Rate Of RNA Synthesis Research Articles

Metabolic labeling of RNA using ribonucleoside analogs enables the evaluation of RNA synthesis and degradation rates.

Long noncoding RNAs (lncRNAs) are transcripts exceeding 200 nucleotides that do not encode proteins. Despite lacking protein-coding capabilities, lncRNAs play crucial roles in cellular processes, including gene-expression modulation and structural maintenance. The study of lncRNAs has evolved significantly since 2009, with advancements in analytical methodologies providing new insights into their functions and dynamics. Key developments include BRIC-Seq, SLAM-Seq, TUC-Seq, TimeLapse-seq, and Dyrec-Seq. These methodologies have enabled researchers to investigate lncRNA behavior under various conditions, including cellular stress responses and complex biologic systems. Future challenges include developing comprehensive techniques for identifying lncRNA-interacting proteins and advancing in vivo methodologies using model organisms. As the field progresses, integrating these technologies will enhance our understanding of lncRNA biology, potentially leading to novel therapeutic strategies and deeper insights into gene-regulation mechanisms.

The first chicken oocyte nucleus whole transcriptomic profile defines the spectrum of maternal mRNA and non-coding RNA genes transcribed by the lampbrush chromosomes.

Lampbrush chromosomes, with their unusually high rate of nascent RNA synthesis, provide a valuable model for studying mechanisms of global transcriptome up-regulation. Here, we obtained a whole-genomic profile of transcription along the entire length of all lampbrush chromosomes in the chicken karyotype. With nuclear RNA-seq, we obtained information about a wider set of transcripts, including long non-coding RNAs retained in the nucleus and stable intronic sequence RNAs. For a number of protein-coding genes, we visualized their nascent transcripts on the lateral loops of lampbrush chromosomes by RNA-FISH. The set of genes transcribed on the lampbrush chromosomes is required for basic cellular processes and is characterized by a broad expression pattern. We also present the first high-throughput transcriptome characterization of miRNAs and piRNAs in chicken oocytes at the lampbrush chromosome stage. Major targets of predicted piRNAs include CR1 and long terminal repeat (LTR) containing retrotransposable elements. Transcription of tandem repeat arrays was demonstrated by alignment against the whole telomere-to-telomere chromosome assemblies. We show that transcription of telomere-derived RNAs is initiated at adjacent LTR elements. We conclude that hypertranscription on the lateral loops of giant lampbrush chromosomes is required for synthesizing large amounts of transferred to the embryo maternal RNA for thousands of genes.

Conformational Dynamics of Influenza A Virus Ribonucleoprotein Complexes during RNA Synthesis.

Viral ribonucleoproteins (vRNPs) are the cornerstones of viral proliferation, as they form the macromolecular complexes that are responsible for the transcription and replication of most single-stranded RNA viruses. The influenza A virus (IAV) polymerase catalyzes RNA synthesis within the context of vRNPs where genomic viral RNA (vRNA) is packaged by the viral nucleoprotein (NP). We used high-speed atomic force microscopy and electron microscopy to study the conformational dynamics of individual IAV recombinant RNPs (rRNPs) during RNA synthesis. The rRNPs present an annular organization that allows for the real-time tracking of conformational changes in the NP-vRNA template caused by the advancing polymerase. We demonstrate that the rRNPs undergo a well-defined conformational cycle during RNA synthesis, which can be interpreted in light of previous transcription models. We also present initial estimations of the average RNA synthesis rate in the rRNP and its dependence on the nucleotide concentration and stability of the nascent RNA secondary structures. Furthermore, we provide evidence that rRNPs can perform consecutive cycles of RNA synthesis, accounting for their ability to recycle and generate multiple copies of RNA.

Genome-scale chromatin binding dynamics of RNA Polymerase II general transcription machinery components

A great deal of work has revealed, in structural detail, the components of the preinitiation complex (PIC) machinery required for initiation of mRNA gene transcription by RNA polymerase II (Pol II). However, less-well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining how rates of in vivo RNA synthesis are established. We used competition ChIP in budding yeast to obtain genome-scale estimates of the residence times for five general transcription factors (GTFs): TBP, TFIIA, TFIIB, TFIIE and TFIIF. While many GTF-chromatin interactions were short-lived ( < 1 min), there were numerous interactions with residence times in the range of several minutes. Sets of genes with a shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. The datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism, offering a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription.

Comparison of Transcription Elongation Rates of Three RNA Polymerases in Real Time.

