scholarly journals Genome-wide dynamics of RNA synthesis, processing and degradation without RNA metabolic labeling

2019 ◽  
Author(s):  
Mattia Furlan ◽  
Eugenia Galeota ◽  
Nunzio Del Gaudio ◽  
Erik Dassi ◽  
Michele Caselle ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Steady State ◽  
Time Course ◽  
Rna Synthesis ◽  
Computational Method ◽  
Metabolic Labeling ◽  
Experimental Conditions ◽  
Rna Dynamics ◽  
Kinetic Rates ◽  
Post Transcriptional Regulation

AbstractThe kinetic rates of RNA synthesis, processing and degradation determine the dynamics of transcriptional regulation by governing both the abundance and the responsiveness to modulations of premature and mature RNA species. The study of RNA dynamics is largely based on the integrative analysis of total and nascent transcription, with the latter being quantified through RNA metabolic labeling. We describe here INSPEcT-, a computational method based on mathematical modeling of intronic and exonic expression, able to derive the dynamics of transcription from steady state or time course profiling of just total RNA, without requiring any information on nascent transcripts. Our approach closely recapitulates the kinetic rates obtained through RNA metabolic labeling, improves the ability to detect changes in transcripts half-lives, reduces the cost and complexity of the experiments, and can be adopted to study experimental conditions where nascent transcription cannot be readily profiled. Finally, we applied INSPEcT- to the characterization of post-transcriptional regulation landscapes in dozens of physiological and disease conditions. This approach was included in the INSPEcT Bioconductor package, which can now unveil RNA dynamics from steady state or time course data, with or without the profiling of nascent RNA.

Post-transcriptional regulation of bovine parathyroid hormone synthesis

1993 ◽  
Vol 10 (1) ◽  
pp. 43-49 ◽  
Author(s):  
N S Hawa ◽  
J L H O'Riordan ◽  
S M Farrow
Keyword(s):  
Transcriptional Regulation ◽  
Steady State ◽  
Actinomycin D ◽  
Mrna Levels ◽  
Dot Blot ◽  
Hormone Synthesis ◽  
Membrane Bound ◽  
Post Transcriptional Regulation ◽  
Bovine Parathyroid Hormone ◽  
In Cells

ABSTRACT Incubation of bovine parathyroid cells for 48 h in 0·4 mmol calcium/l had no significant effect on steady-state preproparathyroid hormone (preproPTH) mRNA levels when compared with cells incubated in 1·0 mmol calcium/l, but low calcium concentrations increased the membrane-bound polysomal content of preproPTH mRNA by 200±16% (mean±s.d.). No preproPTH mRNA was detected on free polysomes. Actinomycin D (5 and 10 μg/ml) had no effect on steady-state preproPTH mRNA levels measured in dot-blot assays after 24 h, but reduced levels in cells incubated in 1·0 mmol calcium/l to 54±16% and 39±12% of control values respectively after 48 h of incubation. Similarly, in cells incubated in 0·4 mmol calcium/l, actinomycin D (5 and 10μg/ml) reduced steady-state preproPTH mRNA levels to 57±13% and 45±5% of control values respectively. Actinomycin D did not prevent the rise in polysomal content of preproPTH mRNA induced in cells by incubation in 0·4 mmol calcium/l, but increased polysomal content in cells incubated in 0·4 and 1·0mmol calcium/l by 159±9% and 164±13% respectively after 48 h. These results demonstrate post-transcriptional regulation of PTH synthesis in cultured bovine parathyroid cells, and suggest that this control involves a protein which may be calcium-sensitive.

Analysis of post-transcriptional regulation using the FunREG method

2010 ◽  
Vol 38 (6) ◽  
pp. 1608-1614 ◽  
Author(s):  
Benoît Laloo ◽  
Marion Maurel ◽  
Sandra Jalvy-Delvaille ◽  
Francis Sagliocco ◽  
Christophe F. Grosset
Keyword(s):  
Transcriptional Regulation ◽  
Translation Efficiency ◽  
Histone Deacetylase 6 ◽  
Reporter System ◽  
Rna Sequences ◽  
Experimental Conditions ◽  
Fluorescent Reporter ◽  
Wide Range ◽  
Transcriptional Deregulation ◽  
Post Transcriptional Regulation

