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RNAs undergo phase transitions with lower critical solution temperatures

Nature Chemistry volume 15, pages 1693–1704 (2023)Cite this article

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Abstract

Co-phase separation of RNAs and RNA-binding proteins drives the biogenesis of ribonucleoprotein granules. RNAs can also undergo phase transitions in the absence of proteins. However, the physicochemical driving forces of protein-free, RNA-driven phase transitions remain unclear. Here we report that various types of RNA undergo phase separation with system-specific lower critical solution temperatures. This entropically driven phase separation is an intrinsic feature of the phosphate backbone that requires Mg2+ ions and is modulated by RNA bases. RNA-only condensates can additionally undergo enthalpically favourable percolation transitions within dense phases. This is enabled by a combination of Mg2+-dependent bridging interactions between phosphate groups and RNA-specific base stacking and base pairing. Phase separation coupled to percolation can cause dynamic arrest of RNAs within condensates and suppress the catalytic activity of an RNase P ribozyme. Our work highlights the need to incorporate RNA-driven phase transitions into models for ribonucleoprotein granule biogenesis.

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Fig. 1: Temperature-controlled phase separation of RNA homopolymers.
Fig. 2: Desolvation and Mg2+-induced bridging leads to compaction of individual poly(P) chains.
Fig. 3: Heat-induced PSCP of CAG repeat RNAs.
Fig. 4: Purine-to-uracil substitution suppresses LCST-type phase separation and percolation of repeat RNAs.
Fig. 5: Phase behaviour of RPRs.
Fig. 6: Phase separation inhibits the activity of Pfu RPR.

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All data supporting this study are included in the Article and/or the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R35 GM138186 to P.R.B., GM120582 to V.G.), the Air Force Office of Scientific Research (FA9550-20-1-0241 to R.V.P.), the St. Jude Research Collaborative on Biophysics of RNP granules (to P.R.B. and R.V.P.) and the Henry M. Jackson Foundation for the Advancement of Military Medicine (USUHS subaward 5516 to V.G.). W.J.Z. gratefully acknowledges a Pelotonia postdoctoral fellowship from the OSU Comprehensive Cancer Center. The authors acknowledge members of the Banerjee, Gopalan and Pappu labs for valuable discussions during different stages of the manuscript preparation. X.Z. thanks A. A. Chen and F.-Y. Dupradeau for helpful discussions on the forcefield parameters used in this work, and S. Tahan for support in the use of the RIS cluster at Washington University in St. Louis.

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Author notes

  1. These authors contributed equally: Gable M. Wadsworth, Walter J. Zahurancik, Xiangze Zeng.

Authors and Affiliations

  1. Department of Physics, The State University of New York at Buffalo, Buffalo, NY, USA

    Gable M. Wadsworth, Paul Pullara & Priya R. Banerjee

  2. Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH, USA

    Walter J. Zahurancik, Lien B. Lai, Vaishnavi Sidharthan & Venkat Gopalan

  3. Department of Biomedical Engineering and Center for Biomolecular Condensates, Washington University in St. Louis, St. Louis, MO, USA

    Xiangze Zeng & Rohit V. Pappu

  4. Department of Physics, Hong Kong Baptist University, Hong Kong, China

    Xiangze Zeng

  5. Teaching and Research Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China

    Xiangze Zeng

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  1. Gable M. Wadsworth
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Contributions

P.R.B. conceived the idea for this study. P.R.B. and G.M.W. designed the study with input from L.B.L., W.J.Z., V.G., X.Z. and R.V.P. G.M.W. performed the RNA phase separation experiments and data analysis with assistance from P.P. L.B.L, V.S. and W.J.Z. synthesized the different RNAs and modified them with fluorescent labels. W.J.Z. performed all the RNase P activity experiments. X.Z. and R.V.P. performed the all-atom molecular dynamics simulations, analysed the results and developed the framework to explain the observed phenomenology. All authors contributed to the writing and revision of the paper.

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Correspondence to Rohit V. Pappu, Venkat Gopalan or Priya R. Banerjee.