RNA polymerases (RNAPs) across the bacterial kingdom have retained a conserved structure and function. In spite of the remarkable similarity of the enzyme in different bacteria, a wide variation is found in the promoter-polymerase interaction, transcription initiation, and termination. However, the transcription elongation was considered to be a monotonic process, although the rate of elongation could vary in different bacteria. Such variations in RNAP elongation rates could be important to fine-tune the transcription, which in turn would influence cellular metabolism and growth rates. Here, we describe a quantitative study to measure the transcription rates for the RNAPs from three bacteria, namely, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, which exhibit different growth kinetics. The RNA synthesis rates of the RNAPs were calculated from the real-time elongation kinetic profile using surface plasmon resonance through a computational flux flow model. The computational model revealed the modular process of elongation, with different rate profiles for the three RNAPs. Notably, the transcription elongation rates of these RNAPs followed the trend in the growth rates of these bacteria.

GrandR: a comprehensive package for nucleotide conversion RNA-seq data analysis

Metabolic labeling of RNA is a powerful technique for studying the temporal dynamics of gene expression. Nucleotide conversion approaches greatly facilitate the generation of data but introduce challenges for their analysis. Here we present grandR, a comprehensive package for quality control, differential gene expression analysis, kinetic modeling, and visualization of such data. We compare several existing methods for inference of RNA synthesis rates and half-lives using progressive labeling time courses. We demonstrate the need for recalibration of effective labeling times and introduce a Bayesian approach to study the temporal dynamics of RNA using snapshot experiments.

Learning consistent subcellular landmarks to quantify changes in multiplexed protein maps

Highly multiplexed imaging holds enormous promise for understanding how spatial context shapes the activity of the genome and its products at multiple length scales. Here, we introduce a deep learning framework called CAMPA (Conditional Autoencoder for Multiplexed Pixel Analysis), which uses a conditional variational autoencoder to learn representations of molecular pixel profiles that are consistent across heterogeneous cell populations and experimental perturbations. Clustering these pixel-level representations identifies consistent subcellular landmarks, which can be quantitatively compared in terms of their size, shape, molecular composition and relative spatial organization. Using high-resolution multiplexed immunofluorescence, this reveals how subcellular organization changes upon perturbation of RNA synthesis, RNA processing or cell size, and uncovers links between the molecular composition of membraneless organelles and cell-to-cell variability in bulk RNA synthesis rates. By capturing interpretable cellular phenotypes, we anticipate that CAMPA will greatly accelerate the systematic mapping of multiscale atlases of biological organization to identify the rules by which context shapes physiology and disease.

Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the Drosophila larval brain.

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggest the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.

An approach to learn regulation to maximize growth and entropy production rates in metabolism

Elucidating cell regulation remains a challenging task due to the complexity of metabolism and the difficulty of experimental measurements. Here we present a method for prediction of cell regulation to maximize cell growth rate while maintaining the solvent capacity of the cell. Prediction is formulated as an optimization problem using a thermodynamic framework that can leverage experimental data. We develop a formulation and variable initialization procedure that allows for computing solutions of the optimization with an interior point method. The approach is applied to photoheterotrophic growth of Rhodospirilium rubrum using ethanol as a carbon source, which has applications to biosynthesis of ethylene production. Growth is captured as the rate of synthesis of amino acids into proteins, and synthesis of nucleotide triphoshaptes into RNA and DNA. The method predicts regulation that produces a high rate of protein and RNA synthesis while DNA synthesis is reduced close to zero in agreement with production of DNA being turned off for much of the cell cycle.

Escherichia coli protein synthesis is limited by mRNA availability rather than ribosomal capacity during phosphate starvation.

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. Escherichia coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Feedback from nuclear RNA on transcription promotes robust RNA concentration homeostasis in human cells.

RNA concentration homeostasis involves coordinating RNA abundance and synthesis rates with cell size. Here, we study this in human cells by combining genome-wide perturbations with quantitative single-cell measurements. Despite relative ease in perturbing RNA synthesis, we find that RNA concentrations generally remain highly constant. Perturbations that would be expected to increase nuclear mRNA levels, including those targeting nuclear mRNA degradation or export, result in downregulation of RNA synthesis. This is associated with reduced abundance of transcription-associated proteins and protein states that are normally coordinated with RNA production in single cells, including RNA polymerase II (RNA Pol II) itself. Acute perturbations, elevation of nuclear mRNA levels, and mathematical modeling indicate that mammalian cells achieve robust mRNA concentration homeostasis by the mRNA-based negative feedback on transcriptional activity in the nucleus. This ultimately acts to coordinate RNA Pol II abundance with nuclear mRNA degradation and export rates and may underpin the scaling of mRNA abundance with cell size.

