ScDUAL-seq Simultaneously Measures RNA Synthesis and Degradation Rates in Single Cells
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PROVIDER: PRJNA1209856 | ENA |
REPOSITORIES: ENA
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Project description:The interplay between RNA transcription and degradation tightly regulates the dynamic changes of RNA abundance. To unveil the underlying regulatory mechanism of RNA in shaping gene expression patterns, it is essential yet challenging to profile the coordinated regulation of RNA synthesis and degradation rates at the single-cell level during dynamic programs such as cell differentiation and cell state transition. Herein, we propose scDUAL-seq, a double metabolic RNA labeling-based single-cell RNA sequencing method to simultaneously measure RNA synthesis and degradation rates in thousands of single cells during dynamic processes. In a cortisol response model of colorectal cancer cells, scDUAL-seq identifies the RNA regulatory strategies for 5,930 genes, discovers key regulons driving the strategy transitions, and disentangles the interplay between RNA synthesis and degradation that control distinct strategy transitions during cortisol response. We anticipate that scDUAL-seq will be broadly applied to record RNA kinetic rates and the underlying regulatory mechanism in diverse dynamic processes.
2025-07-01 | GSE286521 | GEO
Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis.
2014-04-24 | GSE53767 | GEO
Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis. 4 samples of E. coli ribosome profiling and mRNA-seq, including biological replicates
2014-04-24 | E-GEOD-53767 | biostudies-arrayexpress
Project description:Long-read Ribo-STAMP simultaneously measures transcription and translation at full length isoform resolution
| PRJNA1076859 | ENA
Project description:We developed a pulse-chase variant of SILAC (stable isotope labeling by amino acids in cell culture pcSILAC). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Methods : two cultures of Jurkat cells to be compared were fully labeled with, respectively, “medium” (13C6-L-arginine, R6) and “heavy” (13C615N4-L-arginine, R10) Arg, while Lys was left unlabeled (“light”, K0) for both. At the start of the experiment, both cultures were transferred to new media containing light Arg (R0) to perform the chase of R-labeled pre-existing proteins. In turn, the new media contained, respectively, “medium” (“M”, 2H4-Lysine, K4) and “heavy” (“H”, 13C615N2-L-lysine, K8) Lys, to label all newly synthesized proteins for the pulse measurements. At the same time or after medium exchange, one of the cultures was subjected to a perturbation (here, treatment with 1 uM Geldanamycin ). Extracts from control and drug-treated cells were then harvested at defined time points and mixed equimolarly. Proteins were digested with the FASP protocol, separated into 24 fractions by off-gel isoelectric focusing and analysed by nano-LC-MS/MS on an Orbitrap Velos Instrument. Data were analysed as triplex SILAC using MaxQuant and peptide spectrum matches were filtered at 1% FDR. Output tables were processed further with custom-made perl scripts and then used as input for the mathematical model. All subsequent data treatment steps were done with MatLab as described in the article. RAW DATA FILES: 1) pcSILAC experiment 2, replicate 2 (data presented in article) : 3 time points t=6,12,20h ID Description 4957 Mix= M/L (DMSO) + H/L(Geldanamycin); t=6h 5052 Mix= M/L (DMSO) + H/L(Geldanamycin); t=12h 4884 Mix= M/L (DMSO) + H/L(Geldanamycin); t=20h 2) pcSILAC experiment 1, replicate 2 (data presented in supplementary files to article) ID Description 4733 Mix= M/L (DMSO) + H/L(Geldanamycin); t=6h 4912 Mix= M/L (DMSO) + H/L(Geldanamycin); t=12h 4803 Mix= M/L (DMSO) + H/L(Geldanamycin); t=20h 3) stSILAC experiment 1 (internal stSILAC control done within pcSILAC exp. 1 ; data presented in supplementary files to article) ID Description 4731 Mix= M (DMSO) + H(Geldanamycin); t=6h 4734 Mix= M (DMSO) + H(Geldanamycin); t=20h
2013-12-10 | PXD000538 | Pride
Project description:Fragile X syndrome (FXS) is caused by inactivation of FMR1 gene and loss of its encoded product the RNA binding protein FMRP, which generally represses translation of its target transcripts in the brain. In mouse models of FXS (i.e., Fmr1 knockout animals; Fmr1 KO), deletion of Cpeb1, which encodes a translational activator, mitigates nearly all pathophysiologies associated with the disorder. We have observed that the wide-spread dys-regulation of RNA abundance in Fmr1 KO brain cortex and its rescue to normal levels in Fmr1/Cpeb1 double KO mice were the driver of the observed dys-regulation and rescue of translation as measured by whole transcriptome ribosome occupany in the brain. We hypothesize that in Fragile X brain there is wide spread dys-regulation at RNA stability level. Here we test this hypothesis by profiling RNA synthesis, processing and degradation rates in Fragile X and wild type neurons, by taking advantage of short 5-EU labeling and computational modeling. We show that, while RNA synthesis and processing rates were barely changed, there is wide-spread evelated RNA degradation rates in the Fragile X neurons, particularly for genes using optimal codons.
