Repeat expansion disorders such as Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are caused by an increase in the number of short nucleotide repeats within coding or noncoding regions of otherwise unrelated genes. A common pathological characteristic of these diseases is the formation of aberrant ribonucleoprotein (RNP) granules, which show features of phase-separated liquid-like compartments. Although numerous studies have characterized the ability of proteins to assemble these RNP granules, Ankur Jain and Ron Vale now demonstrate that sequence-specific multivalent base-pairing of RNAs can lead to phase separation, without necessarily requiring protein components (Nature http://dx.doi.org/10.1038/nature22386).

The authors show that RNAs with a critical number of CAG or CUG triplet repeats form viscous droplets in vitro (pictured). These can be dissolved by RNase A, monovalent cations and short complementary antisense oligonucleotides (ASOs), indicating that multivalent base-pairing and electrostatic interactions play a role in RNA clustering. CAG-repeat RNAs also form nuclear foci when expressed in cells. In contrast to their gel-like behavior in vitro, however, cellular foci show liquid-like properties. This may reflect the influence of cellular helicases that can remodel RNA base-pairing. The RNA foci colocalize with nuclear speckles, cellular compartments that are enriched in pre-mRNA splicing factors. They also sequester an endogenous RNA-binding protein, which has been implicated in repeat-expansion pathogenicity. Interestingly, agents that destabilize RNA base-pairing, such as ASOs or the nucleic acid intercalator doxorubicin, disrupt these cellular RNA foci without dissolving nuclear speckles. Guanine-rich nucleotide repeats that can form G-quadruplexes also show the propensity to form clusters, if the number of repeats is sufficient to induce multimolecular RNA interactions. This is particularly relevant because GGGGCC repeat expansion in the C9orf72 locus is one of the most common mutations associated with familial FTD and ALS.

Collectively, these data suggest that intermolecular base-pairing can trigger aggregation of RNA into nuclear foci, which may disrupt cellular homeostasis and contribute to disease-associated toxicity. While the precise pathological role of these foci remains to be established, it is curious that RNA foci and disease symptoms manifest only when the number of nucleotide repeats reach a certain threshold, an observation that may be related to the requirement of multivalency to promote phase transition.