mRNA Production Rate Research Articles

Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos

An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. We systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, we quantify the rate of RNA Pol II elongation in live Drosophila embryos. We find that promoters and exonic gene lengths have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, we observe in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. Our study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production.

A Comparison Between Stochastic and Deterministic Models of a Biological Oscillator

We look at the behavior of biological oscillators, specifically analyzing a genetic circuit that has oscillatory behavior. Implementing the system in deterministic and stochastic models, we compare the two models using various tests and analyze the effect of stochastic noise on these oscillations. We first investigate the effect of volume and find that at small system volumes, stochastic events cause the system to exhibit more sporadic oscillations and a longer period of oscillation. Next, we develop classification systems for discerning the boundary in the parameter space where the model begins to show oscillations. This is more challenging in the stochastic model, due to persistent fluctuations. We compare different methods of analysis of our deterministic and stochastic models and visualize the conditions which lead to oscillation in each model. We find that the parameter range of oscillation is larger in the stochastic model than in the deterministic model. Finally, we find an increased rate of mRNA production can create greater noise in a system and amplify the difference between a stochastic and deterministic system.

Exploring the role of eRNA in regulating gene expression.

eRNAs as the products of enhancers can regulate gene expression via various possible ways, but which regulation way is more reasonable is debatable in biology, and in particular, how eRNAs impact gene expression remains unclear. Here we introduce a mechanistic model of gene expression to address these issues. This model considers three possible regulation ways of eRNA: Type-I by which eRNA regulates transcriptional activity by facilitating the formation of enhancer-promoter (E-P) loop, Type-II by which eRNA directly promotes the mRNA production rate, and mixed regulation (i.e., the combination of Type-I and Type-II). We show that with the increase of the E-P loop length, mRNA distribution can transition from unimodality to bimodality or vice versa in all the three regulation cases. However, in contrast to the other two regulations, Type-II regulation can lead to the highest mean mRNA level and the lowest mRNA noise, independent of the E-P loop length. These results would not only reveal the essential mechanism of how eRNA regulates gene expression, but also imply a new mechanism for phenotypic switching, namely the E-P loop can induce phenotypic switching.

R-loop induced G-quadruplex in non-template promotes transcription by successive R-loop formation

G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.

An in-silico human cell model reveals the influence of spatial organization on RNA splicing.

Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.

Multimodal transcriptional control of pattern formation in embryonic development

Predicting how interactions between transcription factors and regulatory DNA sequence dictate rates of transcription and, ultimately, drive developmental outcomes remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, we dissect the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, we found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, we discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these regulatory strategies (bursting and the time window) respond in different ways to input transcription factor concentrations, suggesting that the stripe is shaped by the interplay of 2 distinct underlying molecular processes.

A Computational Human Whole-Cell Model Reveals the Effects of Spatial Organization on RNA Splicing

Spatial organization is a fundamental characteristic of cells, achieved by utilizing both membrane-bound and non-bound organelles. We construct a spatially resolved human whole-cell (HeLa) model from experiment-based structural, morphological and reaction network data to describe the mRNA splicing process and dynamics of splicing particles. We performed stochastic simulations for up to 15 minutes of biological time of the entire cell. We find that the number of nuclear pore complexes controls the number of assembled splicing particles; that even a slight increase of splicing particle localization in nuclear speckles (non-membrane-bound organelles) leads to a disproportionate enhancement of mRNA splicing and reduction in the transcript noise; and that compartmentalization is critical for the yield of correctly assembled splicing particles. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, further emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and subcompartments, provides an adaptable foundation to study many cellular processes which are strongly modulated by spatio-temporal heterogeneity.

Linear Integration of ERK Activity Predominates over Persistence Detection in Fra-1 Regulation.

ERK signaling regulates the expression of target genes, but it is unclear how ERK activity dynamics are interpreted. Here, we investigate this question using simultaneous, live, single-cell imaging of two ERK activity reporters and expression of Fra-1, a target gene controlling epithelial cell identity. We find that Fra-1 is expressed in proportion to the amplitude and duration of ERK activity. In contrast to previous "persistence detector" and "selective filter" models in which Fra-1 expression only occurs when ERK activity persists beyond a threshold duration, our observations demonstrate that the network regulating Fra-1 expression integrates total ERK activity and responds to it linearly. However, exploration of a generalized mathematical model of the Fra-1 coherent feedforward loop demonstrates that it can perform either linear integration or persistence detection, depending on the basal mRNA production rate and protein production delays. Our data indicate that significant basal expression and short delays cause Fra-1 to respond linearly to integrated ERK activity.