Step-by-Step Regulation of Productive and Abortive Transcription Initiation by Pyrophosphorolysis

An understanding of the kinetics and mechanism of bacterial transcription initiation is needed to understand regulation of gene expression and advance fields from antibiotic discovery to promoter design. The step-by-step forward kinetics and mechanism of initiation and RNA-DNA hybrid growth, made irreversible by omitting pyrophosphate (PPi) byproduct, were determined recently for E. coli RNA polymerase (RNAP)-λPR promoter complexes. Strong position-dependences of overall rate constants (kcat/Km analogs) for each nucleotide-addition step were observed because of coupling of hybrid growth to disruption of promoter contacts, bubble closing, and RNAP escape. Here we investigate reversal of these steps (pyrophosphorolysis) at PPi concentrations ([PPi]) found in exponentially-growing cells. We quantify [PPi] effects on the amount and rate of synthesis of long (>10-mer, post-escape) and short (stalled, abortive) RNA to determine how PPi regulates initiation. Physiological [PPi] makes uridine incorporation and some other initiation steps significantly reversible. Physiological [PPi] reduces the fraction of RNAP-promoter complexes that productively initiate and the rate of RNA synthesis per productive complex, while increasing the fraction of complexes that abortively initiate, affecting abortive rates, and shifting the abortive-product distribution to shorter RNAs. Pyrophosphorolysis rates for some initiation complexes are orders of magnitude larger than for removal of the same nucleotide from elongation complexes because of the strong bias toward the pre-translocated state in initiation, and exhibit even stronger dependences on nucleotide identity (pyrimidine ≫ purine). Because cytoplasmic [PPi] is much higher in exponential-phase than stationary-phase cells, these [PPi] effects on initiation rates and amounts of RNA synthesis must be physiologically-relevant.

Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy

Older individuals undergo more frequent inpatient hospitalizations and bedrest compared to younger persons, causing rapid losses of muscle mass and strength. Evidence suggests that only aged muscle fails to completely recover following disuse despite aged and adult muscle having similar levels of muscle loss. Current efforts to improve muscle recovery in older individuals are commonly aimed at increasing myofibrillar protein synthesis via mTOR stimulation despite recent evidence demonstrating that old muscle has chronically elevated levels of mTORC1 activity. We hypothesized that old muscle fails to fully recover muscle mass due to impaired proteostasis and not due limitations in protein synthesis.MethodsAdult (10 month) and old (30 month) F344BN rats were hindlimb unloaded (HU) for 14‐days to induce atrophy, followed by subsequent reloading (RE) to study muscle regrowth. During RE the rats were labeled with deuterium oxide (D2O) for 7, 15, 30, 45 or 60 days (n=4‐6 per timepoint per group) to determine bulk and individual protein synthesis and RNA synthesis. We also assessed muscle mass, fiber size by immunohistochemistry, mTORC1 signaling by western blot, and aggregate formation by tissue fractionation.ResultsAdult and old gastrocnemius (GA) muscle had significant losses of muscle mass (359 mg ±55 and 724 mg ±60 loss respectively,p&lt;0.05) and fiber size (1296 um2±202 and 1480 um2 ±142 loss respectively, p&lt;0.01) with HU. While adult muscle fully recovered GA mass and fiber size by day 15, old GA muscle did not fully recover during the entire 60 days of RE (18% CSA and 11% mass loss still present). Despite limited mass and fiber size recovery in old muscle, GA myofibrillar protein synthesis rates (1.45 ±0.18 vs 2.28 ±0.24, FSR%, p&lt;0.05) and mTORC1‐related signaling were higher in old muscle compared to adult. Additionally, GA RNA synthesis rates (2.79 ±0.139 vs 5.18 ±0.684, p&lt;0.05) and RNA concentration (183 ng ±3.6 vs 249 ng ±13.9, p&lt;0.05), which are markers of translational capacity, were also elevated in old muscle relative to adult during RE. Old muscle had higher levels of insoluble protein aggregates, a marker of impaired proteostasis, during RE compared to adult (1.2 ±0.09 vs 1.5 ±012 fold increase, p&lt;0.05). Lastly, we assessed individual protein synthesis rates of the whole muscle proteome and discovered that old GA muscle had a larger number of proteins with increased synthesis rates compared to adult. In conclusion, these data strongly suggest that limitations in old muscle to recover after disuse are not due to limitations in protein synthesis but are instead due to impaired proteostasis with age. Therefore, understanding how proteostasis responds during these periods surrounding unloading in old muscle are critical to improve muscle recovery after disuse.

