key: cord-0882009-louivjim
authors: Tauber, Devin; Tauber, Gabriel; Parker, Roy
title: Mechanisms and Regulation of RNA Condensation in RNP Granule Formation
date: 2020-05-11
journal: Trends Biochem Sci
DOI: 10.1016/j.tibs.2020.05.002
sha: f93ee6b4bfbe21346fd7bfea31b7d4dc95a2886f
doc_id: 882009
cord_uid: louivjim

Abstract Ribonucleoprotein (RNP) granules are RNA-protein assemblies involved in multiple aspects of RNA metabolism and are linked with memory, development and disease. Some RNP granules form, in part, through formation of intermolecular RNA–RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. Herein, we assess 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 to maintain the proper distribution of RNA molecules between dispersed and condensed forms.

J o u r n a l P r e -p r o o f 7 compartmentalize long untranslating mRNAs, such as SGs [51] or bacterial RNA (BR)-bodies in C.

crescentus [52] [53] , may simply assemble through relatively random interactions between long RNAs.

Random interactions could also result from conformational diversity (and thus diversity of potential trans interactions), increasing the probability of kinetic traps. In contrast, some RNP granules assemble, at least in part, by genetically programmed RNA-RNA interactions, which are likely tied to particular RNA folds. For example, during Drosophila oogenesis, defined base-pairing interactions between the oskar or bicoid 3ʹ untranslated regions (UTRs) target them to specific maternal mRNP granules [54] [55] .

Evolutionary pressure might also lead to the evolution of mRNA sequences and structures that limit their ability of base-pair with RNAs present in a particular RNP granule. For instance, in Ashbya gosypii, cis-RNA duplexes are suggested to limit the ability of the CLN3 mRNA to interact with the BNI1 mRNA and thereby allow the CLN3 mRNA to be physically segregated from the BNI1 mRNA in different RNP granules [18] . Finally, non-canonical RNA-RNA interactions may also contribute to RNA condensation in biological contexts. For example, the packaged genomes of several dsRNA viruses are dsRNA liquid crystalline condensates [56] [57] [58] [59] .

The available evidence suggests that RNAs will generally fold in cis first, and then form intermolecular RNA-RNA interactions between surface regions of each RNA that promote RNA condensation. Specifically, it is observed that mRNAs fold in cis into a compact structure as ribosomes run-off, and then later accumulate in RNP granules [37] . Moreover, estimates of the amount of proteins on mRNAs suggests there will be substantial regions of RNA that are not coated with proteins, particularly when untranslating and in the cytosol (see Box 2) . This suggests the formation of J o u r n a l P r e -p r o o f 10 1). Similarly, in bacteria, the protein CspA ameliorates the formation of RNA structures, which could be in cis or trans, during the cold shock response [70] . Like YB-1, CspA is abundant, with an intracellular concentration of ~30 µM, and has high affinity for RNA [70] . The role of abundant RBPs in limiting RNA condensation may explain why RBPs are at substantially higher concentration in the nucleus relative to RNA, thereby limiting the condensation of nascent transcripts [71] .

In a second manner, kinetic RNA decondensers, such as some DEAD-box proteins, can act to lower the activation energy barriers between folded and unfolded RNA conformations and decrease the valency of RNAs by resolving trans interactions and/or promoting cis RNA refolding ( Figure 2B ). For example, the translation initiation factor eIF4A functions to limit RNA condensation both in vitro and in cells [16] . In this mechanism, eIF4A and other related proteins, such as DDX19A/DBP5 [16, 39, 72] , lower the activation energies between folded RNA and unfolded RNA secondary structure through ATPdependent RNA binding, which disrupts several nucleotides in the structured RNA. By decreasing the stability of RNA-RNA interactions, this increases the rate at which the given RNA can unfold or undergo conformational transitions. In the absence of ATP hydrolysis, eIF4A acts as a thermodynamic decondenser by competing for trans RNA-RNA interactions. However, ATP hydrolysis and P i release promotes eIF4A dissociation [73] [74] and allows for RNA refolding either in cis or in trans, with the former favored by the proximity effect. ATP hydrolysis and P i release also frees the eIF4A protein to engage in additional cycles of RNA structural rearrangements [73] [74] [75] [76] [77] , which facilitates more effective RNA decondensation [16] . This indicates that the key difference between a kinetic and thermodynamic decondenser is the off rate for RNA. Other DBPs likely function to limit RNA condensation. For example, knocking down UAP56/DDX39B, which is related to eIF4A and consists of the conserved DBP core domain [78] , increases nuclear speckle size and traps mRNAs in speckles [79] [80] .

Mechanisms to promote RNA condensation J o u r n a l P r e -p r o o f 3). Thermodynamic RNA condensers would reduce the ∆G of RNP condensation through RNA binding.

Potential mechanisms include binding intermolecular RNA structures with high affinity, shielding phosphate backbone repulsions to stabilize RNA-RNA interactions, or binding exposed RNA with high affinity and crosslinking individual mRNPs through protein-protein interactions ( Figure 3A ).