Robust kinetics of an RNA virus: Transcription rates are set by genome levels.

In order to persist in nature, RNA viruses have evolved strategies to grow in diverse host environments. To better understand how such strategies might work, we used qRT-PCR to measure viral RNA species during cellular infections by a model RNA virus, vesicular stomatitis virus (VSV). Absolute levels of the VSV major transcript and genome were measured for infections in BHK and PC3 cells, across different multiplicities of infection (MOI 1, 10, 100), in the absence or presence of protein synthesis, as well as in cells in an interferon-activated anti-viral state. While viral genome replication was delayed in more resistant host cells, kinetic modeling of these data revealed a simple linear relationship between the mRNA production rate and genome levels under all tested conditions. These results indicate that while viral transcription and genome replication both depend on the availability of the viral RNA-dependent RNA polymerase and host cellular resources, transcription proceeds without apparent limits on these resources.

Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq.

4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes ∼4 d to complete, with deep sequencing requiring an additional ∼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.

Quantitative Imaging of Transcription in Living Drosophila Embryos Links Polymerase Activity to Patterning

Spatiotemporal patterns of gene expression are fundamental to every developmental program. The resulting macroscopic domains have been mainly characterized by their levels of gene products. However, the establishment of such patterns results from differences in the dynamics of microscopic events in individual cells such as transcription. It is unclear how these microscopic decisions lead to macroscopic patterns, as measurements in fixed tissue cannot access the underlying transcriptional dynamics. In vivo transcriptional dynamics have long been approached in single-celled organisms, but never in a multicellular developmental context. Here, we directly address how boundaries of gene expression emerge in the Drosophila embryo by measuring the absolute number of actively transcribing polymerases in real time in individual nuclei. Specifically, we show that the formation of a boundary cannot be quantitatively explained by the rate of mRNA production in each cell, but instead requires amplification of the dynamic range of the expression boundary. This amplification is accomplished by nuclei randomly adopting active or inactive states of transcription, leading to a collective effect where the fraction of active nuclei is modulated in space. Thus, developmental patterns are not just the consequence of reproducible transcriptional dynamics in individual nuclei, but are the result of averaging expression over space and time.

Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate

Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation.

Mechanistic population modeling of diabetes disease progression in Goto‐Kakizaki rat muscle

Pyruvate dehydrogenase kinase 4 (PDK4) is a lipid status responsive gene involved in muscle fuel selection. Evidence is mounting in support of the therapeutic potential of PDK4 inhibitors to treat diabetes. Factors that regulate PDK4 mRNA expression include plasma corticosterone, insulin and free fatty acids. The objective was to determine the impact of those plasma factors on PDK4 mRNA and to develop and validate a population mathematical model to differentiate aging, diet and disease effects on muscle PDK4 expression. The Goto-Kakizaki (GK) rat, a polygenic non-obese model of type 2 diabetes, was used as the diabetic animal model. Muscle PDK4 mRNA expression was examined by real-time QRTPCR. Groups of GK rats along with controls fed with either a normal or high fat diet were killed at 4, 8, 12, 16 and 20 weeks of age. Plasma corticosterone, insulin and free fatty acids were measured. The proposed mechanism-based model successfully described the age, disease and diet effects and the relative contribution of these plasma regulators on PDK4 mRNA expression. Muscle growth reduced the PDK4 mRNA production rate by 14% per gram increase. The high fat diet increased the initial production rate constant in GK rats by 2.19-fold. The model indicated that corticosterone had a moderate effect and PDK4 was more sensitive to free fatty acid than insulin fluxes, which was in good agreement with the literature data.

Gene Regulation: Global Transcription Rates Scale with Size

Is bigger better? Scientists have long puzzled over the potential relationship between cell size and the rate of mRNA production. A recent report builds a strong case that global transcription rates scale with size.

