Hu2020 - E. coli translation model
Ontology highlight
ABSTRACT:
OTHER RELATED OMICS DATASETS IN: PRJNA312619
SUBMITTER: 
Xiao-Pan Hu
PROVIDER: MODEL2006210001 | BioModels | 2020-07-01
REPOSITORIES: BioModels
Publications
Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates.
Dong H H Nilsson L L Kurland C G CG
Journal of molecular biology 19960801 5
We have used two-dimensional polyacrylamide gel electrophoresis to fractionate tRNAs from Escherichia coli. A sufficiently high degree of resolution was obtained for 44 out of 46 tRNA species in E. coli to be resolved into individual electrophoretic components. These isolated components were identified by hybridization to tRNA-specific oligonucleotide probes. Systematic measurements of the abundance of each individual tRNA isoacceptor in E. coli, grown at rates varying from 0.4 to 2.5 doublings ...[more]
Similar Datasets
Project description: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.
2020-05-31 | GSE136091 | GEO
Project description:Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic by two mechanisms: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.
2021-12-08 | GSE184331 | GEO
Project description:To validate the sequence motifs identified by our multi-task learning model MTtrans, a new 5' UTR library with around 8,000 synthetic 5'UTRs was built to express EGFP. The reads count was used as a proxy of translation rate here to validate the estimated regulatory effect of motifs that we inferred from multiple datasets, proving the robustness of the sequence motifs.
2022-04-28 | GSE201766 | GEO
Project description:Firczuk2013 - Eukaryotic mRNA translation machinery
This is a model of Saccharomyces cerevisiae
mRNA translation which includes the initiation, elongation and termination phases. The model is for 20 condon mRNAs. The building of a multi-factor complex in initiation and also the different processes in elongation and termination are modelled in detail. The model takes into account that ribosomes cover more than one codon of mRNA so that the movement of ribosomes are effectively blocked by other ribosomes several codons downstream. It is assumed that 15 codons are occupied by each ribosome. This blocking effect is considered in reaction R18 in initiation and also reaction R26, the reaction where translocation of ribosomes takes place in elongation. The kinetic functions of these two reactions are based on MacDonald et al. 1968 and Heinrich & Rapaport 1980. All other kinetic functions follow mass-action kinetics. The concentrations of transfer RNA species (Met-tRNA, aa-tRNA and tRNA in the model) are kept constant, while the other species' concentrations can change in the course of the simulation. The model describes the translation of a short mRNA with 20 codons. Therefore, all reactions in the elongation cycle (R22, R23, R25, R26, R28 and R29) and the corresponding species are replicated accordingly to model the species with ribosomes bound at different positions. In summary, the model contains 165 different species and 141 reactions.
The value of the 56 rate constant parameters were estimated by fitting the model against a series of experimental data consisting of modulation of the various translation factors (Figures 2, 3 and S3). Overall the parameter estimation was carried out over 212 different data points (steady states).
This model is described in the article:
An in vivo control map for the eukaryotic mRNA translation machinery
Helena Firczuk, Shichina Kannambath, Jürgen Pahle, Amy Claydon, Robert Beynon, John Duncan, Hans Westerhoff, Pedro Mendes and John EG McCarthy
Molecular Systems Biology. 9:635
Abstract:
Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.
This model is hosted on BioModels Database
and identified by: BIOMD0000000457
.
To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource
for published quantitative kinetic models
.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to the public
domain worldwide. Please refer to CC0 Public Domain
Dedication
for more information.
2011-04-30 | BIOMD0000000457 | BioModels
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to LPS (1 micg/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, LPS monosome and LPS polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (LPS polysome/LPS monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. HUVECs were stimulated with LPS (1 micg/mL) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 ïL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 ïL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4ï° C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 ïL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 ïL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4ï° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.
2010-07-01 | E-GEOD-5352 | biostudies-arrayexpress
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to TNF-alpha (100 units/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, TNF monosome and TNF polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (TNF polysome/TNF monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. HUVECs were stimulated with TNF-alpha (100 U/mL) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 micL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 micL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 deg C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 micL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 micL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4 deg C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.
2010-07-01 | E-GEOD-5354 | biostudies-arrayexpress
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to histamine (100 mM) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments, control monosome, control polysome, histamine monosome and histamine polysome. Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (histamine polysome/histamine monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. HUVECs were stimulated with histamine (100 mM) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 ïL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 ïL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4ï° C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 ïL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 ïL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4ï° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.
2010-07-01 | E-GEOD-5385 | biostudies-arrayexpress
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to histamine (100 mM) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments, control monosome, control polysome, histamine monosome and histamine polysome. Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (histamine polysome/histamine monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. HUVECs were stimulated with Serum (20% pooled human serum) for 6 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 ïL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 micL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 deg C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 ïL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 micL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4 deg C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.
2010-07-01 | E-GEOD-5386 | biostudies-arrayexpress
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Insulin (100 micM) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, Insulin monosome and Insulin polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (Insulin polysome/Insulin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. HUVECs were stimulated with Insulin (100 micM) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 ïL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 ïL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4ï° C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 ïL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 ïL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4ï° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.
2010-07-01 | E-GEOD-5353 | biostudies-arrayexpress
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits. Ribosomal profiling of large and small polysomal fractions in Drosophila S2 cells to assess translation of smORFs
2014-09-11 | E-GEOD-60384 | biostudies-arrayexpress