RNase E biomolecular condensates stimulate PNPase activity

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

doi:10.1101/2022.06.06.495039

Cited by 3 publications

(4 citation statements)

References 46 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 23 . Instead, the organization of BR bodies appears to modulate degradosome activity 24,25 . 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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“…We also observed previously that RNase E condensates are observable within seconds after the addition of RNA, suggesting that phase separation of BR-bodies is also a rapid process (20). Importantly, the RNA degradosome binding partner PNPase, which is a 3’ to 5’ exoribonuclease, was also shown to have increased exonucleolytic activity in the presence of RNase E droplets (23). This suggests that BR-bodies help coordinate the multi-step RNA decay process by concentrating essential mRNA decay enzymes with their mRNA substrates, not only to accelerate the rate-limiting step of RNA decay by RNase E, but also to promote the subsequent 3’ to 5’ exonucleolytic steps that complete mRNA decay, thereby preventing the pre-mature release of RNA decay intermediates.…”

Section: Discussionmentioning

confidence: 99%

Caulobacter crescentusRNase E condensation contributes to autoregulation and fitness

Nandana,

Al-Husini,

Vaishnav

et al. 2023

Preprint

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RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E fromEscherichia coliand neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA’s 5’ UTR. While the 5’ UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacteriumCaulobacter crescentusRNase E contains a similar 5’ UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal IDR of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Usingin vitropurified RNase E, we find that the IDR’s ability to promote phase-separation correlates with enhanced 5’ UTR cleavage, suggesting that phase-separation of RNase E with the 5’ UTR enhances autoregulation. Finally, using growth competition experiments we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5’ UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

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“…An important goal working forward will be to define the role of BR-body localization to each of the core enzymes involved in the RNA decay process. Indeed, by using a two protein BR-body system using only RNase E and PNPase, PNPase activity was observed to be directly stimulated via condensation with RNase E 17 , suggesting organization in BR-bodies may help to stimulate RNA degradosome activity. By testing the full complement of "core" BR-body proteins and also the set of "accessory" BR-body associated proteins, we will likely be able to delineate the normal vs specialized functions of BR-bodies.…”

Section: A Complex Protein Interaction Network Impacts Br-body Conden...mentioning

confidence: 99%

“…Importantly, molecules inside of condensates can often show diffusive dynamics, suggesting biomolecular condensates can facilitate biochemical processes within them 15,16 . Models have been suggested for how condensates might help accelerate enzyme catalyzed processes, including acceleration of reaction rates by local concentration, increased reaction specificity by selective recruitment of certain substrates and avoidance of others, and the ability to prevent release of pathway intermediates [17][18][19] . Conversely, they have been proposed to sequester substrate molecules from their enzymes leading to a reduction in reaction rates 20,21 .…”

Section: Introductionmentioning

confidence: 99%

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

Nandana

Rathnayaka-Mudiyanselage

Muthunayake

et al. 2023

Preprint

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Bacterial RNP bodies (BR-bodies) are non membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein substrates of BR-bodies through enrichment of the bodies followed by liquid chromatography-mass spec (LC-MS) proteomic analysis. We found 111 BR-body enriched proteins, including many RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, suggesting BR-bodies are more complex than previously assumed. While most BR-body enriched proteins cannot phase separate, we identified five which can undergo RNA dependent phase separation in vitro, suggesting other RNP condensates interface with BR-bodies. The RNA degradosome protein clients are recruited more strongly to RNase E droplets than that of the other RNP condensates, suggesting that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble unidirectionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the observed dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation while coordinating this mode of organization with RNA processing.

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RNase E biomolecular condensates stimulate PNPase activity

Cited by 3 publications

References 46 publications

Bacterial stress granule protects mRNA through ribonucleases exclusion

Bacterial stress granule protects mRNA through ribonucleases exclusion

Caulobacter crescentusRNase E condensation contributes to autoregulation and fitness

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

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