An increasing number of arguments, including altered microRNA expression, support the idea that post-transcriptional deregulation participates in gene disturbances found in diseased tissues. To evaluate this hypothesis, we developed a method which facilitates post-transcriptional investigations in a wide range of human cells and experimental conditions. This method, called FunREG (functional, integrated and quantitative method to measure post-transcriptional regulation), connects lentiviral transduction with a fluorescent reporter system and quantitative PCR. Using FunREG, we efficiently measured post-transcriptional regulation mediated either by selected RNA sequences or regulatory factors (microRNAs), and then evaluated the contribution of mRNA decay and translation efficiency in the observed regulation. We demonstrated the existence of gene-specific post-transcriptional deregulation in liver tumour cells, and also reported a molecular link between a transcript variant abrogating HDAC6 (histone deacetylase 6) regulation by miR-433 and a rare familial genetic disease. Because FunREG is sensitive, quantitative and easy to use, many applications can be envisioned in fundamental and pathophysiological research.

scholarly journals Dynamics of transcriptional and post-transcriptional regulation

2020 ◽  
Author(s):  
Mattia Furlan ◽  
Stefano de Pretis ◽  
Mattia Pelizzola
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Metabolic Labeling ◽  
Degradation Kinetic ◽  
Regulatory Processes ◽  
Transcriptional Regulatory ◽  
Kinetic Rates ◽  
Complex Scenario ◽  
Post Transcriptional Regulation ◽  
Transcriptional Output

Abstract Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and degradation kinetic rates collectively determine the abundance of each gene’s RNA. It has become clear that the same transcriptional output can correspond to different combinations of the kinetic rates. This underscores the fact that markedly different modes of gene expression regulation exist, each with profound effects on a gene’s ability to modulate its own expression. This review describes the development of the experimental and computational approaches, including RNA metabolic labeling and mathematical modeling, that have been disclosing the mechanisms underlying complex transcriptional programs. Current limitations and future perspectives in the field are also discussed.

scholarly journals 54Mn2+ as a tracer of the polymerization of actin. Intermediate oligomers condense to give F-actin

1984 ◽  
Vol 223 (3) ◽  
pp. 571-576 ◽  
Author(s):  
E Grazi
Keyword(s):  
Steady State ◽  
Association Constant ◽  
Time Course ◽  
Protein Analysis ◽  
Effective Mechanism ◽  
Experimental Conditions ◽  
Initial Concentration ◽  
Direct Condensation

Mg2+, at submicromolar concentrations, is needed for the nucleation of actin [Maruyama (1981) J. Biol. Chem. 256, 1060-1062]. I show here that Mn2+ fulfils the same function. It binds to oligomers present in the ATP-G-actin solutions with a ratio of 2-3 Mn2+ ions per 100 actin monomers and with an association constant of 0.66 × 10(10) M-1 at pH 8.2 at 25 degrees C. The time course of the binding of Mn2+ to polymerizing actin is not affected by the initial concentration of the protein. Analysis of the distribution of the binding shows that, both in the large oligomeric species and in the polymers, 1 Mn2+ ion is bound for every 14-25 actin monomers, whereas in the smaller oligomeric species 1 Mn2+ ion is bound for every 4 actin monomers. The proposal is made that Mn2+ stabilizes actin nuclei and decreases the concentration of the monomers at the steady state. It is also proposed that, at least in some experimental conditions, the direct condensation of oligomers of intermediate length is an effective mechanism of F-actin formation.

scholarly journals Transcriptional Regulation of MECP2E1-E2 Isoforms and BDNF by Metformin and Simvastatin through Analyzing Nascent RNA Synthesis in a Human Brain Cell Line

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1253
Author(s):  
Marjorie Buist ◽  
David Fuss ◽  
Mojgan Rastegar
Keyword(s):  
Transcriptional Regulation ◽  
Steady State ◽  
Human Brain ◽  
Cell Line ◽  
Rna Synthesis ◽  
Binding Protein ◽  
Brain Cell ◽  
Serum Starvation ◽  
Nascent Rna ◽  
The Impact

Methyl CpG binding protein 2 (MeCP2) is the main DNA methyl-binding protein in the brain that binds to 5-methylcytosine and 5-hydroxymethyl cytosine. MECP2 gene mutations are the main origin of Rett Syndrome (RTT), a neurodevelopmental disorder in young females. The disease has no existing cure, however, metabolic drugs such as metformin and statins have recently emerged as potential therapeutic candidates. In addition, induced MECP2-BDNF homeostasis regulation has been suggested as a therapy avenue. Here, we analyzed nascent RNA synthesis versus steady state total cellular RNA to study the transcriptional effects of metformin (an anti-diabetic drug) on MECP2 isoforms (E1 and E2) and BNDF in a human brain cell line. Additionally, we investigated the impact of simvastatin (a cholesterol lowering drug) on transcriptional regulation of MECP2E1/E2-BDNF. Metformin was capable of post-transcriptionally inducing BDNF and/or MECP2E1, while transcriptionally inhibiting MECP2E2. In contrast simvastatin significantly inhibited BDNF transcription without significantly impacting MECP2E2 transcripts. Further analysis of ribosomal RNA transcripts confirmed that the drug neither individually nor in combination affected these fundamentally important transcripts. Experimental analysis was completed in conditions of the presence or absence of serum starvation that showed minimal impact for serum deprival, although significant inhibition of steady state MECP2E1 by simvastatin was only detected in non-serum starved cells. Taken together, our results suggest that metformin controls MECP2E1/E2-BDNF transcriptionally and/or post-transcriptionally, and that simvastatin is a potent transcriptional inhibitor of BDNF. The transcriptional effect of these drugs on MECP2E1/E2-BDNF were not additive under these tested conditions, however, either drug may have potential application for related disorders.

