Yoshizawa T, Ali R, Jiou J, Fung HY, Burke KA, Kim SJ, Lin Y, Peeples WB, Saltzberg D, Soniat M, Baumhardt JM, Oldenbourg R, Sali A, Fawzi NL, Rosen MK, Chook YM. Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. Cell. 2018 Apr 19;173(3):693-705.e22. PubMed.
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Massachusetts General Hospital, Harvard Medical School, German Center for Neurodegenerative Diseases (DZNE Berlin)
Six new papers in Cell and Science on droplets in very different eukaryotic systems—from mammalian cells to fungi—and involved in different cellular mechanisms. What a week!
These new works all point at the complexity of LLPS in living systems and show us the mechanisms beyond protein concentrations and modifications; they present us examples of how heteromeric interactions of biomolecules in liquid droplets can determine droplet function and how this is used by cells as solutions to regulate different essential processes in the cell, such as the nuclear-cytoplasmic transport, spatial organization and restriction of droplet formation, and RNA selectivity and droplet specification.
The two papers published in Science focus on the so far underappreciated (or at least under-addressed) role of RNA in the process of mammalian stress granule formation (Maharana et al., from the Alberti Lab) and in the dynamics and spatial separation of droplets involved in distinct cell functions in fungi (Langdon et al., from the Gladfelter Lab). Although addressing two absolutely different processes, the two papers do have a lot in common: They show that not only proteins and protein modifications play a role in RNA-binding protein biology and pathology, but introduce also the pivotal role of RNA kind, structure, and sequence in these processes. That the specifics of RNA play a big role in RNA-binding protein LLPS is not surprising, however, we now learn that not necessarily the proteins and their structure “select” the RNA, but that RNA structure and hybridization has important scaffolding activity for protein droplets and “select” the proteins—we may as well call them “RNA-protein droplets.”
One very important message delivered by these two new RNA-protein LLPS papers is the notion that the complex interplay between proteins and RNA in cellular LLPS exceeds our often protein-central view. They connect the protein world with the RNA world beyond translation and transcription, and thereby offer a fresh view on the role of different RNA species beyond encoding (mRNA) and facilitating (tRNA) protein production. With this, RNA homeostasis gets a fresh look in biology and health/disease, and a novel role of the vast amount of noncoding RNAs evolves. I am excited to see how we can embed these concepts in the field of neurodegenerative diseases such as ALS and Alzheimer’s disease, in which LLPS has been suggested to play a role for disease initiation and progression.
Thank you for these inspiring works!
View all comments by Susanne WegmannNeurobiology, KU Leuven & VIB
Overall, the different Cell papers nicely document how nuclear import proteins function as chaperones: not only to transport disease-related RNA-binding proteins through the nuclear pore, but also to suppress unwanted liquid-liquid phase separations of these proteins into stress granules (for a recent review on liquid-liquid phase separations, see Boeynaems et al., 2018). Both defects in nucleocytoplasmic transport, as well as disturbances in liquid-liquid phase separations, have recently been linked by many research groups to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Defects in both processes not only can explain how certain important disease-related proteins become mislocalized into the cytoplasm, but also provide a framework to understand the generation of aggregates. Proteins present in the droplets can undergo an irreversible liquid-to-solid phase transition, which ultimately results in the formation of solid, fibrous aggregates observed in postmortem tissue of patients.
The concept that nuclear import receptors function specifically as chaperones for wild-type and disease-linked RNA-binding proteins with a nuclear localization signal is convincingly shown by Guo et al. Karyopherin-β2/Transportin-1 interacts with the PY-nuclear localization signal (NLS) to inhibit and/or reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization. Most remarkably, Karyopherin-β2/Transportin-1 can also restore the nuclear localization of these proteins in yeast and patient fibroblasts. Interestingly, Importin-α in combination with Karyopherin-β1 performs a similar function in relation to TDP-43, and these proteins can also prevent and reverse TDP-43 fibrillization.
