Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization

Abstract

SUMMARY Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia and multisystem proteinopathy - diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. Here we demonstrate that the RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…The maturation of liquid droplets into aggregates has been reported for the ALS‐associated proteins FUS (Patel et al , 2015), TDP‐43 (Schmidt & Rohatgi, 2016), and hnRNPA1 (Molliex et al , 2015). After LLPS, these proteins transition from an initial liquid droplet state, through a gel‐like state of high viscosity, to a final stable aggregated form (Lin et al , 2015).…”

“…Recent studies revealed that the RNA binding and stress granule‐associated proteins FUS (Patel et al , 2015), hnRNPA1 (Molliex et al , 2015), and TDP43 (Conicella et al , 2016) have the ability to reversibly form intracellular membrane‐less organelles. These reversible droplets represent physiologically active protein or protein‐nucleic acid “bioreactors” (Hyman et al , 2014), which form through a process known as LLPS (Brangwynne et al , 2015).…”

“…These reversible droplets represent physiologically active protein or protein‐nucleic acid “bioreactors” (Hyman et al , 2014), which form through a process known as LLPS (Brangwynne et al , 2015). Such transient membrane‐less organelles have multiple cellular functions, such as p‐granule formation to establish intracellular gradients of RNA transcription (Brangwynne et al , 2009), the enrichment of RNA binding proteins in stress granules (Lin et al , 2015; Molliex et al , 2015), concentrating transcription factors in nucleoli (Berry et al , 2015), and the initiation of microtubule spindle formation (Jiang et al , 2015). However, the functional phase separation of nuclear proteins was shown to be disrupted by C9orf72 GR/PR dipeptide repeats (Lee et al , 2016) and is related to protein aggregation in neurodegeneration (Schmidt & Rohatgi, 2016).…”

“…By analogy to hnRNPA1 (Molliex et al , 2015)‐, TDP‐43 (Conicella et al , 2016)‐, FUS (Murakami et al , 2015)‐, and C9orf72‐derived dipeptide repeats (Lee et al , 2016), tau LLPS could act to initiate tau aggregation in AD and FTD. We suggest that protein LLPS may be a biophysical mechanism underlying multiple protein aggregation and neurodegenerative diseases.…”

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…These bodies, in contrast to classic organelles, locally enrich components within defined boundaries despite the lack of an outer membrane. Nucleoli (Brangwynne et al , 2011), P granules (Brangwynne et al , 2009), and stress granules (Molliex et al , 2015; Patel et al , 2015), three membrane‐less organelles, were recently reported to have liquid droplet properties (Brangwynne, 2013), supporting a role for liquid–liquid phase separation in their formation (Li et al , 2012b; Nott et al , 2015; Patel et al , 2015). The recruitment of additional components to these bodies is not well understood but must involve protein/protein interactions (Tourrière et al , 2003).…”