Mechanisms and Regulation of RNA Condensation in RNP Granule Formation

Abstract

Ribonucleoprotein (RNP) granules are RNA-protein assemblies that are involved in multiple aspects of RNA metabolism and are linked to memory, development, and disease. Some RNP granules form, in part, through the formation of intermolecular RNA-RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. We assess the mechanisms of RNA condensation and how cells modulate this phenomenon. We propose that cells control RNA condensation through ATP-dependent processes, static RNA buffering, and dynamic post-translational mechanisms. Moreover, perturbations in these mechanisms can be involved in disease. This reveals multiple cellular mechanisms of kinetic and thermodynamic control that maintain the proper distribution of RNA molecules between dispersed and condensed forms.

Keywords: DEAD-box protein; P-body; RNA chaperone; RNA-binding protein; RNA–RNA interaction; biomolecular condensate; ribonucleoprotein granule; stress granule.

Figure 1

Ribonucleoprotein (RNP) Granules Form from a Diversity of Different Interactions. RNP granules form from a summation of multivalent protein–protein, RNA–RNA, and protein–RNA interactions that each imparts biochemical properties to define the characteristics of the granule. Different RNP granules most likely have different requirements for each type of interaction for the respective functions of the granule or for cellular regulation. In addition, each interaction type may be specific or promiscuous, as well as weak or strong, further contributing to defining an RNP granule.

Figure I

The RNA Folding Problem and RNA Chaperones. Kinetic and thermodynamic RNA chaperones function to modulate RNA folding. (A) Energy diagram of kinetic RNA chaperone function. Compared with unassisted RNA melting and refolding (solid black curve), kinetic RNA chaperones utilize dynamic binding to destabilize structures and facilitate structural transitions by partial RNA unfolding and strand displacement, thereby lowering the activation energy of the structural transition (dashed blue curve). Depending on the structure and context, kinetic RNA chaperones may promote different degrees of unfolding. For example, DEAD-box proteins can completely melt small RNA duplexes [75,76,137]. (B) Reaction diagram of RNA chaperone function. (Left) Kinetic RNA chaperones accelerate RNA unfolding through dynamic binding. Although the bracketed structure is depicted as being completely unfolded for clarity, kinetic RNA chaperones can accelerate RNA refolding through partially unfolded intermediates. The energy of these complexes determines the activation energy (red asterisks). (Right) Thermodynamic RNA chaperones utilize high-affinity binding to reduce the free energy of an RNA structure. One means of doing so is by binding unstructured regions to prevent them from interacting (blue protein). A second means is to bind to and stabilize particular RNA structures (purple protein).

Figure 2

Mechanisms by Which RNA Chaperones Limit RNA Condensation. RNA chaperones have multiple mechanisms to limit RNA condensation. (A) Thermodynamic decondensers (red) use high-affinity RNA binding to compete for RNA–RNA interaction sites so as to limit RNA condensation. Thermodynamic decondensers therefore lower the valency of RNA and prevent RNA conformational changes by locking RNA conformers in ΔG wells. (B) Kinetic dencondensers prevent RNA condensation by destabilizing trans RNA–RNA interactions, therefore lowering the activation energy barrier between trans interacting RNAs and dispersed states. Kinetic decondensers could also promote cis RNA refolding, thereby reducing the valency of a given RNA. Because kinetic RNA decondensers such as DEAD-box proteins destabilize RNA secondary structure by RNA binding and not by ATP hydrolysis, the key difference between a thermodynamic and kinetic RNA decondenser is the relative off-rate for RNA, with kinetic decondensers binding dynamically. Kinetic decondensers such as eIF4A (orange) can function as thermodynamic decondensers in the absence of ATP hydrolysis [16].

Figure 3

Mechanisms by Which RNA Chaperones Promote RNA Condensation. RNA chaperones can promote RNA condensation through kinetic and thermodynamic modes. (A) Thermodynamic condensers increase the valency of RNA by contributing stable protein–protein interactions to a ribonucleoprotein (RNP). In addition, high-affinity RNA binding results in an RNA–protein co-condensate that reduces the exchange of RNP granule components. (B) Kinetic condensers raise the rate of forming trans RNA–RNA interactions, for example, by imparting a proximity effect between RNAs. Such kinetic condensers utilize protein–protein interactions to dimerize, coupled with tandem RNA binding that positions RNAs for interactions in trans. Because kinetic condensers increase the frequency of properly orientated collisions between RNA molecules for trans interactions, kinetic condensers generate rate enhancements by increasing the pre-exponential factor of the Arrhenius equation. Coupled with a high off-rate for RNA, kinetic condensers can recycle on dispersed RNPs. Therefore, the key difference between a kinetic and thermodynamic RNA condenser is the relative off-rate for RNA binding.