scholarly journals Post-Transcriptional Circadian Regulation in Macrophages Organizes Temporally Distinct Immunometabolic States

2020 ◽  
Author(s):  
Emily J Collins ◽  
Mariana P Cervantes-Silva ◽  
George A Timmons ◽  
James R O’Siorain ◽  
Annie M Curtis ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Metabolic Regulation ◽  
Time Course ◽  
Mitochondrial Morphology ◽  
Circadian Regulation ◽  
Atp Production ◽  
Post Transcriptional Regulation ◽  
The Impact

SUMMARYOur core timekeeping mechanism, the circadian clock, regulates an astonishing amount of cellular physiology and behavior, playing a vital role in organismal fitness. While the mechanics of circadian control over cellular regulation can in part be explained by the transcriptional activation stemming from the positive arm of the clock’s transcription-translation negative feedback loop, research has shown that extensive circadian regulation occurs beyond transcriptional activation in fungal species and data suggest that this post-transcriptional regulation may also be preserved in mammals. To determine the extent to which circadian output is regulated post-transcriptionally in mammalian cells, we comprehensively profiled the transcriptome and proteome of murine bone marrow-derived macrophages in a high resolution, sample rich time course. We found that only 15% of the circadian proteome had corresponding oscillating mRNA and this regulation was cell intrinsic. Ontological analysis of oscillating proteins revealed robust temporal enrichment for protein degradation and translation, providing potential insights into the source of this extensive post-transcriptional regulation. We noted post-transcriptional temporal-gating across a number of connected metabolic pathways. This temporal metabolic regulation further corresponded with rhythms we observed in ATP production, mitochondrial morphology, and phagocytosis. With the strong interconnection between cellular metabolic states and macrophage phenotypes/responses, our work demonstrates that post-transcriptional circadian regulation in macrophages is broadly utilized as a tool to confer time-dependent immune function and responses. As macrophages coordinate many immunological and inflammatory functions, an understanding of this regulation provides a framework to determine the impact of circadian regulation on a wide array of disease pathologies.

The time course of transcriptional regulation of the ‘constitutive’ cyclooxgenase-1 gene by Helicobacter pylori†

2001 ◽  
Vol 120 (5) ◽  
pp. A659-A659 ◽  
Author(s):  
M BYRNE ◽  
P CORCORAN ◽  
K SHEEHAN ◽  
J ATHERTON ◽  
D FITZGERALD ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Transcriptional Regulation ◽  
Time Course

A Simple Method for Estimation of Lipoxygenase and Cyclo-Oxygenase Pathways in Human Platelets - the Use of Thiobarbituric Acid Reaction

1980 ◽  
Vol 44 (02) ◽  
pp. 111-114 ◽  
Author(s):  
Hiroshi Takayama ◽  
Minoru Okuma ◽  
Haruto Uchino
Keyword(s):  
Time Course ◽  
Normal Values ◽  
Human Platelet ◽  
Thiobarbituric Acid ◽  
Human Platelets ◽  
Optimal Conditions ◽  
Experimental Conditions ◽  
Simple Method ◽  
Acid Reaction ◽  
Optimal Ph

SummaryTo develop a simple method for estimation of platelet lipoxygenase (PLO) and cyclo-oxygenase (PCO) pathways, the arachidonic acid (AA) metabolism of human platelet was investigated under various experimental conditions by the use of the thiobarbituric acid (TBA) reaction and a radioisotope technique. A TBA-reactive substance different from malondialdehyde (MDA) via PCO pathway was detected and shown to be derived from the PLO pathway. Since the optimal pH and time course of its formation were different from those of MDA formation via PCO pathway, PLO and PCO pathways were estimated by quantitating the TBA-reactive substances produced by the incubation of AA either with aspirin-treated platelets or with untreated ones, respectively, each under optimal conditions. Normal values expressed in terms of nmol MDA/108 platelets were 1.17±0.34 (M±SD, n = 31) and 0.79±0.15 (n = 31) for PLO and PCO pathways, respectively.

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