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Nature Chemistry thanks Hue Sun Chan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phase separation of homopolymeric RNAs.

(a) A schematic of temperature-controlled microscopy assay to probe RNA phase separation. (b) Phase separation and arrest of poly(rA) upon heating. Brightfield images of 1.5 mg/ml−1 poly(rA) in 25 mM Tris-HCl (pH 7.5 at 25 °C), 5 mM MgCl2 (25T-5M buffer) during heating (red arrow) and cooling (cyan arrow) as indicated; observed LCPT of this sample is 39.3 ± 8.5 °C. Upon cooling, poly(rA) droplets did not dissolve. (c) Brightfield images of 1.5 mg/ml−1 poly(rG) in 25 mM Tris-HCl (pH 7.5 at 25 °C), 1 mM MgCl2 (25T-1M buffer) at two different temperatures. Extensive irreversible aggregation is evident. (d) LCST-type phase separation of poly(P). Brightfield images of 1.5 mg/ml−1 poly(P) in 25 mM Tris-HCl (pH 7.5 at 25 °C), 250 mM MgCl2 (25T-250M buffer) during heating (red arrow) and cooling (cyan arrow) as indicated; observed LCPT of this sample is 35.3 ± 5.0 °C. Buffer notation used: the number in front of ‘T’ indicates [Tris-HCl] and the number in front of ‘M’ indicates [Mg2+] in mM in each buffer. Error bars represent s.e.m. for n = 3 replicates.

Extended Data Fig. 2 An emerging model for RNA phase separation coupled percolation (PSCP) behaviour.

(a) The observed hierarchy of LCST-type phase transition propensity of RNA bases and the phosphate backbone. (b) An enthalpic model of RNA phase separation. Here, RNA in an ensemble of minimum free energy structures (left) is heated, thereby denaturing the RNA. Subsequent cooling below Tph should enable the RNA to undergo a UCST transition. This model implies that enthalpic interactions such as hydrogen bonding and base-stacking drive RNA phase separation. (c) PSCP model of RNA condensation. In this model desolvation entropy drives the self-association of RNA to form phase-separated condensates upon heating. During subsequent cooling, the rank order of the phase separation temperature (LCPT or Tph) and the percolation temperature (Tprc) determines refolding vs. condensate arrest. When Tph>Tprc, the system undergoes reversible phase separation whereas in cases of Tph< Tprc, the system shows hysteretic phase behaviour. Our experimental and computational results clearly show that RNA condensation proceeds via this pathway.

Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Tables 1 and 2, legends for Videos 1–19 and references.

Reporting Summary

Supplementary Video 1

Phase separation of poly(rU). A sample of 1.5 mg ml−1 poly(rU) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 400 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy. The poly(rU) phase separated reversibly with a UCPT of 24.2 ± 0.5 °C and an LCPT of 1.1 ± 0.3 °C (n = 3 replicates).

Supplementary Video 2

Phase separation of poly(rU). A sample of 1.5 mg ml−1 poly(rU) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 500 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy. The poly(rU) phase separated reversibly with a UCPT of 25.2 ± 1.2 °C (n = 3 replicates) and no LCST was observed.

Supplementary Video 3

Phase separation of poly(rC). A sample of 1.5 mg ml−1 poly(rC) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 100 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy. The poly(rC) phase separated reversibly with an LCPT of 57.6 ± 1.9 °C (n = 3 replicates).

Supplementary Video 4

Phase separation of poly(rA). A sample of 1.5 mg ml−1 poly(rA) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 5 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy going to 80 °C. The poly(rA) phase separated irreversibly with an LCPT of 39.3 ± 8.5 °C (n = 3 replicates).

Supplementary Movie 5

Phase separation of poly(rA). A sample of 1.5 mg ml−1 poly(rA) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 5 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy going to 34 °C. The poly(rA) phase separated irreversibly with an LCPT of 32.7 °C in this trial.

Supplementary Video 6

Thermal cycling of poly(rG). A sample of 1.5 mg ml−1 poly(rG) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 1 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy going to 80 °C. The poly(rG) remained aggregated at all temperatures.