What makes these studies extremely interesting is the fact that a better insight is also provided into the way the nuclear import proteins are interacting with their cargos. Again, the focus is mainly on FUS and Karyopherin-β2/Transportin-1. Apart from the tight binding to the C-terminal nuclear localization signal of FUS, Yoshizawa et al. show that Karyopherin-β2/Transportin-1 disturbs the liquid-liquid phase separation by weak interactions of this importin with sequences distributed throughout FUS. Not unexpectedly, the N-terminal low-complexity domain (also called the prion-like domain) of FUS is involved in these weak interactions. Interestingly, also other domains like the RGG regions, the RRM and the zinc finger (ZnF) domain play important roles in these interactions. The effect of Karyopherin-β2/Transportin-1 on the liquid-liquid phase separation of FUS seems to be due to a competition with the FUS-FUS interactions.
Further important mechanistic insights are provided by Hofweber et al. and Qamar et al. They show that not only Karyopherin-β2/Transportin-1 but also arginine methylation of FUS have a function beyond nuclear import. This could be extremely important in the context of FTD as mislocalization and aggregation of FUS is observed in a subpopulation of FTD patients in the absence of FUS mutations. Both Karyopherin-β2/Transportin-1 and arginine methylation suppress liquid-liquid phase separation and stress granule association of FUS. These papers also add further convincing evidence to the concept that the N-terminal prion-like domain of FUS is not only important for liquid-liquid phase separation. As is clearly shown, the RGG domains and especially the arginines in these motifs also play crucial roles in liquid-liquid phase separation of FUS. These findings are in line with previous reports that the RGG-ZnF-RGG domains of FUS can form fibrous assemblies in vitro (Schwartz et al., 2013) and with our recent observation that synthetic FUS-RGG peptide forms droplets in the presence of a molecular crowder or polyU RNA (Boeynaems et al., 2017).
Overall, the observations reported here nicely complement our recent observations that the arginine-rich dipeptide repeat proteins (polyPR and polyGR) which are translated from the hexanucleotide repeats in C9ORF72 can also undergo liquid-liquid phase separations (Boeynaems et al., 2017). These dipeptide repeat proteins can bind to many proteins with low-complexity domains and can influence the liquid-liquid phase separation of arginine-rich proteins with low complexity domains, including FUS.
Now that the role of nuclear import receptors in the liquid-liquid phase separation of different disease-related RNA-binding proteins is firmly established, the next step will be to obtain more evidence that these mechanisms are also at play in more complex disease-related in vivo models. However, the biggest challenge will be to interfere therapeutically with the liquid-liquid phase separation process. As well as nucleocytoplasmic transport, it is a fundamental biological process. A controlled and cell-specific interference with such general and crucial processes will be extremely challenging. One doesn’t want to make things worse!
References:
Boeynaems S, Alberti S, Fawzi NL, Mittag T, Polymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, Van Den Bosch L, Tompa P, Fuxreiter M. Protein Phase Separation: A New Phase in Cell Biology. Trends Cell Biol. 2018 Mar 27; PubMed.
Boeynaems S, Bogaert E, Kovacs D, Konijnenberg A, Timmerman E, Volkov A, Guharoy M, De Decker M, Jaspers T, Ryan VH, Janke AM, Baatsen P, Vercruysse T, Kolaitis RM, Daelemans D, Taylor JP, Kedersha N, Anderson P, Impens F, Sobott F, Schymkowitz J, Rousseau F, Fawzi NL, Robberecht W, Van Damme P, Tompa P, Van Den Bosch L. Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics. Mol Cell. 2017 Mar 16;65(6):1044-1055.e5. PubMed.
Schwartz JC, Wang X, Podell ER, Cech TR. RNA seeds higher-order assembly of FUS protein. Cell Rep. 2013 Nov 27;5(4):918-25. Epub 2013 Nov 21 PubMed.
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