Quantitative reconstitution of yeast RNA processing bodies

Currie, Simon L.; Xing, Wenmin; Muhlrad, Denise; Decker, Carolyn J.; Parker, Roy; Rosen, Michael K.

doi:10.1101/2022.08.13.503854

Cited by 4 publications

(7 citation statements)

References 112 publications

(263 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S1). We compared PF127 treatment with traditional passivation techniques, including BSA, mPEG, and combined mPEG/BSA, across three condensate systems composed of: Dhh1 (7), representing IDR-mediated phase separation; Nck/N-WASP (6), generated by multivalent modular domain interactions and characterized by high wettability; and polySUMO/polySIM (17), a distinct modular domain system with low wettability. PF127 passivation yielded the lowest adhesion (highest contact angles) for all three condensates (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Many condensates form through liquid-liquid phase separation (LLPS) of multivalent macromolecules (1, 3). Significant advancements in our understanding of condensates have derived from biochemical reconstitution with fluorescence microscopy detection (4-7). Undesirable interactions between condensates and glass surfaces represent a significant methodological challenge in these experiments.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

Yao,

Rosen

2024

Preprint

View full text Add to dashboard Cite

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, generically applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multi-point attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time FRAP imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

Yao,

Rosen

2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Genes , Plasmids, and DNA polyPRM, polySH3, polySUMO, polySIM, Dhh1, GFP-Dhh1, and human cGAS were described previously 6,21,22,33 . For detailed information, see supporting information.…”

Section: Lead Contact and Materials Availabilitymentioning

confidence: 99%

Small Molecule Properties Define Partitioning into Biomolecular Condensates

Thody

Clements

Baniasadi

et al. 2022

Preprint

View full text Add to dashboard Cite

Biomolecular condensates regulate cellular function by compartmentalizing molecules without a surrounding membrane1,2. Condensate functions are believed to arise from the specific exclusion or enrichment of molecules3-5. Thus, understanding the principles governing condensate composition is critical to characterizing condensate function. The molecular bases of macromolecular composition have been studied in detail for several condensates6-9, but partitioning of small molecules into condensates remains poorly understood. Using mass spectrometry with validation by fluorescence microscopy, we quantified partitioning of ~1700 metabolites and FDA approved drugs into four condensates composed of different DNA and/or protein scaffolds. We found that partitioning spanned from ~100-fold exclusion to ~10,000-fold enrichment. Strong correlations between the different condensates suggest an underlying physical similarity despite disparate macromolecular components. Strongly partitioning molecules generally did not bind condensate forming proteins with high affinity under conditions where condensates do not form, suggesting only a minor role for stereospecific interactions in partitioning. We developed a machine learning model that accurately predicts partitioning using only physicochemical properties of the compounds. The strongest predictors of partitioning were features related to aqueous solubility and hydrophobicity. Small molecule partitioning was similar for condensates in aqueous buffer and in concentrated cell extracts, suggesting analogous behaviors in vivo. Together, the data and model suggest that small molecule partitioning is not generally based on high-affinity, stereospecific interactions with scaffolds, but rather on physical properties of the compounds, and their compatibility with an emergent hydrophobic environment within the condensate. Our results will aid design of chemical compounds that target biomolecular condensates and reveal unexpected physical and functional similarities of distinct condensates.

show abstract

“…Together with RNApol condensates identified previously 5 , this suggests that bacteria contain many RNP condensates likely help coordinate multi-step reactions in RNA processing 3 . As observed for yeast P-bodies, a set of “core” proteins were found to be quantitatively enriched 27 and likely homogeneously represented in these structures, were successfully reconstituted in vitro with similar dynamics properties and stoichiometries identified 28 . We observed that a subset of BR-body enriched proteins known to stoichiometrically assemble the RNA degradosome (RNase E, RhlB, Aconitase, and PNPase), appear to be uniformly enriched in BR-bodies in vivo 4 and selectively recruited to RNase E condensates in vitro (Fig 6).…”

Section: Discussionmentioning

confidence: 69%

“…Stress granules have been found to contain >300 proteins and >1800 RNA transcripts [23][24][25] , often including a partially overlapping set of components with P-bodies 22,26 , yet the differences in composition and slower dynamics 27 suggests that these structures likely have distinct functions. Through careful quantitative measurements, the core set of P-body proteins has recently been reconstituted in vitro, with similar dynamic propertiess 28 . In bacteria, BR-bodies were found to be enriched in long, poorly translated cellular mRNAs and sRNAs, a class of translational repressors, together with the RNA degradosome proteins 4,29 .…”

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

View full text Add to dashboard Cite

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.

show abstract

Quantitative reconstitution of yeast RNA processing bodies

Cited by 4 publications

References 112 publications

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

Small Molecule Properties Define Partitioning into Biomolecular Condensates

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

Contact Info

Product

Resources

About