Phase-specific rna accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles

ABSTRACT Liquid–liquid phase separation has emerged as an important means of intracellular RNA compartmentalization. Some membraneless organelles host two or more compartments serving

different putative biochemical roles. The mechanisms for, and functional consequences of, this subcompartmentalization are not yet well understood. Here we show that adjacent phases of

decapeptide-based multiphase model membraneless organelles differ markedly in their interactions with RNA. Single- and double-stranded RNAs preferentially accumulate in different phases

within the same droplet, and one phase is more destabilizing for RNA duplexes than the other. Single-phase peptide droplets did not capture this behaviour. Phase coexistence introduces new

thermodynamic equilibria that alter RNA duplex stability and RNA sorting by hybridization state. These effects require neither biospecific RNA-binding sites nor full-length proteins. As

such, they are more general and point to primitive versions of mechanisms operating in extant biology that could aid understanding and enable the design of functional artificial membraneless

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BEING VIEWED BY OTHERS DETERMINANTS THAT ENABLE DISORDERED PROTEIN ASSEMBLY INTO DISCRETE CONDENSED PHASES Article 05 February 2024 ACTIVE COACERVATE DROPLETS AS A MODEL FOR MEMBRANELESS

ORGANELLES AND PROTOCELLS Article Open access 14 October 2020 ANXA11 BIOMOLECULAR CONDENSATES FACILITATE PROTEIN-LIPID PHASE COUPLING ON LYSOSOMAL MEMBRANES Article Open access 21 March 2025

DATA AVAILABILITY All data supporting the findings of this study are available within the Article and its Supplementary Information, and also from the corresponding authors upon request.

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fluorescence microscope. _Biophys. J._ 78, 3260–3274 (2000). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the NASA

Exobiology programme grant no. 80NSSC17K0034 (S.C., M.O.M., P.C.B. and C.D.K.). S.C. was also supported by Future Investigators in NASA Earth and Space Science and Technology (FINESST) under

grant no. 80NSSC19K1531 and no. 80NSSC22K0553. We thank F. Pir Cakmak and H. Fares for helpful discussions, and T. Mal for help with NMR analysis. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Chemistry, The Pennsylvania State University, University Park, PA, USA Saehyun Choi, Philip C. Bevilacqua & Christine D. Keating * Center for RNA Molecular

Biology, The Pennsylvania State University, University Park, PA, USA McCauley O. Meyer & Philip C. Bevilacqua * Department of Biochemistry and Molecular Biology, The Pennsylvania State

University, University Park, PA, USA McCauley O. Meyer & Philip C. Bevilacqua Authors * Saehyun Choi View author publications You can also search for this author inPubMed Google Scholar

* McCauley O. Meyer View author publications You can also search for this author inPubMed Google Scholar * Philip C. Bevilacqua View author publications You can also search for this author

inPubMed Google Scholar * Christine D. Keating View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.O.M. performed the radiolabelled RNA

partitioning experiments. S.C. performed all other experiments. All authors conceived and designed the experiments and analysed the data. S.C. and C.D.K. wrote the manuscript, with input

from P.C.B. and M.O.M. CORRESPONDING AUTHORS Correspondence to Philip C. Bevilacqua or Christine D. Keating. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Chemistry_ thanks Pilong Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL

INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY

INFORMATION Supplementary Figs. 1–18, Discussions 1–7 and Tables 1–17. SUPPLEMENTARY DATA 1 Calculation of radiolabelled RNA partitioning in coacervate phases. SOURCE DATA SOURCE DATA FIG. 1

Estimated labelled peptide concentration for all trials. SOURCE DATA FIG. 2 Estimated RNA concentration for all trials. SOURCE DATA FIG. 3 FRET values and its intensities for all trials.

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accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles. _Nat. Chem._ 14, 1110–1117 (2022). https://doi.org/10.1038/s41557-022-00980-7 Download

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