Cotranscriptional Splicing Potentiates the mRNA Production from a Subset of Estradiol-Stimulated Genes

While early steps of gene expression, such as transcription preinitiation, are known to often be rate limiting and to be regulated by such stimuli as steroid hormones, the potential impact of downstream steps, including splicing, on the mRNA production rate is unknown. In this work, we studied the effects of the transcriptional stimulus estradiol on cyclin D1, PS2, and c-fos gene expression by measuring the levels of RNA polymerase II on the DNA templates, the levels of nascent transcripts associated with RNA polymerase II, and the levels of unspliced, partially spliced, and fully spliced RNAs. We demonstrated that the efficiency of cotranscriptional splicing of the first intron was higher in the case of cyclin D1 than with PS2 and potentiated the cyclin D1 mRNA production rate. The mechanism involved in cotranscriptional splicing depended on the level of serine 5 phosphorylation of RNA polymerase II at the gene 5' end and on the recruitment of CBP80, one of the two subunits of the cap binding complex, which stimulates splicing of the promoter-proximal intron. Our data indicate that mRNA production from a subset of estradiol-stimulated genes, such as cyclin D1, could occur in a very efficient "assembly line." In contrast, we demonstrated for the first time that despite a strong transcriptional activation of the PS2 gene, the production of mRNA is not optimized owing to inefficient cotranscriptional RNA processing.

Modeling stochastic gene expression under repression

Intrinsic transcriptional noise induced by operator fluctuations is investigated with a simple spin-like stochastic model. The effects of transcriptional fluctuations in protein synthesis are probed by coupling transcription and translation by an amplificative interaction. In the presence of repression a new term contributes to the noise, which depends on the rate of mRNA production. If the switch decay time is small compared with the mRNA life time, the noise is also small. In general the damping of protein production by a repressive agent occurs linearly but fluctuations can show a maximum at intermediate repression. The discrepancy among the switch decay time, the mRNA degradation, and protein degradation is crucial for the repressive control in translation without large fluctuations. The noise profiles obtained here are in quantitative agreement with recent experiments.

Regulation of ornithine aminotransferase mRNA levels in rat kidney by estrogen and thyroid hormone.

The regulation of the mitochondrial matrix enzyme, ornithine aminotransferase, by estrogen and triiodothyronine (T3) in rat kidney was examined using a cloned cDNA probe and in vitro translation of poly(A+) RNA. After a single, acute dose of either 17 beta-estradiol or T3, the rate of enzyme synthesis and the levels of translatable and hybridizable ornithine aminotransferase mRNA all increase in parallel. Levels of hybridizable mRNA were estimated by hybridization of randomly 32P-labeled RNA to filter-bound plasmid DNA. Maximal levels of induction by estrogen and T3 were about 15- and 3-fold, respectively. Lag times of at least 5 h and less than 3 h were observed for induction by T3 and estrogen. T3 and estrogen exert a synergistic effect in increasing ornithine aminotransferase mRNA levels. 16 h after T3 administration and 24 h after estrogen administration, a 1.6- and 13-fold increase in mRNA levels were observed. Both of these treatments in combination for the indicated time periods resulted in a 21-fold increase in ornithine aminotransferase mRNA. From the mRNA accumulation curves, half-lives of 10 to 14 h and 12 to 16 h were estimated for the mRNA after estrogen and T3 induction, respectively. These similar half-lives suggest that an increase in the rate of mRNA production is primarily responsible for the induction observed after estrogen administration.

Regulation of dihydrofolate reductase gene transcription in methotrexate-resistant mouse fibroblasts.

We have used a methotrexate-resistant mouse 3T6 cell line (M50L3) that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of about 300 to study the regulation of DHFR hnRNA synthesis. We have previously shown that when resting (G0) M50L3 cells are serum stimulated to reenter the cell cycle, the amount and rate of synthesis of DHFR and the content of DHFR mRNA all begin to increase as the cells enter the S phase of the cell cycle. The increase in DHFR mRNA content is due to an increase in the rate of mRNA production. In the presnt study, we have used the technique of DNA-excess filter hybridization to determine the rate of synthesis of DHFR hnRNA relative to total hnRNA at various times following serum stimulation. We found that the relative rate of DHFR hnRNA synthesis began to increase at about the same time (6 hours), and increased to about the same extent (three to fourfold by 15 hours following stimulation) as we observed previously for DHFR mRNA production. This suggests that the increase in DHFR mRNA production (and consequently DHFR gene expression) is controlled primarily, if not exclusively, at the level of transcription. We also studied the effect of addition of high concentrations of dibutyryl cAMP and theophylline on DHFR gene transcription. We found that addition of these drugs at the time of stimulation completely blocked the increase in DHFR hnRNA synthesis as well as entry into S phase. Addition of the drugs at either 13 or 20 hours following stimulation led to a rapid decrease in DHFR hnRNA synthesis. The drugs were found to have little effect on the ability of the cells to complete S phase when they were added at 13 hours following stimulation. Our results suggest that high intracellular concentrations of cAMP may affect DHFR gene expression not only by preventing the progression of cells through the G1 phase of the cell cycle but also by affecting the rate of DHFR gene transcription in a more direct manner.