Differential Centrifugation to Enrich Bacterial Ribonucleoprotein Bodies (BR bodies) from Caulobacter crescentus

Muthunayake, Nisansala S.; Al-Husini, Nadra; Schrader, Jared M.

doi:10.1016/j.xpro.2020.100205

Cited by 3 publications

(6 citation statements)

References 12 publications

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“…The tighter apparent affinity with RNA may explain why ribosomal protein S1 preferentially dissolves RNase E condensates in the presence of RNA. We also used a coIP assay with MBP-RNase E to test MetK and ribosomal protein S1 association with MBP-RNase E. Indeed, as compared to the core BR-body protein aconitase which is thought to stoichiometrically bind to RNase E 30 , we found that both ribosomal protein S1 and MetK showed lower CoIP than aconitase. However, MetK showed a faint band of protein retained on the resin, while ribosomal protein S1 showed no detectable retention (Fig 5C).…”

Section: Resultsmentioning

confidence: 99%

“…In order to define the C. crescentus BR-body associated proteome, BR-body enrichment was performed as in 30 and subjected to bottom-up proteomics preparation and analysis (Fig 1A). Here we compared enriched BR-body samples to a mock treated lysate using a mutant that is unable to assemble BR-bodies due to the deletion of the C-terminal IDR of RNase E 30 . 100 μg protein was prepared in triplicate for either condition and converted into peptides via enzymatic digestion using S-Traps 32 .…”

Section: Resultsmentioning

confidence: 99%

“…BR-body enrichment proteomics identifies BR-body associated proteins. A) Caulobacter crescentus BR-body enrichment 30 was performed, followed by LC-MS/MS proteomics to identify BR-body associated proteins. JS299 (an RNase E active site mutant) was used to isolate BR-bodies, and JS221 (an RNase E mutant lacking the C-terminal disordered region that cannot form BR-bodies) was used as a negative control.…”

Section: Resultsmentioning

confidence: 99%

“…To define the proteome of Caulobacter crescentus BR-bodies, we performed a cellular enrichment by differential centrifugation 30 followed by quantitative liquid chromatography tandem mass spectrometry (LC-MS). Similar to eukaryotic condensates, we identified over 100 proteins which were enriched in BR-bodies.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…The method achieves the effect of separation by continuously increasing the relative centrifugal force, controlling the centrifugation time, and conducting multiple centrifugations ( Figure 3 ). Muthunayake et al (2020) used differential centrifugation to enrich Bacterial Ribonucleoprotein Bodies (BR bodies) from Caulobacter crescentus . Ma et al (2020) isolated the apoptotic bodies by differential centrifugation after inducing the apoptosis of osteoclasts.…”

Section: Rough Fractionationmentioning

confidence: 99%

1Progress, applications, challenges and prospects of protein purification technology

Hou

Liu

et al. 2022

Front. Bioeng. Biotechnol.

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Protein is one of the most important biological macromolecules in life, which plays a vital role in cell growth, development, movement, heredity, reproduction and other life activities. High quality isolation and purification is an essential step in the study of the structure and function of target proteins. Therefore, the development of protein purification technologies has great theoretical and practical significance in exploring the laws of life activities and guiding production practice. Up to now, there is no forthcoming method to extract any proteins from a complex system, and the field of protein purification still faces significant opportunities and challenges. Conventional protein purification generally includes three steps: pretreatment, rough fractionation, and fine fractionation. Each of the steps will significantly affect the purity, yield and the activity of target proteins. The present review focuses on the principle and process of protein purification, recent advances, and the applications of these technologies in the life and health industry as well as their far-reaching impact, so as to promote the research of protein structure and function, drug development and precision medicine, and bring new insights to researchers in related fields.

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

Nandana,

Rathnayaka-Mudiyanselage,

Muthunayake

et al. 2023

Cell Reports

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Differential Centrifugation to Enrich Bacterial Ribonucleoprotein Bodies (BR bodies) from Caulobacter crescentus

Cited by 3 publications

References 12 publications

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

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

1Progress, applications, challenges and prospects of protein purification technology

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

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