Estimating RNA dynamics using one time point for one sample in a single-pulse metabolic labeling experiment

BackgroundOver the past decade, experimental procedures such as metabolic labeling for determining RNA turnover rates at the transcriptome-wide scale have been widely adopted and are now turning to single cell measurements. Several computational methods to estimate RNA synthesis, processing and degradation rates from such experiments have been suggested, but they all require several RNA sequencing samples. Here we present a method that can estimate those three rates from a single sample.MethodsOur method relies on the analytical solution to the Zeisel model of RNA dynamics. It was validated on metabolic labeling experiments performed on mouse embryonic stem cells. Resulting degradation rates were compared both to previously published rates on the same system and to a state-of-the-art method applied to the same data.ResultsOur method is computationally efficient and outputs rates that correlate well with previously published data sets. Using it on a single sample, we were able to reproduce the observation that dynamic biological processes tend to involve genes with higher metabolic rates, while stable processes involve genes with lower rates. This supports the hypothesis that cells control not only the mRNA steady-state abundance, but also its responsiveness, i.e., how fast steady state is reached. Moreover, degradation rates obtained with our method compare favourably with the other tested method.ConclusionsIn addition to saving experimental work and computational time, estimating rates for a single sample has several advantages. It does not require an error-prone normalization across samples and enables the use of replicates to estimate uncertainty and assess sample quality. Finally the method and theoretical results described here are general enough to be useful in other contexts such as nucleotide conversion methods and single cell metabolic labeling experiments.

Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol—a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

Distinct transcription kinetics of pluripotent cell states

Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT‐seq) to define both coding and non‐coding transcription units (TUs) in these three pluripotent states and combine TT‐seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome‐wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome‐wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome‐wide changes in transcription kinetics invoke ground and paused pluripotent states.

Determining the contributions of protein synthesis and breakdown to muscle atrophy requires non-steady-state equations.

BackgroundAgeing and cachexia cause a loss of muscle mass over time, indicating that protein breakdown exceeds protein synthesis. Deuterium oxide (D2O) is used for studies of protein turnover because of the advantages of long‐term labelling, but these methods introduce considerations that have been largely overlooked when studying conditions of protein gain or loss. The purpose of this study was to demonstrate the importance of accounting for a change in protein mass, a non‐steady state, during D2O labelling studies while also exploring the contribution of protein synthesis and breakdown to denervation‐induced muscle atrophy.MethodsAdult (6 months) male C57BL/6 mice (n = 14) were labelled with D2O for a total of 7 days following unilateral sciatic nerve transection to induce denervation of hindlimb muscles. The contralateral sham limb and nonsurgical mice (n = 5) were used as two different controls to account for potential crossover effects of denervation. We calculated gastrocnemius myofibrillar and collagen protein synthesis and breakdown assuming steady‐state or using non‐steady‐state modelling. We measured RNA synthesis rates to further understand ribosomal turnover during atrophy.ResultsGastrocnemius mass was less in denervated muscle (137 ± 9 mg) compared with sham (174 ± 15 mg; P < 0.0001) or nonsurgical control (162 ± 5 mg; P < 0.0001). With steady‐state calculations, fractional synthesis and breakdown rates (FSR and FBR) were lower in the denervated muscle (1.49 ± 0.06%/day) compared with sham (1.81 ± 0.09%/day; P < 0.0001) or nonsurgical control (2.27 ± 0.04%/day; P < 0.0001). When adjusting for change in protein mass, FSR was 4.21 ± 0.19%/day in denervated limb, whereas FBR was 4.09 ± 0.22%/day. When considering change in protein mass (ksyn), myofibrillar synthesis was lower in denervated limb (2.44 ± 0.14 mg/day) compared with sham (3.43 ± 0.22 mg/day; P < 0.0001) and non‐surgical control (3.74 ± 0.12 mg/day; P < 0.0001), whereas rate of protein breakdown (kdeg, 1/t) was greater in denervated limb (0.050 ± 0.003) compared with sham (0.019 ± 0.001; P < 0.0001) and nonsurgical control (0.023 ± 0.000; P < 0.0001). Muscle collagen breakdown was completely inhibited during denervation. There was a strong correlation (r = 0.83, P < 0.001) between RNA and myofibrillar protein synthesis in sham but not denervated muscle.ConclusionsWe show conflicting results between steady‐ and non‐steady‐state calculations on myofibrillar protein synthesis and breakdown during periods of muscle loss. We also found that collagen accumulation was largely from a decrease in collagen breakdown. Comparison between sham and non‐surgical control demonstrated a crossover effect of denervation on myofibrillar protein synthesis and ribosomal biogenesis, which impacts study design for unilateral atrophy studies. These considerations are important because not accounting for them can mislead therapeutic attempts to maintain muscle mass.

