The small non-coding RNA Vaultrc5 is dispensable to mouse development.

Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they have since also been described as noncanonical miRNA precursors and as riboregulators of autophagy. As central molecules in these processes, vRNAs have been attributed numerous biological roles including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss of function allele. Because Vaultrc5 is the sole murine vRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts pointing to a potential role for vRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.

AGO2 silences mobile transposons in the nucleus of quiescent cells.

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway, with essential roles in development and disease. Yet little is known about its regulation in vivo. Here we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells in which mTOR signaling is low, AGO2 accumulates in the nucleus, where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

Added value of autoregulation and multi-step kinetics of transcription initiation.

Bacterial gene expression regulation occurs mostly during transcription, which has two main rate-limiting steps: the close complex formation, when the RNA polymerase binds to an active promoter, and the subsequent open complex formation, after which it follows elongation. Tuning these steps' kinetics by the action of e.g. transcription factors, allows for a wide diversity of dynamics. For example, adding autoregulation generates single-gene circuits able to perform more complex tasks. Using stochastic models of transcription kinetics with empirically validated parameter values, we investigate how autoregulation and the multi-step transcription initiation kinetics of single-gene autoregulated circuits can be combined to fine-tune steady state mean and cell-to-cell variability in protein expression levels, as well as response times. Next, we investigate how they can be jointly tuned to control complex behaviours, namely, time counting, switching dynamics and memory storage. Overall, our finding suggests that, in bacteria, jointly regulating a single-gene circuit's topology and the transcription initiation multi-step dynamics allows enhancing complex task performance.

Logic of two antagonizing intra-species quorum sensing systems in bacteria.

Bacteria release signaling molecules into the surrounding environment and sense them when present in their proximity. Using this strategy, a cell estimates the number of neighbors in its surrounding. Upon sensing a critical number of individuals, bacteria coordinate a number of cellular processes. This density-dependent control of gene expression and physiology is called quorum sensing (QS). Quorum sensing controls a wide variety of functions in bacteria, including those related to motility, growth, virulence etc. Quorum sensing has been widely observed in bacteria while the individuals of the same species or different species compete and cooperate each other. Interestingly, many species possess more than one QS system (intra-species) and these QS systems interact each other to perform quorum sensing. Thus, several logical arrangements can be possible based on the interaction among intra-species QS systems - parallel, series, antagonizing, and agonizing. In this work, we perform simulations to understand the logic of interaction between two antagonizing intra-species QS systems. In such an interaction, one QS system gets fully expressed and the other only gets partially expressed. This is found to be dictated by the interplay between autoinducer's diffusivity and antagonizing strength. In addition, we speculate an important role of the intracellular regulators (eg. LuxR) in maintaining the uniform response among the individual cells from the different localities. We also expect the interplay between the autoinducer's diffusivity and distribution of cells in fine tuning the collective response. Interestingly, in a localized niche with a heterogeneous cell distribution, the cells are expected to perform a global quorum sensing via fully expressed QS system and a local quorum sensing via partially expressed QS system.

Analysis of a strategy for cooperating cells to survive the presence of cheaters.

Cooperation benefits individual cells in a microbial population by helping accomplish tasks which are difficult or non-beneficial for individuals in the population to carry out by themselves. Hence, numerous examples exist of bacteria cooperating and working towards a common objective. The sharing of a common public good via quorum sensing is one of the ways of cooperation among individuals of many microbial populations. However, cheaters exploit cooperators in a population by not contributing to the production of the common goods but enjoy benefits from goods secreted by cooperating individuals. Thus, compared to cooperators, cheaters exhibit a fitness advantage. This suggests that in a population of cooperators invaded by cheaters, the cheaters should be naturally selected for. Instead, however, cooperation is ubiquitous and occurs in many species at various levels of biological organization. So, the question thus arises that what sort of strategies do these microorganisms employ to survive in the presence of cheaters? We try to answer this question here by mathematical analysis of a strategy used in microbial populations where public benefit received by cheaters is restrained to limit cheater invasion. Our results suggest that individuals exhibiting a little selfishness while still contributing to the population are best suited to resist cheater invasion.

Revisiting demand rules for gene regulation.

Starting with Savageau's pioneering work regarding demand rules for gene regulation from the 1970s, here, we choose the simplest transcription network and ask: how does the cell choose a particular regulatory topology from all available possibilities? According to the demand rules, a cell chooses an activator based regulation of a target if the target protein is required for most of the time. On the other hand, if the target protein is only required sporadically, its control tends to be via a repressor-based regulatory topology. We study the natural distribution of topologies at genome, systems, and micro-levels in E. coli and observe deviations from demand rules. Analyzing the regulation of amino acid biosynthesis, transport, and carbon utilization in E. coli and B. subtilis, and comparing choice of topology with demand, we observe an alternate pattern emerging. Simulations of networks are used to help explain the natural distribution of topologies in nature. Overall, our results indicate that the choice of topology is drawn randomly from a pool of all networks which satisfy the dynamic requirements of the cell, as dictated by physiology. In short, our results suggest that the cell picks "whatever works".

Control of MarRAB Operon in Escherichia coli via Autoactivation and Autorepression.

Choice of network topology for gene regulation has been a question of interest for a long time. How do simple and more complex topologies arise? In this work, we analyze the topology of the marRAB operon in Escherichia coli, which is associated with control of expression of genes associated with conferring resistance to low-level antibiotics to the bacterium. Among the 2102 promoters in E. coli, the marRAB promoter is the only one that encodes for an autoactivator and an autorepressor. What advantages does this topology confer to the bacterium? In this work, we demonstrate that, compared to control by a single regulator, the marRAB regulatory arrangement has the least control cost associated with modulating gene expression in response to environmental stimuli. In addition, the presence of dual regulators allows the regulon to exhibit a diverse range of dynamics, a feature that is not observed in genes controlled by a single regulator.

Role of feedback and network architecture in controlling virulence gene expression in Bordetella.

Bordetella is a Gram-negative bacterium responsible for causing whooping cough in a broad range of host organisms. For successful infection, Bordetella controls expression of four distinct classes of genes (referred to as class 1, 2, 3, and 4 genes) at distinct times in the infection cycle. This control is executed by a single two-component system, BvgAS. Interestingly, the transmembrane component of the two-component system, BvgS, consists of three phospho-transfer domains leading to phosphorylation of the response regulator, BvgA. Phosphorylated BvgA then controls expression of virulence genes and also controls bvgAS transcription. In this work, we perform simulations to characterize the role of the network architecture in governing gene expression in Bordetella. Our results show that the wild-type network is locally optimal for controlling the timing of expression of the different classes of genes involved in infection. In addition, the interplay between environmental signals and positive feedback aids the bacterium identify precise conditions for and control expression of virulence genes.

Interplay between Fur and HNS in controlling virulence gene expression in Salmonella typhimurium.

Salmonella enterica is responsible for a large number of diseases in a wide-range of hosts. Two of the global regulators involved in controlling gene expression during the infection cycle of the bacterium are Fur and HNS. In this paper, we demonstrate computationally that Fur and HNS have disproportionately high density of binding sites in the Pathogenicity Islands on the Salmonella chromosome. Moreover, the frequency of binding sites for the two proteins is correlated throughout the genome of the organism. These results indicate a complex interplay between Fur and HNS in regulating cellular global behavior.