RNA modulates physiological and neuropathological protein phase transitions

Abstract

Aggregation of RNA-binding proteins (RBPs) is a pathological hallmark of neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In these diseases, TDP-43 and FUS RBPs are depleted from the nuclear compartment, where they are normally localized, and found within cytoplasmic inclusions in degenerating regions of affected individuals' postmortem tissue. The mechanisms responsible for aggregation of these proteins has remained elusive, but recent studies suggest liquid-liquid phase separation (LLPS) might serve as a critical nucleation step in formation of pathological inclusions. The process of phase separation also underlies the formation and maintenance of several functional membraneless organelles (MLOs) throughout the cell, some of which contain TDP-43, FUS, and other disease-linked RBPs. One common ligand of disease-linked RBPs, RNA, is a major component of MLOs containing RBPs and has been demonstrated to be a strong modulator of RBP phase transitions. Although early evidence suggested a largely synergistic effect of RNA on RBP phase separation and MLO assembly, recent work indicates that RNA can also antagonize RBP phase behavior under certain physiological and pathological conditions. In this review, we describe the mechanisms underlying RNA-mediated phase transitions of RBPs and examine the molecular properties of these interactions, such as RNA length, sequence, and secondary structure, that mediate physiological or pathological LLPS.

Figure 1.. Examples of biophysical state dictating structure function in vivo.

(a) Within the central channel of the nuclear pore complex (NPC), hydrophobic interactions between FG-repeat regions of the FG-nucleoporins (i.e. Nup98, Nup62) contribute to the formation of a hydrogel-like assembly. This gel-like state forms a permeability barrier to block the translocation of nonspecific cargo in and out of the nucleus, while allowing for specific interactions between nuclear transport receptors and nucleoporins that drive cargo tranport. (b) The nucleolar protein NPM1 utilizes electrostatic interactions provided by acidic and basic tracts within its central IDR to undergo LLPS as part of the the granular component (GC) of the nucleolus. The liquid-like state of the GC allows for rapid exchange of newly-transcribed and processed rRNA, from the dense fibrillar component (DFC) and the fibrillar core (FC) subcompartments of the nucleolus, and ribosomal subunits, from the surrounding nucleoplasm, that is required for proper ribosomal assembly and export.

Figure 2.. RNA concentration is an important determinant of RBP phase behavior in vitro and in vivo.

(a) In vitro LLPS of RBPs can be stimulated by the addition of low concentrations of RNA, wherein excess concentrations of RBPs are allowed to multimerize on single RNA molecules and undergo phase transitions. In contrast, in vitro phase separation of RBPs resulting from molecular crowding or high RBP concentrations can be buffered by the addition of RNA at high/excess concentrations. The increased availability of RNA binding partners can prevent RBP multimerization on single RNAs and thus may prevent/reverse LLPS. (b) The effects of these molar ratios of RBP:RNA can be observed in the cell, where high RNA:RBP ratios in the nucleus maintain RBP solubility and low RNA:RBP ratios in the cytoplasm can promote physiological/pathological phase transitions.

Figure 3.. RNA length contributes to regulation of RBP LLPS and MLO dynamics.

(a) RNA length seemingly inversely correlates with its capacity to buffer phase transitions of RBPs (i.e. FUS). The low binding occupancy of short oligonucleotides and/or tRNA molecules may prevent multiple RBP binding, while longer RNAs (i.e. NEAT1_2 lncRNA, rRNA) may contain many binding sites that enable RNA scaffolding of RBP LLPS. Longer RNA length also may promote RNA:RNA interactions that contribute to LLPS and MLO formation, while shorter RNAs have less capacity to form these multivalent RNA/RBP networks. (b) Examples of different intracellular condensates/MLOs that contain RNAs of different lengths. While short RNA oligonucleotides are capable of effectively buffering RBP phase transitions in the cell (i.e. bait RNA oligonucleotides), longer RNAs tend to promote the formation of MLOs and tune their characteristics. MLOs with longer RNA components may exhibit a less dynamic biophysical state (i.e. myogranules, Titin mRNA) than those containing shorter RNA components (i.e. stress granules, non-translating mRNA) due to an increased propensity for RNA self-interaction and reduced molecular exchange.