Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics

Abstract

SummaryLiquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

ALS/FTD-Associated C9ORF72 Repeat RNA Promotes Phase Transitions In Vitro and in Cells

“…Molecular crowding affects phase separation and RNA granule assembly both in vitro and in cells (Boeynaems et al, 2017; Bounedjah et al, 2012; Kedersha et al, 2016; Molliex et al, 2015). The molecular crowding agent Ficoll causes a ten-fold reduction in r(G4C2)4 and b-isox required to promote condensation (Fig 4E, lanes 2, 6, 10, 13, 17).…”

Protein Phase Separation: A New Phase in Cell Biology

“…The range of sequence biases associated with IDRs that mediate phase separation indicates that there may be a range of underlying driving forces. These likely include electrostatic, dipole–dipole, pi–pi, cation–pi, hydrophobic, and hydrogen bonding interactions [9,49,50,52–56] (Figure 2A,B). Indeed, mutational studies have demonstrated that phase separation of different LCDs can be prevented by interfering with a variety of residue types [9,10,14,16,57].…”

“…A growing understanding of the underlying molecular principles and the physicochemical forces that drive the formation of membraneless organelles (see Glossary) has enabled the elucidation of their diverse functions in a variety of cellular processes, including the stress response, the regulation of gene expression, and the control of signal transduction [5–8]. In the past few years, there has been increasing evidence for the involvement of membraneless organelles in age-related disorders, such as amyotrophic lateral sclerosis (ALS) [9–16]. Together, these discoveries have created a new field in cell biology, focused on understanding how organization of cellular matter into membraneless organelles contributes to function, and how their dysregulation leads to disease.…”

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons

“…This propensity has been localised to a low-complexity domain in the C-terminal region that may play a role in RNA binding, granule formation, and/or liquid–liquid phase separation to subserve transcription dynamics and mRNA transport [6, 12, 32, 34, 55, 56]. It is hypothesised that inherited or spontaneous mutation of the encoding gene, TARDBP , or of an interacting partner, upsets the balance between aggregation and dissolution of TDP-43 [14, 71], which may be differentially regulated under changing conditions, including during the cellular stress/death response [7, 30]. …”