For better or worse, liquid-liquid phase separation (LLPS) happens. It’s beneficial when RNA and RNA-binding proteins (RBPs) form dynamic liquid assemblies required for many cell functions. It’s pathological when the same assemblies yield the toxic fibrils that mark many neurodegenerative diseases. In a leap forward, six papers illuminate new pathways that promote the better, and prevent the worse. It’s all about protein chaperones, posttranslational modifications, and RNA structures.
- Nuclear-import receptors and arginine methylation regulate phase separation and aggregation of RNA-binding proteins in the cytosol.
- These receptors dissolve fibrils and hydrogels, and mitigate neurodegeneration in model systems.
- In the nucleus, high RNA concentrations prevent aggregation of FUS, other RBPs.
Four papers in the April 19 Cell reveal that nuclear import receptors (NIRs) potently inhibit and even reverse phase transition and fibril formation of several pathogenic RBPs. Two studies, one from the labs of Yuh Min Chook and Michael Rosen, University of Texas Southwestern Medical Center, Dallas, and another led by James Shorter at the University of Pennsylvania in Philadelphia and Paul Taylor, St. Jude’s Children’s Research Hospital, Memphis, Tennessee, show that transportin proteins, best known for shuttling RBPs into the nucleus, appear to have another function. In the cytosol, they protect against aberrant aggregation of disease-related RBPs, including FUS, TDP-43, and others. Alzforum first reported on this data from the Phase Transitions in Biology and Disease meeting in Leuven last May (May 2017 conference news). Independently, Peter St George-Hyslop of the University of Toronto and Dorothee Dormann at Ludwig-Maximilians-University in Munich uncovered that transportin tames FUS. They also report that arginine methylation inhibits phase transition of FUS, and that these pathways go awry in some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
“All these papers converge on the same findings, showing two new pathways that regulate aggregation,” said Ben Wolozin of Boston University. “They establish that nuclear import proteins also function as disaggregases, acting like the Jaws of Life to untangle protein wreckage, and that methylation at least partly regulates that process. That gives us an extra target to try to interfere with these processes,” he told Alzforum.
All this occurs in the cytosol, but what about in the nucleus, where RPBs concentrate and cytosolic chaperones are taboo? What keeps FUS and other RPBs from undergoing LLPS or aggregating there? In the April 12 Science, Simon Alberti, Anthony Hyman, and colleagues at the Max Planck Institute, Dresden, Germany, report that high concentrations of RNA do the trick. And finally, to add texture to it all, researchers led by Amy Gladfelter, University of North Carolina, Chapel Hill, report in the same issue that the shape of RNA determines the content, biophysical properties, and phase separation of RNA-protein assemblies, proving that there is much more to know about these particles and their regulation in health and disease.
Fibrils Out. Kapβ2, aka TNPO1, binds to the PY-NLS of FUS, dissolves fibrils by interfering with weak FUS-FUS interactions, and ushers soluble FUS monomers back to the nucleus. [Courtesy of Guo et al., Cell 2018.]
The connection of nuclear import receptors to LLPS casts new light on an old relationship. Chook has long studied NIR interactions with FUS (Zhang and Chook, 2012). One of them, transportin-1 (TNPO1, aka Karyopherin-β2 or Kapβ2) recognizes a proline- and tyrosine-containing nuclear localization sequence (PY-NLS) on the C-terminus of FUS and on other RNA-binding proteins. Once bound to this sequence, TNPO1 directs translocation of proteins into the nucleus. Some disease-causing FUS mutations disrupt nuclear-cytoplasmic shuttling, leading to mislocalization and aggregation of the protein in the cytosol. But could there be more to the story? Chook and Shorter wondered if a tightly bound NIR protein might affect the ability of proteins like FUS to undergo phase transition in the cytoplasm. Rosen was already studying the physiological role of phase transition (Li et al., 2012), and they teamed up to tackle the problem. To start, co-first authors Takuya Yoshizawa in the Chook lab and Rustam Ali in the Rosen lab did a simple experiment: They added TNPO1 to purified FUS. The result was clear: TNPO1 potently prevented LLPS, and could even dissolve droplets of already liquefied FUS (see movie). This required an intact NLS, they showed.
Fading FUS. Liquid droplets formed from purified FUS protein dissipate within five minutes of TNPO1 addition. [Courtesy of Yoshizawa et al., Cell 2018.]
From there, the investigators probed the interaction of the FUS and TNPO1. After TNPO1 bound tightly to the FUS NLS, it made multiple weak interactions with other parts of the FUS protein. TNPO1 contacted multiple tyrosine-containing motifs in the N-terminal low-complexity prion-like domain, including the SYSGY sequence that forms reversible, amyloid-like structures, and the solid core of FUS fibers (Feb 2018 news; Apr 2018 news; Sep 2017 news). The protein interacted with portions of the FUS C-terminal RNA-binding and arginine-rich domains, which the investigators showed also contribute to LLPS. Though weak, collectively these multivalent interactions appear sufficient to disrupt FUS-FUS assembly, and prevent or reverse LLPS.
