Nonmodular oscillator and switch based on RNA decay drive regeneration of multimodal gene expression

Periodic gene expression dynamics are key to cell and organism physiology. Studies of oscillatory expression have focused on networks with intuitive regulatory negative feedback loops, leaving unknown whether other common biochemical reactions can produce oscillations. Oscillation and noise have been proposed to support the capacity of mammalian progenitor cells to restore heterogenous, multimodal expression from extreme subpopulations, but underlying networks and specific roles of noise remained elusive. We use mass-action-based models to show that regulated RNA degradation involving as few as two RNA species, applicable to nearly half of human protein-coding genes, can generate sustained oscillations without imposed feedback. Diverging oscillation periods synergize with noise to robustly restore bimodal expression in cell populations. The global bifurcation organizing this divergence relies on an oscillator and bistable switch which cannot be decomposed into two structural modules. Our work reveals surprisingly rich dynamics of post-transcriptional reactions and a potentially widespread mechanism useful for development and regeneration.

Regenerated crustacean limbs are precise replicas

Animals can regenerate complex organs, yet this frequently results in imprecise replicas of the original structure. In the crustacean Parhyale, embryonic and regenerating legs differ in gene expression dynamics but produce apparently similar mature structures. We examine the fidelity of Parhyale leg regeneration using complementary approaches to investigate microanatomy, sensory function, cellular composition and cell molecular profiles. We find that regeneration precisely replicates the complex microanatomy and spatial distribution of external sensory organs, and restores their sensory function. Single-nuclei sequencing shows that regenerated and uninjured legs are indistinguishable in terms of cell type composition and transcriptional profiles. This remarkable fidelity highlights the ability of organisms to achieve identical outcomes via distinct processes.

Tunable Transcription Factor Library for Robust Quantification of Gene Expression Dynamics in E. coli

Predicting the quantitative regulatory function of a TF based on factors such as binding sequence, binding location and promoter type is not possible. The interconnected nature of gene networks and the difficulty in tuning individual TF concentrations makes the isolated study of TF function challenging. Here we present a library of E. coli strains designed to allow for precise control of the concentration of individual TFs enabling the study of the role of TF concentration on physiology and regulation. We demonstrate the usefulness of this resource by measuring the regulatory function of the zinc responsive TF, ZntR and the paralogous TF pair, GalR/GalS. For ZntR, we find that zinc alters ZntR regulatory function in a way that enables activation of the regulated gene to be robust with respect to ZntR concentration. For GalR and GalS, we are able to demonstrate that these parlogous TFs have fundamentally distinct regulatory roles beyond differences in binding affinity.

Implementation of a novel optogenetic tool in mammalian cells based on a split T7 RNA polymerase

Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to orthogonally control gene expression in mammalian cells. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize and optimize a new orthogonal optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7, mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate gene expression burden when compared to other optogenetic constructs. These properties make mOptoT7 a new powerful tool to use when orthogonality and viral-like RNA species are desired in both synthetic biology and basic science applications.

Transcriptome dynamics during metamorphosis of imaginal discs into wings and thoracic dorsum in Apis mellifera castes

Background Much of the complex anatomy of a holometabolous insect is built from disc-shaped epithelial structures found inside the larva, i.e., the imaginal discs, which undergo a rapid differentiation during metamorphosis. Imaginal discs-derived structures, like wings, are built through the action of genes under precise regulation. Results We analyzed 30 honeybee transcriptomes in the search for the gene expression needed for wings and thoracic dorsum construction from the larval wing discs primordia. Analyses were carried out before, during, and after the metamorphic molt and using worker and queen castes. Our RNA-seq libraries revealed 13,202 genes, representing 86.2% of the honeybee annotated genes. Gene Ontology analysis revealed functional terms that were caste-specific or shared by workers and queens. Genes expressed in wing discs and descendant structures showed differential expression profiles dynamics in premetamorphic, metamorphic and postmetamorphic developmental phases, and also between castes. At the metamorphic molt, when ecdysteroids peak, the wing buds of workers showed maximal gene upregulation comparatively to queens, thus underscoring differences in gene expression between castes at the height of the larval-pupal transition. Analysis of small RNA libraries of wing buds allowed us to build miRNA-mRNA interaction networks to predict the regulation of genes expressed during wing discs development. Conclusion Together, these data reveal gene expression dynamics leading to wings and thoracic dorsum formation from the wing discs, besides highlighting caste-specific differences during wing discs metamorphosis.

Improving the robustness of engineered bacteria to nutrient stress using programmed proteolysis

The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and the freeing of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-messenger RNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately seven times more efficient. Based on these results, we then show how proteolysis can be used to improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.

Global Analysis of the Zinc Homeostasis Network in Pseudomonas aeruginosa and Its Gene Expression Dynamics

Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptive homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.

Large-scale comparative small RNA analyses reveal genomic structural variants in driving expression dynamics and differential selection pressures on distinct small RNA classes during tomato domestication

Tomato has undergone extensive selections during domestication. Recent progress has shown that genomic structural variants (SVs) have contributed to gene expression dynamics during tomato domestication, resulting in changes of important traits. Here, through comprehensive analyses of small RNAs (sRNAs) from nine representative tomato accessions, we demonstrate that SVs substantially contribute to the dynamic expression of the three major classes of plant sRNAs: microRNAs (miRNAs), phased secondary short interfering RNAs (phasiRNAs), and 24-nt heterochromatic siRNAs (hc-siRNAs). Changes in the abundance of phasiRNAs and 24-nt hc-siRNAs likely contribute to the alteration of mRNA gene expression during recent evolution of tomato, particularly for genes associated with biotic and abiotic stress tolerance. We also observe that miRNA expression dynamics are associated with imprecise processing, alternative miRNA-miRNA* selections, and SVs. SVs mainly affect the expression of less-conserved miRNAs that do not have established regulatory functions or low abundant members in highly expressed miRNA families, highlighting different selection pressures on miRNAs compared to phasiRNAs and 24-nt hc-siRNAs. Our findings provide insights into plant sRNA evolution as well as SV-based gene regulation during crop domestication. Furthermore, our dataset provides a rich resource for mining the sRNA regulatory network in tomato.