2020-06-01 | GSE140642 | GEO
Project description:Steady-state RNA levels are a result of RNA synthesis and degradation. The importance of transcription-factor mediated induction or repression of mRNA synthesis is well established, but the role and mechanisms of RNA degradation are less well understood. We globally evaluated the RNA decay rates in proliferating and differentiated mouse myoblasts on whole-genome Affymetrix exon arrays, allowing for the assessment of directionality of RNA degradation and the detection of splice variant-specific differences in RNA decay rates. We found large differences in decay rates. mRNAs coding for proteins involved in signal transduction and transcriptional regulation have shortest half lives, whereas mRNAs coding for DNA replication enzymes and muscle contraction proteins are among the most stable. Many genes differentially expressed between proliferating and differentiated myoblasts demonstrate major differences in RNA decay rates. Quantitative PCR experiments confirmed the higher stability of transcripts with increased expression levels. RNA degradation has no apparent preferential directionality. RNA degradation appears to affect the ratio of different splice variants. For example, Itga7 isoforms with higher abundance in differentiated than in proliferating cells are more stable in differentiated cells, despite the sharing of a common 3’ untranslated region. Thus, where it was previously thought that the abundance of different splice isoforms was mainly controlled by tissue-specific splicing factors, we now demonstrate that isoforms may be produced at comparable levels but degraded with different efficiencies, depending on the differentiation status of the cells. Our results indicate that control of RNA degradation rates contributes significantly to the differentiation stage-dependent differences in abundance of transcripts and splice variants. Keywords: total RNA expression profiling; cultured cells We analyzed proliferating C2C12 cells and C2C12 cells differentiated into myotubes by serum starvation (8 days after induction of differentiation). We analyzed 7 time points after addition of actionmycin D (0, 10, 20, 30, 60, 150, 480 minutes). There were two biological replicates per condition.
2010-09-22 | E-GEOD-14387 | biostudies-arrayexpress
Project description:Degradation rates in bioelectrochemical systems
| PRJEB80810 | ENA
Project description:The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative Dynamic Transcriptome Analysis (cDTA) that uses non-perturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network. For this purpose, cDTA was carried out as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). S. cerevisiae cultures were grown at 30¡ãC in 50 ml aliquots of YPD medium. S. pombe cultures were grown at 32¡ãC in YES medium. For inhibitor perturbation, BY4741 cells (OD600=0.1) were inoculated from a saturated overnight culture in two 100 ml aliquots of YPD liquid medium, incubated at 30¡ãC and grown to early-log phase (OD600=0.8). Cells were labeled for 6 minutes (sample 0 min). Then cycloheximide was added and cells were labeled for 10 minutes and 60 minutes. Cells were harvested and treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). 1,10-Phenanthroline was added to the culture to a final concentration of 25 _g/ml as described (Dori-Bachash et al., 2012), and labeled for 6 minutes after 18 minutes treatment. Cells were treated as above. For the anchor-away analysis, S. cerevisiae cells (OD600=0.1) were cultured in two 200 ml aliquots of YPD containing 1_g/ml rapamycin and incubated at 30¡ãC untill OD600 reached 0.8. Due to the instability of rapamycin, we added 1_g/ml rapamycin every two hours. Then a 20 ml sample was taken and labeled with 4sU. The cells were then treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).
2013-10-18 | E-MTAB-1525 | biostudies-arrayexpress
Project description:Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a thousand ORFs in exponentially-growing wildtype yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and tRNA adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared to other transcripts with equally high ribosome densities.
2019-09-10 | PXD014357 | Pride