Dynamics and functional roles of splicing factor autoregulation

Non-spliceosomal splicing factors are essential, conserved regulators of alternative splicing. They provide concentration-dependent control of diverse pre-mRNAs. Many splicing factors direct unproductive splicing of their own pre-mRNAs through negative autoregulation. However, the impact of such feedback loops on splicing dynamics at the single cell level remains unclear. We developed a system to dynamically, quantitatively analyze negative autoregulatory splicing by the SF2 splicing factor in response to perturbations in single HEK293 cells. Here, we show that negative autoregulatory splicing provides critical functions for gene regulation, establishing a ceiling of SF2 protein concentration, reducing cell-cell heterogeneity in SF2 levels, and buffering variation in SF2 transcription. Most importantly, it adapts SF2 splicing activity to variations in demand from other pre-mRNA substrates. A minimal mathematical model of autoregulatory splicing explains these experimentally observed features, and provides values for effective biochemical parameters. These results reveal the unique functional roles that splicing negative autoregulation plays in homeostatically regulating transcriptional programs.

Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitioned malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing hybrid promoters to control leucine synthesis, this flavonoid addiction phenotype confers a selective growth advantage to the naringenin-producing cell. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield.

Improvement of the memory function of a mutual repression network in a stochastic environment by negative autoregulation

Background Cellular memory is a ubiquitous function of biological systems. By generating a sustained response to a transient inductive stimulus, often due to bistability, memory is central to the robust control of many important biological processes. However, our understanding of the origins of cellular memory remains incomplete. Stochastic fluctuations that are inherent to most biological systems have been shown to hamper memory function. Yet, how stochasticity changes the behavior of genetic circuits is generally not clear from a deterministic analysis of the network alone. Here, we apply deterministic rate equations, stochastic simulations, and theoretical analyses of Fokker-Planck equations to investigate how intrinsic noise affects the memory function in a mutual repression network. Results We find that the addition of negative autoregulation improves the persistence of memory in a small gene regulatory network by reducing stochastic fluctuations. Our theoretical analyses reveal that this improved memory function stems from an increased stability of the steady states of the system. Moreover, we show how the tuning of critical network parameters can further enhance memory. Conclusions Our work illuminates the power of stochastic and theoretical approaches to understanding biological circuits, and the importance of considering stochasticity when designing synthetic circuits with memory function.

A negative autoregulation network motif is required for synchronized Myxococcus xanthus development

Transcription factor autoregulation is a simple network motif (recurring circuit) built into genetic regulatory networks that direct cell behavior. Negative autoregulation (NAR) network motifs are particularly abundant in bacteria and provide specific functions, such as buffering against transcriptional noise. Here, we investigate the phenotypic consequence of perturbing NAR of a major transcription factor, MrpC, that controls the multicellular development program of the bacterium Myxococcus xanthus. Launch of the developmental program directs certain cells in the population to first aggregate into haystack-shaped mounds, and then to differentiate into environmentally resistant spores to form mature fruiting bodies. Perturbation of MrpC NAR causes a striking phenotype in which cells lose synchronized transition from aggregation to sporulation. Instead, some cells abruptly exit aggregation centers and remain locked in a cohesive swarming state, while the remaining cells transition to spores inside residual fruiting bodies. As predicted, disruption of MrpC NAR led to an increased and broadened population distribution of mrpC expression. Examination of MrpC levels in developmental subpopulations during in situ development demonstrated cells locked in the swarms contained intermediate MrpC levels insufficient to promote sporulation. These results suggest an inherent property of NAR motifs that function in multicellular developmental programs is to facilitate synchronized responses.Significance StatementAll organisms use regulatory networks for cellular homeostasis, mediating appropriate responses to environmental changes, or to direct animal development. Understanding how the basic building blocks (motifs) of regulatory networks contribute to these processes is essential to mitigate the effects of mutations in regulatory networks (i.e. cancers) or to synthesize beneficial organisms. In this study, we demonstrate that a common regulatory motif, a transcription factor that represses its own expression, helps synchronize cells that engage in collective behaviors.

Toxin–antitoxin operon kacAT of Klebsiella pneumoniae is regulated by conditional cooperativity via a W-shaped KacA–KacT complex

Bacterial toxin–antitoxin pairs play important roles in bacterial multidrug tolerance. Gcn5-related N-acetyltransferase (GNAT) toxins inhibit translation by acetylation of aminoacyl-tRNAs and are counteracted by direct contacts with cognate ribbon–helix–helix (RHH) antitoxins. Our previous analysis showed that the GNAT toxin KacT and RHH antitoxin KacA of Klebsiella pneumoniae form a heterohexamer in solution and that the complex interacts with the cognate promoter DNA, resulting in negative autoregulation of kacAT transcription. Here, we present the crystal structure of DNA-bound KacAT complex at 2.2 Å resolution. The crystal structure revealed the formation of a unique heterohexamer, KacT–KacA2–KacA2–KacT. The direct interaction of KacA and KacT involves a unique W-shaped structure with the two KacT molecules at opposite ends. Inhibition of KacT is achieved by the binding of four KacA proteins that preclude the formation of an active KacT dimer. The kacAT operon is auto-regulated and we present an experimentally supported molecular model proposing that the KacT:KacA ratio controls kacAT transcription by conditional cooperativity. These results yield a profound understanding of how transcription GNAT–RHH pairs are regulated.