Determinants of the temperature adaptation of mRNA degradation.

The rate of chemical reactions increases proportionally with temperature, but the interplay of biochemical reactions permits deviations from this relation and adaptation. The degradation of individual mRNAs in yeast increased to varying degrees with temperature. We examined how these variations are influenced by the translation and codon composition of mRNAs. We developed a method that revealed the existence of a neutral half-life above which mRNAs are stabilized by translation but below which they are destabilized. The proportion of these two mRNA subpopulations remained relatively constant under different conditions, even with slow cell growth due to nutrient limitation, but heat shock reduced the proportion of translationally stabilized mRNAs. At the same time, the degradation of these mRNAs was partially temperature-compensated through Upf1, the mediator of nonsense-mediated decay. Compensation was also promoted by some asparagine and serine codons, whereas tyrosine codons promote temperature sensitization. These codons play an important role in the degradation of mRNAs encoding key cell membrane and cell wall proteins, which promote cell integrity.

Tuning up Transcription Factors for Therapy.

The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, transcription activator-like effector (TALE) and CRISPR-dCas9 revealed common principles behind their efficacy, which can aid the optimization of transcriptional activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor natural and synthetic transcription factors to the needs of specific applications.

An open-loop approach to calculate noise-induced transitions.

Bistability permits the co-existence of two distinct cell fates in a population of genetically identical cells. Noise induced transitions between two fates of a bistable system are difficult to calculate due to the intricate interplay between nonlinear dynamics and noise in bistable positive feedback loops. Here we opened multivariable feedback loops at the slowest variable to obtain the open-loop function and the fluctuations in the open-loop output. By the subsequent reclosing of the loop, we calculated the mean first passage time (MFPT) using the Fokker-Planck equation in good agreement with the exact stochastic simulation. When an external component interacts with a feedback component, it amplifies the extrinsic noise in the loop. Consequently, the open-loop function is shifted and the transition rates between the two states in the closed loop are increased. Despite this shift, the open-loop output reflects the system faithfully to predict the MFPT in the feedback loop. Therefore, the open-loop approach can help theoretical analysis. Furthermore, the measurement of the mean value, variance, and the reaction time-scale of the open-loop output permits the prediction of MFPT simply from experimental data, which underscores the practical value of the stochastic open-loop approach.

Impact of Methods on the Measurement of mRNA Turnover.

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

Quantification of pre-mRNA escape rate and synergy in splicing.

Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks.

Identification of optimal parameter combinations for the emergence of bistability.

Bistability underlies cellular memory and maintains alternative differentiation states. Bistability can emerge only if its parameter range is either physically realizable or can be enlarged to become realizable. We derived a general rule and showed that the bistable range of a reaction parameter is maximized by a pair of other parameters in any gene regulatory network provided they satisfy a general condition. The resulting analytical expressions revealed whether or not such reaction pairs are present in prototypical positive feedback loops. They are absent from the feedback loop enclosed by protein dimers but present in both the toggle-switch and the feedback circuit inhibited by sequestration. Sequestration can generate bistability even at narrow feedback expression range at which cooperative binding fails to do so, provided inhibition is set to an optimal value. These results help to design bistable circuits and cellular reprogramming and reveal whether bistability is possible in gene networks in the range of realistic parameter values.

Contributions of low molecule number and chromosomal positioning to stochastic gene expression.

The presence of low-copy-number regulators and switch-like signal propagation in regulatory networks are expected to increase noise in cellular processes. We developed a noise amplifier that detects fluctuations in the level of low-abundance mRNAs in yeast. The observed fluctuations are not due to the low number of molecules expressed from a gene per se but originate in the random, rare events of gene activation. The frequency of these events and the correlation between stochastic expressions of genes in a single cell depend on the positioning of the genes along the chromosomes. Transcriptional regulators produced by such random expression propagate noise to their target genes.

Gene choice in cancer cells is exclusive in ion transport but concurrent in DNA replication.

Cancers share common cellular and physiological features. Little is known about whether distinctive gene expression patterns can be displayed at the single-cell level by gene families in cancer cells. The expression of gene homologs within a family can exhibit concurrence and exclusivity. Concurrence can promote all-or-none expression patterns of related genes and underlie alternative physiological states. Conversely, exclusive gene families express the same or similar number of homologs in each cell, allowing a broad repertoire of cell identities to be generated. We show that gene families involved in the cell-cycle and antigen presentation are expressed concurrently. Concurrence in the DNA replication complex MCM reflects the replicative status of cells, including cell lines and cancer-derived organoids. Exclusive expression requires precise regulatory mechanism, but cancer cells retain this form of control for ion homeostasis and extend it to gene families involved in cell migration. Thus, the cell adhesion-based identity of healthy cells is transformed to an identity based on migration in the population of cancer cells, reminiscent of epithelial-mesenchymal transition.

Myoblasts inhibit prostate cancer growth by paracrine secretion of tumor necrosis factor-α.