Supplementary Video 7

Movie 7. Phase separation of poly(P). A sample of 1.5 mg ml−1 poly(P) in 25 mM Tris-HCl (pH 7.5 at 25 °C) and 250 mM Mg2+ underwent thermal cycling via temperature-controlled microscopy. The poly(P) phase separated with an LCPT of 35.3 ± 5.0 °C (n = 3 replicates).

Supplementary Video 8

Phase separation of (CAG)31 RNA. A sample of 10 µM (CAG)31 in 25 mM Tris-HCl (pH 7.5 at 25 °C), 10 mM Mg2+ and 10 mM Na+ underwent thermal cycling via temperature-controlled microscopy. Observed LCPT = 66.8 ± 3.9 °C (n = 3 replicates).

Supplementary Video 9

Dynamic arrest via percolation of (CAG)31 RNA upon phase separation. A sample of 100 µM (CAG)31 in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C), 50 mM Mg2+ and 25 mM Na+ underwent phase separation at 38.0 ± 3.5 °C (n = 3 replicates) followed by the formation of an extensive percolated network of aspherical droplets. These droplets relaxed and merged into spherical droplets as the temperature was increased to 80 °C.

Supplementary Video 10

Rapid fusion of (CAG)31 droplets. A sample of 50 µM (CAG)31 phase separated at 41.1 ± 1.7 °C in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+. The droplets underwent rapid shape relaxation through coalescence as the temperature was increased above 60 °C.

Supplementary Video 11

Dissolution of (CAG)31 droplets upon addition of EDTA. A sample of 10 µM (CAG)31 RNA underwent annealing in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+. The droplets dissolved upon the addition of a small volume of 500 mM EDTA from the left of the imaging area.

Supplementary Video 12

Phase separation and arrest of a scrambled (CAG)31 sequence. A sample of 50 µM scrambled (CAG)31 is shown in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C), 50 mM Mg2+ and 25 mM Na+. An irreversible phase transition was observed with an LCPT at 57.9 ± 4.2 °C (n = 3 replicates).

Supplementary Video 13

Phase separation and arrest of (CAG)20. A sample of 155 µM (CAG)20 was prepared in 10 mM Tris-HCl (pH 7.5 at 25 °C) and 200 mM Mg2+. Irreversible phase separation was observed at 59.9 ± 2.3 °C (n = 3 replicates).

Supplementary Video 14

Reversible phase separation of (CUG)31. A sample of 50 µM (CUG)31 RNA underwent thermal cycling with an LCPT of 72.7 ± 5.4 °C in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C), 50 mM Mg2+ and 25 mM Na+. The droplets dissolved after crossing the LCPT as the temperature decreased. The same conditions for (CAG)31 resulted in irreversible droplet formation.

Supplementary Video 15

Absence of phase separation for (CUU)31. A sample of 50 µM (CUU)31 RNA underwent thermal cycling in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C), 50 mM Mg2+ and 25 mM Na+. No phase separation was observed.

Supplementary Video 16

Phase separation and percolation of Pfu RNase P RNA. A sample of 10 µM Pfu RPR was imaged in a buffer containing 50 mM HEPES–KOH (pH 7.5 at 25 °C) and 50 mM Mg2+. Irreversible phase separation was observed at 68.2 ± 1.3 °C (n = 3 replicates).

Supplementary Video 17

Phase separation and percolation of Mja RNase P RNA. A sample of 10 µM Mja RPR was imaged in a buffer containing 50 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+. Irreversible phase separation was observed at 65.9 ± 3.2 °C (n = 3 replicates).

Supplementary Video 18

Phase separation and percolation of Mma RNase P RNA. A sample of 10 µM Mma RPR was imaged in a buffer containing 50 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+. Irreversible phase separation was observed at 54.2 ± 0.9 °C (n = 3 replicates).

Supplementary Video 19

Melting of arrested Pfu RPR droplets. Droplets were prepared via annealing in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C), 25 mM Mg2+ and 10 mM Na+. The arrested Pfu RPR droplets then underwent thermal cycling via temperature-controlled microscopy showing the subsequent relaxation of the Pfu RPR condensates into spherical droplets.

