Spatial engineering of E. coli with addressable phase-separated RNAs

Guo, Haotian; Ryan, Joseph C.; Song, Xiaohu; Mallet, Adeline; Zhang, Mengmeng; Pabst, Victor; Decrulle, Antoine L.; Ejsmont, Paulina; Wintermute, Edwin H.; Lindner, Ariel B.

doi:10.1016/j.cell.2022.09.016

Cited by 38 publications

(37 citation statements)

References 53 publications

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“…[4] RNA-based synthetic microdomains are also studied as a means to spatialize biochemical reactions in the cytoplasm of Escherichia coli. [5] In the context of synthetic cells, emergent LLPS was used within lipid droplets encapsulating cell-free transcription-translation systems to better understand the compatibility of such phase-separated structures with protein synthesis. [6] In a complementary approach, responsive synthetic organelles constructed inside proteinosomes responded to various stimuli such as light and pH.…”

Section: Introductionmentioning

confidence: 99%

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Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation

2023

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Compartmentalization is key to many cellular processes and a critical bottleneck of any minimal life approach. In cells, a complex chemistry is responsible for bringing together or separating biomolecules at the right place at the right time. Lipids, nucleic acids and proteins self-organize, thereby creating boundaries, interfaces and specialized microenvironments. Exploiting reversible RNA-based liquid-liquid phase separation (LLPS) inside giant unilamellar vesicles (GUVs), we present an efficient system capable of propagating an RNA-based enzymatic reaction across a population of GUVs upon freezing-thawing (FT) temperature cycles. We report that compartmentalization in the condensed RNA-rich phase can accelerate such an enzymatic reaction. In the decondensed state, RNA substrates become homogeneously dispersed, enabling content exchange between vesicles during freeze-thawing. This work explores how a minimal reversible phase separation system in lipid vesicles could help to implement spatiotemporal control in cyclic processes, as required for minimal cells.

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Section: Introductionmentioning

confidence: 99%

“…A recent work used LLPS to enable orthogonal translation of noncanonical amino acids in eukaryotic cells [4] . RNA‐based synthetic microdomains are also studied as a means to spatialize biochemical reactions in the cytoplasm of Escherichia coli [5] …”

Section: Introductionmentioning

confidence: 99%

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation

2023

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“…The development of artificial organelles or subcellular compartments to endow novel functions, regulate gene expression, or explore the biogenesis mechanism of subcellular structures is always one of the focused areas of synthetic biology. − From the perspective of biotechnology, the application of subcellular compartments not only enhances the rate and fidelity of biochemical reactions by concentrating enzymes and substrates − but also sequesters toxic metabolic products to mitigate interference with cellular metabolism. − To this end, many bacterial microcompartments, − elastin-like polypeptides-based compartments, , lumazine synthases-based compartments, or protein crystalline inclusions have been developed.…”

Section: Introductionmentioning

confidence: 99%

Construction of Osmotic Pressure Responsive Vacuole-like Bacterial Organelles with Capsular Polysaccharides as Building Blocks

Wang

et al. 2023

ACS Synth. Biol.

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Many artificial organelles or subcellular compartments have been developed to tune gene expression, regulate metabolic pathways, or endow new cell functions. Most of these organelles or compartments were built using proteins or nucleic acids as building blocks. In this study, we demonstrated that capsular polysaccharide (CPS) retained inside bacteria cytosol assembled into mechanically stable CPS compartments. The CPS compartments were able to accommodate and release protein molecules but not lipids or nucleic acids. Intriguingly, we found that the CPS compartment size responds to osmotic stress and this compartment improves cell survival under high osmotic pressures, which was similar to the vacuole functionalities. By fine-tuning the synthesis and degradation of CPS with osmotic stressresponsive promoters, we achieved dynamic regulation of the size of CPS compartments and the host cells in response to external osmotic stress. Our results shed new light on developing prokaryotic artificial organelles with carbohydrate macromolecules.

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“…5,6 Recent studies demonstrated that specific RNA self-assemblies, particularly among repeat expansions, can also be used to mediate RNA phase separation inside living cells. [7][8][9][10] Compared to protein-based RNA compartmentalization, RNA-RNA interactionmediated condensate formation can be highly sequence-specific, modular, and programmable. 11,12 As a result, powerful RNA devices may be engineered to control cellular compartmentalization of specific cellular RNAs and modulate their local concentrations and functions.…”

Section: Introductionmentioning

confidence: 99%

“…CAG repeats are used here because these trinucleotides can readily form condensation, without the involvement of proteins. 9,10,13,14 We want to test here if these naturally occurring CAG repeats can be used as functional RNA nanodevices, either being a cis-acting RNA element within target RNA transcript or as a trans-acting effector functioning through specific hybridization with target RNAs. Both cis-and trans-mechanisms can be potentially applied to develop general platforms to induce the phase separation of various target RNAs, in vitro and in living systems.…”

Section: Introductionmentioning

confidence: 99%

Targeted RNA Condensation in Living Cells via Genetically Encodable Triplet Repeat Tags

Xue

Sun

et al. 2023

Preprint

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Living systems contain various functional membraneless organelles that can segregate selective proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many synthetic compartments have been engineered in vitro and in living cells, mostly focused on protein-scaffolded systems. Herein, we introduce a nature-inspired genetically encoded RNA tag to program cellular condensate formations and recruit non-phase-transition target RNAs to achieve functional modulation. In our system, different lengths of CAG-repeat tags were tested as the self-assembled scaffold to drive multivalent condensate formation. Various selective target messenger RNAs and noncoding RNAs can be compartmentalized into these condensates. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes, and densities of these cellular RNA condensates. The regulation functions of these CAG-repeat tags on the cellular RNA localization, lifetime, RNA-protein interactions, and gene expression have also been investigated. Considering the importance of RNA condensation in both health and disease conditions, these genetically encodable modular and self-assembled tags can be potentially widely used for chemical biology and synthetic biology studies.

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Spatial engineering of E. coli with addressable phase-separated RNAs

Cited by 38 publications

References 53 publications

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation

Spatiotemporal Propagation of a Minimal Catalytic RNA Network in GUV Protocells by Temperature Cycling and Phase Separation

Construction of Osmotic Pressure Responsive Vacuole-like Bacterial Organelles with Capsular Polysaccharides as Building Blocks

Targeted RNA Condensation in Living Cells via Genetically Encodable Triplet Repeat Tags

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