RNase E biomolecular condensates stimulate PNPase activity

Collins, Michael James; Tomares, Dylan T.; Nandana, Vidhyadhar; Schrader, Jared M.; Childers, W. Seth

doi:10.1038/s41598-023-39565-w

Cited by 9 publications

(3 citation statements)

References 51 publications

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“…Notably, our discovery that ribonucleases responsible for mRNA degradation are excluded from stress granules contrasts with findings in C. crescentus , where the ribonuclease RNaseE is integral to the formation of BR bodies 19 . Instead, the organization of BR bodies appears to modulate degradosome activity 20, 21 . Furthermore, these findings hold promise for informing future strategies aimed at developing tailored synthetic aggresomes for therapeutic applications involving mRNA, such as vaccines and gene silencing.…”

Section: Discussionmentioning

confidence: 99%

Bacterial stress granule protects mRNA through ribonucleases exclusion

Pei,

Xian,

Yan

et al. 2024

Preprint

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Membraneless droplets formed through liquid-liquid phase separation (LLPS) play a crucial role in mRNA storage, enabling organisms to swiftly respond to environmental changes. However, the mechanisms underlying mRNA integration and protection within droplets remain unclear. Here, we unravel the role of bacterial aggresomes as stress granules (SGs) in safeguarding mRNA during stress. We discovered that upon stress onset, mobile mRNA molecules selectively incorporate into individual proteinaceous SGs based on length-dependent enthalpic gain over entropic loss. As stress prolongs, SGs undergo compaction facilitated by stronger non-specific RNA-protein interactions, thereby promoting recruitment of shorter RNA chains. Remarkably, mRNA ribonucleases are repelled from bacterial SGs, due to the influence of protein surface charge. This exclusion mechanism ensures the integrity and preservation of mRNA within SGs during stress conditions, explaining how mRNA can be stored and protected from degradation. Following stress removal, SGs facilitate mRNA translation, thereby enhancing cell fitness in changing environments. These droplets maintain mRNA physiological activity during storage, making them an intriguing new candidate for mRNA therapeutics manufacturing.

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Section: Discussionmentioning

confidence: 99%

Bacterial stress granule protects mRNA through ribonucleases exclusion

Pei,

Xian,

Yan

et al. 2024

Preprint

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“…One example is the sequestration and activity of a client kinase upon phase separation by ATP depletion in C. crescentus, 150 showing that ATP depletion can promote LLPS, enforce protein compartmentalization, and sustain enzyme activity. Another example is the activity of a bacterial polynucleotide phosphorylase, which is enhanced when the enzyme colocalizes with RNase E within biomolecular condensates (in this case ribonucleoprotein bodies 151 ).…”

Section: Macromolecular Composition Of Cytoplasmmentioning

confidence: 99%

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Monterroso,

Margolin,

Boersma

et al. 2024

Chem. Rev.

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Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress. CONTENTS 1910 2.2.5. Fluidization of the Cytoplasm 1911 3. The Bacterial Nucleoid 1911 4. Crowding and Phase Separation in Membrane Environments 4.1. Remodeling of the Membrane by Crowding and Phase Separation 4.

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“…Proteins often serve as scaffold molecules that drive the phase separation, and RNAs usually partition into a condensate as guest molecules 8,19,20 . Cellular functions of RNP granules rely on the selective recruitment of RNA guest molecules and their corresponding processing machineries into condensates 1,2,8,19,20 , as well as the resulting modulation of biochemical reaction rates 21,22 . Since all biological processes are time-dependent, information on how long an RNA molecule resides in a condensate and how the internal architecture of the condensate regulates intra-condensate diffusion are crucial, yet underexplored.…”

Section: Mainmentioning

confidence: 99%

Single molecule tracking reveals nanodomains in biomolecular condensates

Gao,

Sumrall,

Walter

2024

Preprint

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Protein-rich biomolecular condensates formed by phase separation1play a crucial role in cellular RNA regulation by the selective recruitment and processing of RNA molecules2. The functional impact of condensates on RNA biology relies on the residence time of individual RNA molecules within a condensate3, which is governed by intra-condensate diffusion4– the slower or more confined the diffusion, the longer the residence time. However, the spatiotemporal organization of RNA and protein diffusion within a single condensate remains largely unknown due to the challenge of accurately profiling intra-condensate diffusion behaviors down to the single-molecule level. Here we introduced a general condensate-tethering approach that allows single molecule tracking (SMT) of fluorescently labeled proteins and RNAs within non-wetted spherical 3D condensates without the interference of condensate motions. We found that a significant fraction of RNA and protein molecules are locally confined, rather than freely diffusive, within a model condensate formed by full-length, tag-free, RNA-binding protein Fused-in-Sarcoma (FUS), known for its critical roles in RNA biology under both physiological and pathological conditions5. Dynamic Point Accumulation for Imaging in Nanoscale Topography (PAINT)6reconstruction further revealed that RNA and proteins are confined to distinct slow-moving nanometer-scale regions, termed nanodomains, within a single condensate. Remarkably, nanodomains affect the diffusion but not the density of the confined biomolecules, supporting local percolation1,7rather than a secondary phase separation within the condensate as their origin. Beyond their regulatory roles on RNA residence time, nanodomains engender both elevated local connectivity and altered chemical environment, a prerequisite for pathological liquid-to-solid transition of FUS during aging. In summary, our study uncovers a patterned spatial organization of both protein and RNA molecules within a single condensate, revealing distinct diffusion dynamics that affect molecular retention time and interactions underlying cellular function and pathology.

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RNase E biomolecular condensates stimulate PNPase activity

Cited by 9 publications

References 51 publications

Bacterial stress granule protects mRNA through ribonucleases exclusion

Bacterial stress granule protects mRNA through ribonucleases exclusion

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

Single molecule tracking reveals nanodomains in biomolecular condensates

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