PURPOSE: Myoblasts can form muscle fibers after transplantation. Therefore, they are envisioned as a treatment for urinary incontinence after radical prostatectomy. However, to our knowledge the safety of this treatment and the interaction of myoblasts with any remaining neighboring cancer are unknown. We investigated the interactions between myoblasts and prostate carcinoma cells in vitro and in vivo. MATERIALS AND METHODS: Myoblasts isolated from the rectus abdominis were used in a series of co-culture experiments with prostate cancer cells and subcutaneously co-injected in vivo. Cell proliferation, cell cycle arrest and apoptosis of cancer in co-culture with myoblasts were assessed. Tumor volume and metastasis formation were evaluated in a mouse model. Tissue specific markers were assessed by immunohistochemistry, fluorescence activated cell sorting analysis, Western blot and real-time quantitative polymerase chain reaction. RESULTS: Myoblasts in proximity to tumor provided paracrine tumor necrosis factor-α to their microenvironment, decreasing the tumor growth of all prostate cancer cell lines examined. Co-culture experiments revealed induction of cell cycle arrest, tumor death by apoptosis and increased myoblast differentiation. This effect was largely blocked by tumor necrosis factor-α inhibition. The same outcome was noted in a mouse model, in which co-injected human myoblasts also inhibited the tumor growth and metastasis formation of all prostate cancer cell lines evaluated. CONCLUSIONS: Myoblasts restrict prostate cancer growth and limit metastasis formation by paracrine tumor necrosis factor-α secretion in vitro and in vivo.

Spatial epigenetic control of mono- and bistable gene expression.

Bistability in signaling networks is frequently employed to promote stochastic switch-like transitions between cellular differentiation states. Differentiation can also be triggered by antagonism of activators and repressors mediated by epigenetic processes that constitute regulatory circuits anchored to the chromosome. Their regulatory logic has remained unclear. A reaction-diffusion model reveals that the same reaction mechanism can support both graded monostable and switch-like bistable gene expression, depending on whether recruited repressor proteins generate a single silencing gradient or two interacting gradients that flank a gene. Our experiments confirm that chromosomal recruitment of activator and repressor proteins permits a plastic form of control; the stability of gene expression is determined by the spatial distribution of silencing nucleation sites along the chromosome. The unveiled regulatory principles will help to understand the mechanisms of variegated gene expression, to design synthetic genetic networks that combine transcriptional regulatory motifs with chromatin-based epigenetic effects, and to control cellular differentiation.

Polysome propensity and tunable thresholds in coding sequence length enable differential mRNA stability.

The half-life of mRNAs, as well as their translation, increases in proportion to the optimal codons, indicating a tight coupling of codon-dependent differential translation and degradation. Little is known about the regulation of this coupling. We found that the mRNA stability gain in yeast depends on the mRNA coding sequence length. Below a critical length, codon optimality fails to affect the stability of mRNAs although they can be efficiently translated into short peptides and proteins. Above this threshold length, codon optimality-dependent differential mRNA stability emerges in a switch-like fashion, which coincides with a similar increase in the polysome propensity of the mRNAs. This threshold length can be tuned by the untranslated regions (UTR). Some of these UTRs can destabilize mRNAs without reducing translation, which plays a role in controlling the amplitude of the oscillatory expression of cell cycle genes. Our findings help understand the translation of short peptides from noncoding RNAs and the translation by localized monosomes in neurons.

Quantitative models of nuclear transport.

Nuclear pore complexes mediate the rapid trafficking of target macromolecules between the nucleus and the cytoplasm but exclude non-targets. Mathematical modeling helps to define the physical properties of a transport medium that can selectively enhance the permeation of some molecules but block others. Recent pioneering work has established a basis for quantitative modeling of nuclear translocation, and we expect this field to expand rapidly. A second area where modeling of nucleocytoplasmic transport has been prominently employed is in efforts to understand the regulatory networks by which signals pass between the nuclear and cytoplasmic compartments. Recent evidence suggests that the distinctive kinetics and spatial organization of nuclear transport processes can be used to efficiently propagate signals by new and unexpected pathways.

The life and death of RNA across temperatures.

Temperature is an environmental condition that has a pervasive effect on cells along with all the molecules and reactions in them. The mechanisms by which prototypical RNA molecules sense and withstand heat have been identified mostly in bacteria and archaea. The relevance of these phenomena is, however, broader, and similar mechanisms have been recently found throughout the tree of life, from sex determination in reptiles to adaptation of viral RNA polymerases, to genetic disorders in humans. We illustrate the temperature dependence of RNA metabolism with examples from the synthesis to the degradation of mRNAs, and review recently emerged questions. Are cells exposed to greater temperature variations and gradients than previously surmised? How do cells reconcile the conflicting thermal stability requirements of primary and tertiary structures of RNAs? To what extent do enzymes contribute to the temperature compensation of the reaction rates in mRNA turnover by lowering the energy barrier of the catalyzed reactions? We conclude with the ecological, forensic applications of the temperature-dependence of RNA degradation and the biotechnological aspects of mRNA vaccine production.

