Molecular Drivers of RNA Phase Separation

  1. V Ramachandran
  2. DA Potoyan

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Abstract

RNA molecules are essential in orchestrating the assembly of biomolecular condensates and membraneless compartments in cells. Many condensates form via the association of RNA with proteins containing specific RNA binding motifs. However, recent reports indicate that low-complexity RNA sequences can self-assemble into condensate phases without protein assistance. Divalent cations significantly influence the thermodynamics and dynamics of RNA condensates, which exhibit base-specific lower-critical solution temperatures (LCST). The precise molecular origins of these temperatures remain elusive. In this study, we employ atomistic molecular simulations to elucidate the molecular driving forces governing the temperature-dependent phase behavior of RNA, providing new insights into the origins of LCST. Using RNA tetranucleotides and their chemically modified analogs, we map RNA condensates’ equilibrium thermodynamic profiles and structural ensembles across various temperatures and ionic conditions. Our findings reveal that magnesium ions promote LCST behavior by inducing local order-disorder transitions within RNA structures. Consistent with experimental observations, we demonstrate that the thermal stability of RNA condensates follows the Poly(G) > Poly(A) > Poly(C) > Poly(U) order shaped by the interplay of base-stacking and hydrogen bonding interactions. Furthermore, our simulations show that ionic conditions and post-translational modifications can fine-tune RNA self-assembly and modulate condensate physical properties.

Author Summary

RNA molecules are essential for organizing membraneless compartments that play critical roles in cellular processes. While many of these condensates form through interactions between RNA and proteins, recent studies have shown that certain RNA sequences can self-assemble into condensates without protein assistance. This ability is influenced by the sequence composition of RNA and the presence of ions like magnesium. Using detailed molecular simulations we carried out systematic study to reveal how temperature and ionic conditions affect RNA condensation. We discovered that magnesium ions play a key role in driving RNA molecules to condense at lower temperatures by promoting structural changes within the RNA. Our findings also revealed that the stability of RNA condensates varies depending on the RNA sequence, with guanine-rich sequences being the most stable. Additionally, we demonstrated how chemical modifications and ionic conditions can fine-tune the properties of RNA condensates. This study provides new insights into how RNA forms condensates and highlights potential strategies to control their behavior, which could have implications for understanding cellular organization and developing new therapies.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/16728889.

    Preprint

    Molecular Drivers of RNA Phase Separation, https://www.biorxiv.org/content/10.1101/2025.01.20.633842v1

    Authors of the review

    Salvatore Di Marco, Giovanni BussiThis report was written after a journal club given by Salvatore Di Marco in the bussilab group meeting. All the members of the group, including external guests, are acknowledged for participating in the discussion and providing feedback that was useful to prepare this report.The corresponding authors of the original manuscript were consulted before posting this report. This report refers to bioRxiv version 1. Supplementary Information were provided to us by the corresponding author.

    Summary

    The authors perform all-atom molecular dynamics simulations investigate the molecular basis of the temperature-dependent phase behaviour of RNA. They simulate RNA tetranucleotides, under different ionic conditions and temperatures, to visualize the different impact of monovalent and divalent cations on RNA condensation. The main findings of the manuscript are that magnesium ions can induce lower-critical solution temperatures behaviour and that RNA condensation occurs with different strength for different nucleobases. Indeed, it was shown that G tetranucleotide forms denser clusters than A, followed by C and U. The authors also analyse hydrogen bonds, base-stacking and ion contacts to explain the molecular mechanism underlying the phenomena. We found the paper quite interesting and we liked that it was able to qualitatively describe previous experimental work.

    Comments

    • It could be useful for the interpretation of the results if the authors clarified the density of tetranucleotides before the extension of the box. Is the density of the tightly packed tetranucleotides still lower than 500mg/ml?

    • The authors report that convergence of the simulation has been verified on the last 2microseconds of simulation. This suggests that simulations last for at least 2 microseconds, but simulation durations are not clear from the text. A table containing simulation times for each system would enhance clarity

    • The authors employed microMg for Mg2+ simulation, which was optimized for having correct binding free energies and water exchange kinetics, of the order of microseconds. Have the authors considered using nanoMg, which is expected to have also correct binding free energies, but has an accelerated kinetics?

    • Additional details of how the DBSCAN algorithm was used to assess cluster percentage could be helpful. This could clarify questions like: how is the cluster percentage computed if there are multiple clusters? How big are these individual clusters, if multiple clusters participate to the cluster percentage?

    • Clarification on the distance and angle thresolds used by Prolif to compute the number of contacts due to hydrogen bonding, base stacking etc could be beneficial.

    • The authors write that even minimal concentrations of Mg2+ impact condensation (page 5), but they are employing a Mg2+ concentration of 65mM, which is large compared to physiological concentrations, this could be a bit misleading.

    • Force field parameters used for m6A should be discussed explicitly.

    • The definition of the number density definition is unclear from the plots. Is it the number density along the z-direction of the box, averaged over time? How are the lines so constant in some cases? (e.g. Fig. 1(A))

    • An explanation of the reasoning for the choice of the different temperatures could be useful. Have the authors verified that in the systems at 390K water is displaying the same phase behaviour as lower temperatures? For instance, TIP4P water model displays a phase transition at 363K (https://doi.org/10.1063/1.2085031).

    • The authors discuss the temperature-dependent binding of Mg2+ with RNA, and observe increased Mg2+ binding with RNA at higher temperatures. Has the coupling of increased temperature and accelerated kinetics been considered? By a back-of-the-envelop calculations, since the barrier for Mg-phosphate unbinding is ~ 15 kcal/mol, the unbinding kinetics might be accelerated by a factor = exp((1/270-1/390)*15/0.00198) ~ 5600 when going from T=270 K to T=390 K. For instance, have the authors investigated if the clusters stabilized at high T dissolve or persist upon cooling?

    • Writing remarks

      • In the text, sometimes the authors refer to cluster size and cluster percentage in an interchangeable way. The former is used in the text and the latter in the figures. We suggest using only one of the two.

      • Consistency in the colours between nucleobases could aid clarity: e.g in Fig. 4, G4 is orange in (A), but green in (C).

    • Typos

      • Fig.5 (A),(G),(H): "amino acids" should be replaced with "nucleobases". We suggest writing something more standard instead of AM in the plot, such as m6A.

      • Superscripts for Mg2+ or subscripts for NH2 are sometimes written incorrectly.

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2025.01.20.633842 on bioRxiv