Rolling-circle and strand-displacement mechanisms for non-enzymatic RNA replication at the time of the origin of life

It is likely that RNA replication began non-enzymatically, and that polymerases were later selected to speed up the process. We consider replication mechanisms in modern viruses and ask which of these is possible non-enzymatically, using mathematical models and experimental data found in the literature to estimate rates of RNA synthesis and replication. Replication via alternating plus and minus strands is found in some single-stranded RNA viruses. However, if this occurred non-enzymatically it would lead to double-stranded RNA that would not separate. With some form of environmental cycling, such as temperature, salinity, or pH cycling, double-stranded RNA can be melted to form single-stranded RNA, although re-annealing of existing strands would then occur much faster than synthesis of new strands. We show that re-annealing blocks this form of replication at a very low concentration of strands. Other kinds of viruses synthesize linear double strands from single strands and then make new single strands from double strands via strand-displacement. This does not require environmental cycling and is not blocked by re-annealing. However, under non-enzymatic conditions, if strand-displacement occurs from a linear template, we expect the incomplete new strand to be almost always displaced by the tail end of the old strand through toehold-mediated displacement. A third kind of replication in viruses and viroids is rolling-circle replication which occurs via strand-displacement on a circular template. Rolling-circle replication does not require environmental cycling and is not prevented by toehold-mediated displacement. Rolling-circle replication is therefore expected to occur non-enzymatically and is a likely starting point for the evolution of polymerase-catalysed replication.

Day and night regulation of the HO-1/HSP32 synthesis in the harmful dinoflagellate Prorocentrum minimum: response to salinity stress

Potentially toxic dinoflagellate species Prorocentrum minimum (Pavillard) Schiller, 1933 colonized the brackish-water Baltic Sea in the early 1980s, where thereafter it has been forming regular harmful blooms. Heat shock proteins (HSP) play an important role in their ability to adapt to environmental stress. In this study, we evaluated how the synthesis of heme oxygenase-1 (HO-1/HSP32), an important part of cellular machinery for cell protection, changes in P. minimum under the influence of salinity stress during the day-night cycle. We identified sequences of the HO-1 homologs in the unannotated P. minimum transcriptomes, represented in the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database. Phylogenetic analysis showed that this protein clustered in a distinct clade and demonstrated an evolutionary relationship with the HO-1 homologs of other taxonomic groups. To identify P. minimum cells that express the HO-1 protein, we used fluorescent flow cytometry. In control conditions (17‰), the number of cells expressing this protein was 2.2 times higher during the day than at night, with the same average fluorescence intensity (1.7 units). During the daytime, the critical salinity conditions of 8‰ did not lead to significant changes in the number of cells expressing HO-1, while in the night culture this salinity shift caused a 2.7-fold increase in the number of cells expressing HO-1, and led to an increase in the intensity of fluorescence up to 2.0 units. The overall DNA and RNA synthesis rates were estimated by the incorporation of 3H-thymidine and 3H-uridine, respectively. Transcription and replication levels were significantly higher under critical salinity conditions (8‰) compared to the control (17‰) and typical sea salinity conditions (35‰). The greatest increase in the DNA synthesis rates (up to 2.7 times) was observed during the daytime in cells exposed to salinity stress at 8‰. In contrast, the greatest increase in the RNA synthesis rates (2.7 times) was registered in cells exposed to salinity of 8‰ at night. Thus, the replication activity of cells in response to salinity stress increased significantly during the day, while transcription levels reached their maximum values at night. These results indicate that a significant increase in the synthesis of both nucleic acids and the HO-1 stress protein can serve as a biomarker of the impact of environmental stress factors, such as salinity. This allowed concluding that the studied parameters demonstrate a high potential for future modeling of harmful algae blooms, particularly those formed by the invasive potentially toxic species under horohaline conditions of the Baltic Sea.

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

The quantification of the kinetic rates of RNA synthesis, processing, and degradation are largely based on the integrative analysis of total and nascent transcription, the latter being quantified through RNA metabolic labeling. We developed INSPEcT−, a computational method based on the mathematical modeling of premature and mature RNA expression that is able to quantify kinetic rates from steady-state or time course total RNA-seq data without requiring any information on nascent transcripts. Our approach outperforms available solutions, closely recapitulates the kinetic rates obtained through RNA metabolic labeling, improves the ability to detect changes in transcript half-lives, reduces the cost and complexity of the experiments, and can be adopted to study experimental conditions in which 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.