Supplementary Video 20

FRAP of Pfu RPR. A sample of 10 µM Pfu RPR with 10 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+ was used for FRAP experiments (see Methods for additional details).

Supplementary Video 21

FRAP of Mja RPR. A sample of 10 µM Mja RPR with 10 mM Tris-HCl (p 7.5 at 25 °C) and 50 mM Mg2+ was used for FRAP experiments (see Methods for additional details).

Supplementary Video 22

FRAP of Mma RPR. A sample of 10 µM Mma RPR with 10 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+ was used for FRAP experiments (see Methods for additional details).

Supplementary Video 23

Reversible phase separation of Mja RPR in a refolding buffer. A sample of 20 µM RNA was prepared via a refolding protocol and diluted into refolding buffer containing 50 mM HEPES–KOH (pH 8.0 at 25 °C), 10 mM Mg2+ and 800 mM AmAc upon reaching 37 °C. Reversible phase separation was observed with an LCPT of 86.3 ± 3.1 °C (n = 3 replicates).

Supplementary Video 24

Reversible phase separation of Mma RPR in a refolding buffer. A sample of 20 µM RNA was prepared via a refolding protocol and diluted into refolding buffer containing 50 mM Tris-HCl (pH 7.5 at 25 °C), 7.5 mM Mg2+ and 500 mM AmAc upon reaching 37 °C. Reversible phase separation was observed with an LCPT of 49.2 ± 3.2 °C (n = 3 replicates).

Supplementary Video 25

Absence of an apparent phase separation of Pfu RPR in a refolding buffer. A sample of 20 µM RNA was prepared via a refolding protocol and diluted into refolding buffer containing working concentrations of 50 mM HEPES–KOH (pH 8.4 at 25 °C), 10 mM Mg2+ and 800 mM AmAc upon reaching 37 °C.

Supplementary Video 26

Absence of an apparent phase separation of Pfu RPR in a refolding buffer. A sample of 2.5 µM Pfu RPR was prepared in a buffer containing 50 mM HEPES–KOH (pH 7.5 at 25 °C), 10 mM Mg2+ and 800 mM AmAc. The sample was heated to 80 °C.

Supplementary Video 27

Phase separation of Pfu RPR in the activity assay buffer. A sample of 0.625 µM RNA was diluted into buffer containing working concentrations of 50 mM HEPES–KOH (pH 7.5 at 55 °C) and 500 mM Mg2+. During thermal cycling via temperature-controlled microscopy, irreversible phase separation was observed with an LCPT of 70.6 ± 2.6 °C (n = 3 replicates).

Supplementary Video 28

Phase separation of Pfu RPR in the activity assay buffer. A sample of 0.625 µM Pfu RPR was diluted into buffer containing working concentrations of 50 mM HEPES–KOH (pH 7.5 at 55 °C), 500 mM Mg2+ and 2 M AmAc. No phase separation was observed during thermal cycling via temperature-controlled microscopy (n = 3 replicates).

Supplementary Video 29

Phase separation of (CAG)31 RNA in the presence of dimethylsulfoxide (DMSO). A sample of 10 µM (CAG)31 RNA underwent thermal cycling in a buffer containing 10 mM Tris-HCl (pH 7.5 at 25 °C) and 50 mM Mg2+, with the addition of 5% (v/v) DMSO.

Supplementary Code 1

P values and significance for Pfu turnover assay.

Supplementary Data 1

Supplementary Information source data simulations as delimited text files.

Supplementary Data 2

Source data for Supplementary Information figures, for example, statistical source data.

Supplementary Data 3

Raw gel data.

Supplementary Data 4

Raw gel data.

Supplementary Data 5

Source data simulations for Fig. 2.

Source data

Source Data Figs. 1–6

Statistical source data.

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Wadsworth, G.M., Zahurancik, W.J., Zeng, X. et al. RNAs undergo phase transitions with lower critical solution temperatures. Nat. Chem. 15, 1693–1704 (2023). https://doi.org/10.1038/s41557-023-01353-4

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