Multiple Modes of Protein-Protein Interactions Promote RNP Granule Assembly

Abstract

Eukaryotic cells are known to contain a wide variety of RNA-protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA-RNA, protein-RNA, and protein-protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein-protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid-liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.

Keywords: RNA; intrinsically disordered regions; phase separation.

FIGURE 1:

Schematic to show overlap of intrinsically disordered proteins, defined by computational predictions, low complexity domains, defined by a skewed representation of amino acid composition, and prion-like domains, which are computationally predicted to have high probability of forming cross β-sheet structures based on similarities to known prion domains. The size of circles and overlapping regions do not indicate exact proportions.

FIGURE 2:

The figure illustrates the general classes of protein-protein interactions that can contribute to RNP granule assembly including interactions between well-folded domains, SLiMs interacting with the surface of well-folded domains, specific interactions between local structural modules of IDRs such as LARKS, and disordered interactions between IDRs and well-folded domains based on interactions that involve at most transient ordering such as charge-charge, π-π or cation-π interactions. RNA molecules are depicted as black curves, protein molecules as colored shapes and lines. RNA-RNA interactions also play roles in RNP granule assembly and are not depicted explicitly here.

FIGURE 3:. Two archetypes of interactions mediate LLPS.

A. The presence of multiple modular binding domains in one protein, and of multiple SLiMs for these modular binding domains in the binding partner, encodes multivalent domain/motif interactions. The resulting multivalent interactions mediate the formation of large, networked complexes, which phase separate from the solution into a dense phase [53], B. IDRs with “stickers”, i.e. with interacting motifs, can undergo phase separation [16, 68]. They likely remain monomeric or form only small complexes before undergoing a highly cooperative phase transition. Polyelectrolyte IDRs can interact with oppositely charged IDRs (or nucleic acids) and undergo complex coacervation, a type of LLPS.

FIGURE 4:. Protein architectures encoding multivalency.

Multivalent domain/motif interactions can be encoded via (A) repeats of modular binding domains in a single protein chain and repeats of SLiMs in the binding partner, as in the Nck/WASP system [53], via (B) discrete oligomerization of a protein with a modular binding domain, as in Npm1 [55]m, or via (C) dimerization through two interfaces, as in SPOP [48] and TDP-43. Multivalency can also be manifest as (D) multiple “stickers” (blurry dots) on an IDR [16,68]. This multivalency can be enhanced via (E) oligomerization of the IDR, e.g. through coiled coil formation, as in TDP-43 [19].