ATP:Mg2+ shapes material properties of protein-RNA condensates and their partitioning of clients

Abstract

Many cellular condensates are heterotypic mixtures of proteins and RNA formed in complex environments. Magnesium ions (Mg²⁺) and ATP can impact RNA folding, and local intracellular levels of these factors can vary significantly. However, the effect of ATP:Mg²⁺ on the material properties of protein-RNA condensates is largely unknown. Here, we use an in vitro condensate model of nucleoli, made from nucleophosmin 1 (NPM1) proteins and ribosomal RNA (rRNA), to study the effect of ATP:Mg²⁺. While NPM1 dynamics remain unchanged at increasing Mg²⁺ concentrations, the internal RNA dynamics dramatically slowed until a critical point, where gel-like states appeared, suggesting the RNA component alone forms a viscoelastic network that undergoes maturation driven by weak multivalent interactions. ATP reverses this arrest and liquefies the gel-like structures. ATP:Mg²⁺ also influenced the NPM1-rRNA composition of condensates and enhanced the partitioning of two clients: an arginine-rich peptide and a small nuclear RNA. By contrast, larger ribosome partitioning shows dependence on ATP:Mg²⁺ and can become reversibly trapped around NPM1-rRNA condensates. Lastly, we show that dissipative enzymatic reactions that deplete ATP can be used to control the shape, composition, and function of condensates. Our results illustrate how intracellular environments may regulate the state and client partitioning of RNA-containing condensates.

Figure 1

Mg²⁺-induced RNA compaction leads to slow dynamics and gelation. (A–C) NPM1 protein and rRNA, both labeled with different fluorophores, are mixed together to form condensates (A) that show differences in FRAP recovery (B and C), where NPM1 (green) recovers faster than rRNA (red). (D) The partitioning coefficients (Kp) for NPM1 and rRNA within the condensates at different Mg²⁺ concentrations. (E) The recovery of rRNA slows to a halt at Mg²⁺ ion concentrations >7 mM (shaded in yellow) and shows critical scaling behavior (purple fitted line), indicating that it forms a gel, whereas the NPM1 protein remains mobile. (F) The decrease in percentage recovery for both NPM1 and rRNA reflects the gel environment at higher Mg²⁺ concentrations. (G) These droplets become gels at higher Mg²⁺ where the rRNA is fully arrested, indicated by bleached regions not recovering (white arrows). (H–J) In order to test the RNA compaction hypothesis, NPM1-rRNA condensate morphology (H) and FRAP recovery parameters (I and J) were compared with NPM1-pA and NPM1-pU condensates. The errors in this figure are standard deviations from triplicate measurements. Scale bars are all 10 μm.

Figure 2

Temperature and ATP can reverse the effect of Mg²⁺-induced RNA condensation. (A) NPM1-rRNA condensates aging at 20°C with individual τ for rRNA measured over time (dots) and a fitted exponential curve (line). (B) The extracted plateau τ at maturation for rRNA (red) and NPM1 (green) was plotted for aged droplets at different temperatures in 0 mM Mg²⁺ buffer. The errors here are derived from the exponential fits. (C) Forming NPM1-rRNA condensates at 8°C in buffer containing magnesium resulted in irregular gel-like morphologies that changed to spherical droplets at increasing temperatures, with increased circularities. (D) At 20°C, the gels formed in 14 mM Mg²⁺ buffer also liquefied after 11 mM ATP addition, with increasing circularity reflected in the changed morphology of the NPM1-rRNA condensates. (E) The FRAP recovery over times for samples that contain ATP (yellow), ADP (orange), or AMP (blue) compared with just the 5 mM Mg²⁺ buffer (gray). (F and G) The extracted τ (F) and percentage recoveries (G) indicate that the better Mg²⁺-chelating ability of ATP compared with other adenosine nucleotides caused the condensates to liquefy. The errors in (E)–(G) are standard deviations from at least duplicate measurements. Scale bars are all 10 μm. To see this figure in color, go online.

Figure 3

The effect of Mg²⁺ and ATP on partitioning of components, clients, and 70S ribosomes. (A and B) The partitioning coefficients of rRNA (A) and NPM1 (B) at different Mg²⁺ and ATP concentrations. (C) The partitioning coefficients of 5,6-FAM-RP3 peptide (blue) and 5,6-FAM-SNORD52 RNA (orange) at different ATP concentrations in a 5 mM Mg²⁺ base buffer. (D and E) Confocal microscope images of NPM1-rRNA condensates with 70S ribosome as the client. The images in (D) show that rRNA-A647 is excluded from the ribosome fluorescence, whereas (E) shows NPM1-A488 fluorescence as spherical droplets that occupy both locations where there is 70S ribosome and rRNA. (F) The formation of the ribosome halo over time as indicated by an increase in fluorescence pixel intensity as peaks at the edges of the droplet. The errors in this figure are standard deviations from triplicate measurements. Scale bars are all 10 μm.

Figure 4

Enzymatic control of ATP concentrations influences condensate morphology and dynamics. (A) Apyrase enzymes catalyze the removal of phosphate groups from ATP to form AMP, a nucleotide that poorly chelates Mg²⁺. (B–D) The effect of apyrase on the dynamics of rRNA after FRAP is clearly shown, where rRNA recovery in the presence of ATP (yellow) drops when apyrase (purple) is added. (C and D) In fact, the resulting FRAP parameters show that apyrase converts ATP to AMP, which imbues the rRNA with gel-like dynamics, similar to condensates made in 5 mM Mg²⁺ buffer (grey). (E–G) The morphology of NPM1-rRNA condensates at 18°C in 5 mM Mg²⁺ buffer (E) with 5 mM ATP added (F) and when apyrase is also added (G) shows the changes in morphology that is expected from gel-like condensates in buffer containing Mg²⁺ that stabilizes RNA-RNA interactions and spherical morphology in conditions where ATP chelates the Mg²⁺ and liquefies these interactions. (H) This series of confocal images over time of labeled 70S ribosomes shows the disappearance of the ribosome halo and puncta as apyrase was added. ATP depletion results in higher Mg²⁺, which stabilizes the ribosomes and causes them to dissipate back into the dilute phase. The errors in this figure are standard deviations from triplicate measurements. Scale bars are all 10 μm. To see this figure in color, go online.