Amplitude control of cell-cycle waves by nuclear import.

Propagation of waves of biochemical activities through consecutive stages of the cell cycle is essential to execute the steps of cell division in a strict temporal order. Mechanisms that ensure the proper amplitude and timing of these waves are poorly understood. Using a synthetic gene circuit, we show that a transcriptional activator driven by yeast cell-cycle promoters propagates transcriptional oscillations with substantial damping. Although regulated nuclear translocation has been implicated in the timing of oscillatory events, mathematical analysis shows that increasing the rate of nuclear transport is an example of a general regulatory principle, which enhances the fidelity of wave propagation. Indeed, increasing the constitutive import rate of the activator counteracts the damping of waves and concurrently preserves the intensity of the signal. In contrast to the regulatory range of nuclear transport, the range of mRNA turnover considerably limits transcriptional wave propagation. This classification of cellular processes outlines potential regulatory mechanisms that can contribute to faithful transmission of oscillations at different stages of the cell cycle.

Enhancement of cellular memory by reducing stochastic transitions.

On induction of cell differentiation, distinct cell phenotypes are encoded by complex genetic networks. These networks can prevent the reversion of established phenotypes even in the presence of significant fluctuations. Here we explore the key parameters that determine the stability of cellular memory by using the yeast galactose-signalling network as a model system. This network contains multiple nested feedback loops. Of the two positive feedback loops, only the loop mediated by the cytoplasmic signal transducer Gal3p is able to generate two stable expression states with a persistent memory of previous galactose consumption states. The parallel loop mediated by the galactose transporter Gal2p only increases the expression difference between the two states. A negative feedback through the inhibitor Gal80p reduces the strength of the core positive feedback. Despite this, a constitutive increase in the Gal80p concentration tunes the system from having destabilized memory to having persistent memory. A model reveals that fluctuations are trapped more efficiently at higher Gal80p concentrations. Indeed, the rate at which single cells randomly switch back and forth between expression states was reduced. These observations provide a quantitative understanding of the stability and reversibility of cellular differentiation states.

Gene Families With Stochastic Exclusive Gene Choice Underlie Cell Adhesion in Mammalian Cells.

Exclusive stochastic gene choice combines precision with diversity. This regulation enables most T-cells to express exactly one T-cell receptor isoform chosen from a large repertoire, and to react precisely against diverse antigens. Some cells express two receptor isoforms, revealing the stochastic nature of this process. A similar regulation of odorant receptors and protocadherins enable cells to recognize odors and confer individuality to cells in neuronal interaction networks, respectively. We explored whether genes in other families are expressed exclusively by analyzing single-cell RNA-seq data with a simple metric. This metric can detect exclusivity independently of the mean value and the monoallelic nature of gene expression. Chromosomal segments and gene families are more likely to express genes concurrently than exclusively, possibly due to the evolutionary and biophysical aspects of shared regulation. Nonetheless, gene families with exclusive gene choice were detected in multiple cell types, most of them are membrane proteins involved in ion transport and cell adhesion, suggesting the coordination of these two functions. Thus, stochastic exclusive expression extends beyond the prototypical families, permitting precision in gene choice to be combined with the diversity of intercellular interactions.

A system of counteracting feedback loops regulates Cdc42p activity during spontaneous cell polarization.

Cellular polarization is often a response to distinct extracellular or intracellular cues, such as nutrient gradients or cortical landmarks. However, in the absence of such cues, some cells can still select a polarization axis at random. Positive feedback loops promoting localized activation of the GTPase Cdc42p are central to this process in budding yeast. Here, we explore spontaneous polarization during bud site selection in mutant yeast cells that lack functional landmarks. We find that these cells do not select a single random polarization axis, but continuously change this axis during the G1 phase of the cell cycle. This is reflected in traveling waves of activated Cdc42p which randomly explore the cell periphery. Our integrated computational and in vivo analyses of these waves reveal a negative feedback loop that competes with the aforementioned positive feedback loops to regulate Cdc42p activity and confer dynamic responsiveness on the robust initiation of cell polarization.

Control and signal processing by transcriptional interference.

A transcriptional activator can suppress gene expression by interfering with transcription initiated by another activator. Transcriptional interference has been increasingly recognized as a regulatory mechanism of gene expression. The signals received by the two antagonistically acting activators are combined by the polymerase trafficking along the DNA. We have designed a dual-control genetic system in yeast to explore this antagonism systematically. Antagonism by an upstream activator bears the hallmarks of competitive inhibition, whereas a downstream activator inhibits gene expression non-competitively. When gene expression is induced weakly, the antagonistic activator can have a positive effect and can even trigger paradoxical activation. Equilibrium and non-equilibrium models of transcription shed light on the mechanism by which interference converts signals, and reveals that self-antagonism of activators imitates the behavior of feed-forward loops. Indeed, a synthetic circuit generates a bell-shaped response, so that the induction of expression is limited to a narrow range of the input signal. The identification of conserved regulatory principles of interference will help to predict the transcriptional response of genes in their genomic context.