Thermodynamic condensers should generally have a low mobile fraction in RNP granules due to their high affinity for RNA and would produce granules when overexpressed, playing roles in promoting RNP granule formation. In general, thermodynamic condensers should be at low intracellular concentrations to prevent irreversible RNP complex formation. Examples of thermodynamic condensers are seen in yeast PBs where Dcp2, Pat1, and Edc3 all have low mobile fractions, exchange slowly, and promote PB formation [81] [82] [83] . Since RNA binding can be specific, thermodynamic condensers might be important for the compartmentalization of specific RNAs into specialized granules such as neuronal or germ granules. For example, FMRP, Pumilio, and Ataxin2 are slow-exchanging, multivalent RBPs with relatively low intracellular concentrations (Table 1) . These proteins may aid in the formation or integrity of neuronal granules [2, [84] [85] . Additionally, RNA and thermodynamic condensers could undergo cooperative phase transitions [19, 86] .

In a second mechanism, we predict that some RNA chaperones will act as kinetic RNA condensers, which reduce the activation energy of forming a trans RNA-RNA interaction. One mechanism to accomplish this effect is unwinding intramolecular structures to allow for intermolecular interactions to occur. Alternatively, RNA condensation could be promoted by utilizing dynamic protein multimerization to bring RNAs into close proximity and thereby promote the formation of intermolecular RNA-RNA interactions ( Figure 3B ). This mechanism is similar to how the Hfq complex in Such a proximity effect would in turn accelerate the formation of intermolecular RNA-RNA interactions, thus allowing cooperativity between RNA-RNA and protein-protein interactions to promote RNP condensation.

The above principles make predictions about the behavior of kinetic condensers in RNP granules. Kinetic condensers are predicted to have a high mobile fraction in RNP granules since their ability to efficiently and promiscuously condense RNAs relies on having a high off-rate to act in multiple cycles; the key difference between a kinetic and thermodynamic condenser is the off-rate for RNA.

Kinetic condensers are also expected to promote RNP granule formation when overexpressed, reduce it when deleted, and might have a high intracellular concentration to overcome decondensing machinery.

Interestingly, several RBPs in SGs (Table 1) , such as G3BP1, UBAP2L, and TIA-1, meet these criteria since they are highly dynamic in SGs, create SGs when overexpressed, and reduce SG formation when depleted [91-94]. Importantly, SGs require both RNA binding and dimerization of these proteins to form [92] [93] [94] , yet increasing G3BP homodimerization potential does not modify its exchange rate in SGs [12] , suggesting that protein-protein interactions do not control G3BP exchange. In contrast, increasing the RNA binding capacity of G3BP reduces its mobility in SGs, suggesting that WT G3BP has a high off-rate for RNA compared to slow-exchanging, tighter-binding proteins in Drosophila the protein Exu is important for proper oskar and bicoid localization to RNP granules [96] [97] [98] . Exu binds structured elements in the bicoid 3ʹ UTR and Exu RNA binding and dimerization is essential for bicoid's proper localization [99] , which is facilitated in part through homotypic trans RNA-RNA interactions between bicoid 3ʹ UTRs [55, 100] . This is consistent with a model where Exu dimerization catalyzes trans base-pairing between bicoid 3ʹ UTRs.

DEAD-box proteins can also promote RNP granule assembly through additional interactions and the regulation of their ATPase activity. The tail domains can regulate the RNA binding and ATPase activities of DBPs [101] [102] [103] [104] , or engage in protein-protein interactions [103, [105] [106] ] that modulate DBP function and facilitate RNP assembly [80] . Thus, in the ATP-bound state, a DBP bound to RNA and interacting with other proteins can promote assembly of the RNP granule [80, 106] . Subsequently, ATP hydrolysis allows the release from the RNA, and potentially can contribute to transitions or disassembly of the RNP assembly [80, [105] [106] [107] . For example, the DDX3X/Ded1 protein has N-and C-terminal domains that interact with eIF4E, eIF4G, and itself to promote assembly of SG in the ATP bound state, and when ATP is hydrolyzed contributes to release of RNAs from SG [80, [107] [108] [109] J o u r n a l P r e -p r o o f 14 their RGG RNA binding domains, which promotes SG assembly [93, 112] . Methylation of arginine residues can disrupt interactions between arginine and delocalized π-systems like nucleobases [113] , with methylation predicted to interfere with RNA binding. Thus, demethylation might increase RNA binding of these factors, promoting RNA condensation. Additional modifications that can affect RNP granule formation include glycosylation [114] , and therefore should inhibit eIF4A's ability to limit RNA condensation.

Taken together, we anticipate that a dense network of post-translational protein modifications will modulate the function of proteins influencing RNP granule formation.

