-homogeneous ribosome flow model: a convex optimization approach

2014 ◽  
Vol 11 (100) ◽  
pp. 20140713 ◽  
Author(s):  
Gilad Poker ◽  
Yoram Zarai ◽  
Michael Margaliot ◽  
Tamir Tuller
Keyword(s):  
Synthetic Biology ◽  
Convex Optimization ◽  
Production Rate ◽  
Protein Production ◽  
Flow Model ◽  
Protein Translation ◽  
Optimization Approach ◽  
Initiation Rate ◽  
Translation Rate ◽  
Protein Production Rate

Translation is an important stage in gene expression. During this stage, macro-molecules called ribosomes travel along the mRNA strand linking amino acids together in a specific order to create a functioning protein. An important question, related to many biomedical disciplines, is how to maximize protein production. Indeed, translation is known to be one of the most energy-consuming processes in the cell, and it is natural to assume that evolution shaped this process so that it maximizes the protein production rate. If this is indeed so then one can estimate various parameters of the translation machinery by solving an appropriate mathematical optimization problem. The same problem also arises in the context of synthetic biology, namely, re-engineer heterologous genes in order to maximize their translation rate in a host organism. We consider the problem of maximizing the protein production rate using a computational model for translation–elongation called the ribosome flow model (RFM). This model describes the flow of the ribosomes along an mRNA chain of length n using a set of n first-order nonlinear ordinary differential equations. It also includes n + 1 positive parameters: the ribosomal initiation rate into the mRNA chain, and n elongation rates along the chain sites. We show that the steady-state translation rate in the RFM is a strictly concave function of its parameters. This means that the problem of maximizing the translation rate under a suitable constraint always admits a unique solution, and that this solution can be determined using highly efficient algorithms for solving convex optimization problems even for large values of n . Furthermore, our analysis shows that the optimal translation rate can be computed based only on the optimal initiation rate and the elongation rate of the codons near the beginning of the ORF. We discuss some applications of the theoretical results to synthetic biology, molecular evolution, and functional genomics.

Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery

2011 ◽  
Vol 109 (4) ◽  
pp. 983-991 ◽  
Author(s):  
Steven D. Branston ◽  
Cristina F.R.O. Matos ◽  
Robert B. Freedman ◽  
Colin Robinson ◽  
Eli Keshavarz-Moore
Keyword(s):  
Protein Production ◽  
Early Stage ◽  
E Coli ◽  
Export Pathway ◽  
The Impact

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.

scholarly journals RegSNPs-intron: a computational framework for predicting pathogenic impact of intronic single nucleotide variants

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Hai Lin ◽  
Katherine A. Hargreaves ◽  
Rudong Li ◽  
Jill L. Reiter ◽  
Yue Wang ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Evolutionary Conservation ◽  
Random Forest Classifier ◽  
Training Data ◽  
Reporter Assay ◽  
Single Nucleotide Variants ◽  
Excellent Performance ◽  
Computational Framework ◽  
Single Nucleotide ◽  
The Impact

AbstractSingle nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure, and evolutionary conservation features. RegSNPs-intron showed excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of RegSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.

The impact of diversification of a rice–wheat cropping system on crop productivity and soil fertility

2002 ◽  
Vol 139 (4) ◽  
pp. 405-412 ◽  
Author(s):  
V. K. SINGH ◽  
B. B. SHARMA ◽  
B. S. DWIVEDI
Keyword(s):  
Soil Fertility ◽  
Green Manure ◽  
Protein Production ◽  
Field Experiments ◽  
Block Design ◽  
Crop Productivity ◽  
Research Centre ◽  
Wheat Crop ◽  
Cropping Intensity ◽  
The Impact

Field experiments were conducted at the Crop Research Centre of Govind Ballabh Pant University of Agriculture and Technology, Pantnagar during 1996/97 and 1997/98. Each experiment comprised 10 crop sequences: (a) wheat–rice, (b) chickpea–rice, (c) lentil–rice, (d) pea–rice, (e) wheat–mungbean green manure–rice, (f) wheat–Sesbania green manure–rice, (g) wheat–fodder–rice, (h) chickpea–fodder–rice, (i) lentil–fodder–rice and (j) pea–fodder–rice, in a randomized block design with four replications. The crop sequences were compared in terms of economic rice equivalent yield (REY), protein production, apparent nutrient balances and effect on soil fertility status. Amongst crop sequences involving two crops each year (200% cropping intensity), chickpea–rice gave highest REY and protein production. Of the sequences involving three crops each year (300% cropping intensity), chickpea–fodder–rice and wheat–fodder–rice were superior to others. The P balances were positive for all sequences, whereas K balances were generally negative except for sequences involving green manure legumes. Green manuring with Sesbania or mungbean helped restore soil fertility, indicating the advantage of green manure for higher productivity and sustainability of rice–wheat system. Chickpea–rice and chickpea–fodder–rice appeared promising alternatives to rice–wheat crop sequence.

scholarly journals Characterization of Temporal Protein Production in Pseudomonas aeruginosa Biofilms

2005 ◽  
Vol 187 (23) ◽  
pp. 8114-8126 ◽  
Author(s):  
Christopher J. Southey-Pillig ◽  
David G. Davies ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Protein Production ◽  
Developmental Stages ◽  
Developmental Process ◽  
Developmental Cycle ◽  
Specific Proteins ◽  
Biofilm Development ◽  
The Impact

ABSTRACT Phenotypic and genetic evidence supporting the notion of biofilm formation as a developmental process is growing. In the present work, we provide additional support for this hypothesis by identifying the onset of accumulation of biofilm-stage specific proteins during Pseudomonas aeruginosa biofilm maturation and by tracking the abundance of these proteins in planktonic and three biofilm developmental stages. The onset of protein production was found to correlate with the progression of biofilms in developmental stages. Protein identification revealed that proteins with similar function grouped within similar protein abundance patterns. Metabolic and housekeeping proteins were found to group within a pattern separate from virulence, antibiotic resistance, and quorum-sensing-related proteins. The latter were produced in a progressive manner, indicating that attendant features that are characteristic of biofilms such as antibiotic resistance and virulence may be part of the biofilm developmental process. Mutations in genes for selected proteins from several protein production patterns were made, and the impact of these mutations on biofilm development was evaluated. The proteins cytochrome c oxidase, a probable chemotaxis transducer, a two-component response regulator, and MexH were produced only in mature and late-stage biofilms. Mutations in the genes encoding these proteins did not confer defects in growth, initial attachment, early biofilm formation, or twitching motility but were observed to arrest biofilm development at the stage of cell cluster formation we call the maturation-1 stage. The results indicated that expression of theses genes was required for the progression of biofilms into three-dimensional structures on abiotic surfaces and the completion of the biofilm developmental cycle. Reverse transcription-PCR analysis confirmed the detectable change in expression of the respective genes ccoO, PA4101, and PA4208. We propose a possible mechanism for the role of these biofilm-specific proteins in biofilm formation.

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