Perturbations in the cell's machinery for regulating RNA condensation may be involved in human disease. For example, many viruses, including noroviruses, Dengue virus, and the MERS coronavirus, whose replication is reduced by SGs, subvert the formation of SGs [120-122]. In another possibility, failure in the RNA decondensing machinery of the cell could cause aberrant RNA condensation, potentially causing an "RNA entanglement catastrophe" [16] . For instance, mutations affecting the [110, 126] . Interestingly, loss-of-function mutations in the DEAD-box protein regulator Gle1 result in a fatal degenerative motor neuron disease called lethal congenital contracture syndrome (LCCS), which may result from aberrant ribostasis [127] [128] . Moreover, one mechanism of toxicity of J o u r n a l P r e -p r o o f 15 repeat expansion RNAs appears to be the formation of RNA aggregates through RNA-RNA interactions [25] , which can sequester RBPs, thereby altering RNA processing in pathogenic manners [129] [130] [131] [132] .

Notably, expression of repeat expansion RNAs, or promoting RNP granule formation by knockdown of DHX36 can activate the dsRNA-sensing eIF2ɑ kinase PKR and the integrated stress response [133] [134] [135] [136] .

This raises the possibility that aberrant RNA condensation triggers a cellular response, which in some cases may lead to toxic effects. • DEAD-box proteins (DBPs): ATP-dependent RNA binding proteins with high affinity for RNA when complexed with ATP, and low affinity following ATP hydrolysis and P i release. Because they can disrupt RNA-RNA interactions through ATP-dependent RNA binding, they are often referred to as "helicases," though unlike canonical DNA helicases, they are typically nonprocessive and can only resolve duplexes of limited size. Reviewed in [139] .

• Excluded volume effect: Polymer molecules in solution occupy volume (the excluded volume), reducing the effective volume available for other molecules to diffuse and increasing their effective concentration. As the excluded volume of polymers like crowding agents increases, so too do intermolecular collisions and interactions.

• Intrinsically disordered regions: Regions of protein sequence that do not adopt a defined secondary structure or exhibit conformational flexibility and can engage in promiscuous and dynamic interactions.

• Kinetic RNA condensers: Proteins that promote intermolecular RNA interactions by increasing the rate of trans RNA-RNA interaction formation, for example, by bringing RNAs into proximity ( Figure 3B ).

• Kinetic RNA decondensers: Proteins that reduce utilize dynamic binding to promote the dissociation of cis or trans RNA interactions and thereby accelerate RNA refolding ( Figure 2B ). molecules will occur. This is achieved by concentrating reactant surfaces available for interaction and reducing the randomness of molecular orientations.

• RNA self-assembly/condensation: Collectively refers to the processes by which RNA in solution spontaneously forms a condensed assembly through RNA-RNA interactions. dsRNA processes. An RNA aggregate is a nonspecific, stable RNA condensate.

• RNA chaperone: Analogous to protein chaperones, RNA chaperones combat improper interactions, such as misfolding or aggregation, and promote proper RNA interactions. They can act kinetically, by altering the activation energy between RNA conformers, or thermodynamically, by utilizing specific RNA binding to bias RNA folding and interaction equilibria to particular conformers (see Box 1).

• RNA entanglement catastrophe: The hypothesis that aberrant RNA aggregation is driven by spontaneous RNA condensation. Since cis and trans RNA interactions can influence each other [138] , intermolecular RNA-RNA interactions contribute to the RNA folding problem, particularly in the context of RNP granules, which have elevated RNA concentrations and stabilize RNA-RNA interactions [16] . The compartmentalized conditions of condensates may also promote RNA ensemble redistribution [138] . Thus, one would predict that RNA chaperones similarly modulate the kinetics and thermodynamics of intermolecular RNA interactions and RNA self-assembly. 

An important question (reviewed in [71] ) is to what extent is an mRNA coated with protein? Notably, many RBPs bind RNA dynamically, meaning that mRNP composition is continually changing. Both footprinting studies [149-150] and CsCl 2 buoyant density gradient measurements lead to estimates that 50-80% of the mass of nuclear mRNPs is protein, though with lower RBP concentrations and higher RNA compaction in the cytosol, cytosolic mRNPs likely have lower protein compositions [71] . While these are rough estimates, they suggest that much or most of the mRNA in a cytosolic mRNP is not bound by protein and is free to engage in cis or trans interactions [71] .

J o u r n a l P r e -p r o o f 40 Ribonucleoprotein (RNP) granules form from a summation of multivalent protein-protein, RNA-RNA, and protein-RNA interactions that each impart biochemical properties to define the characteristics of the granule. Different RNP granules most likely have different requirements for each type of interaction for the granule's respective functions, or for cellular regulation. Additionally, each interaction type may be specific or promiscuous as well as weak or strong, further contributing to defining an RNP granule. RNA chaperones have multiple mechanisms to limit RNA condensation. (A) Thermodynamic decondensers (red) use high-affininty RNA binding to compete for RNA-RNA interaction sites in order to limit RNA condensation. Thermodynamic decondensers therefore lower the valency of RNA and prevent RNA conformational changes by locking RNA conformers in ∆G 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 also promote cis RNA refolding, thereby reducing the valency of a given RNA. Due to the fact that 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] .

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We thank the Parker lab for comments and discussion. This work was funded by NIH SCR training grant