Shorter and Taylor’s study took the work in a different direction. First authors Lin Guo and Hejia Wang in Philadelphia and Hong Joo Kim in Memphis expanded the range of TNPO1, demonstrating the protein could prevent not just LLPS, but also fibrillization of purified FUS. TNPO1’s actions extended to other RBPs with a PY-NLS, including TAF15, EWSR1, hnRNPA1, and hnRNPA2. The exception was TDP-43, which bears a different NLS and was not affected by TNPO1. However, when researchers swapped in the appropriate transportin, a complex of Importin-α plus karyopherin β1, it successfully prevented aggregation of TDP-43.
Even more, TNPO1 proved to be a potent disaggregase, rapidly dissolving preformed fibrils and fibril-containing hydrogels formed by FUS and other RBPs, in some cases within minutes. That was surprising, Shorter told Alzforum. He had previously identified prion disaggregases in yeast, but none had been found in mammalian cells. “We really didn’t expect that it would reverse aggregation, but we could even see it on a macroscopic level. When we constructed hydrogels and dropped the protein on top, it melted those gels,” he said.
What about mutated, pathogenic forms of FUS? Mutations in the prion-like low-complexity domain often accelerate aberrant phase transition. Those mutants, and many in the NLS, were still susceptible to disaggregation by TNPO1. However, the protein could not solubilize FUS with NLS mutations that severely reduced TNPO1-NLS interactions. In particular, two FUS NLS mutations that destroyed TNPO1 binding partially or entirely resisted disaggregase activity. Interestingly, those two mutants, R495X and P525L, both cause an early, severe form of ALS.
Moving to in vivo studies, when the investigators boosted expression of TNPO1 in yeast, they were able to inhibit RBP aggregation. In mammalian cells, TNPO1 overexpression abrogated accumulation of mutant FUS in stress granules, and restored FUS expression in the nucleus, while preventing its toxicity. In fibroblasts from ALS patients with an R521H FUS mutation, increased expression of TNPO1 reversed a FUS loss-of-function phenotype, namely reduction in specific mRNAs. In flies, TNPO1 lessened neurodegeneration due to mutated FUS or mutated hnRNAP2, another RBP linked to FTLD.
The next step, Shorter said, will be to assess transportins in an animal model, to see if the proteins are protective there, too. “We predict they would be, but we have to do it,” he said.
In addition to nuclear import receptors, posttranslational modification of FUS plays a critical role in phase separation, say two additional papers. The first, from the Dormann lab, builds on her previous work on FUS in ALS and FTLD. She had determined that in ALS-FUS, mutations affecting the NLS disrupt nuclear transport mediated by TNPO1, and thus cellular localization (Jul 2010 news). In FTLD-FUS patients who have no FUS mutations, she found that FUS arginine methylation was reduced, which also disrupted nuclear transport (Sep 2012 news).
In the new study, first authors Mario Hofweber and Saskia Hutten found that either loss of TNPO binding or arginine hypomethylation of C-terminal arginine-rich motifs enhanced phase separation of FUS in vitro and in vivo. The results unify the two disease pathways, Dormann told Alzforum, in that both NLS mutation and hypomethylation lead to increased phase separation, then aberrant FUS accumulation in stress granules.
The fourth and final Cell paper extends that work. St George-Hyslop and colleagues dove deep into the biophysical consequences of FUS hypomethylation and how it enhances phase separation of the protein. In the study, seven first authors—Seema Qamar, GuoZhen Wang, Suzanne Randle, Francesco Simone Ruggeri, Juan Varela, Julie Qiaojin Lin, and Emma Phillips—cover the experimental spectrum from physics to biology. Using a series of physical tools, they establish that in hypomethylated FUS, cation-π bonds between arginine residues in the C-terminal domain and tyrosines in the low-complexity domain likely initiate phase separation, which is then followed by stabilizing intermolecular β-sheet hydrogen bonding.
Those changes appear to contribute to FUS pathogenicity by disrupting protein synthesis. Regulated, local protein synthesis is required for synaptic function and plasticity. The weakening of this activity is a common denominator between ALS-FUS and FTLD-FUS, and likely underpins the cause of neuronal injury in these two FUS-related diseases, St George-Hyslop told Alzforum. The researchers found that arginine hypomethylation of FUS at primary neuron axon tips decreased new protein synthesis, but when they overexpressed TNPO1, the deficits disappeared.
The work emphasizes that some of FUS’s issues, and the actions of TNPO1, have nothing to do with nuclear-cytoplasmic transport pathways. “Many people are still looking at defective cytoplasmic-to-nuclear translocation, which is clearly a problem, but additional problems occur in terminals as well,” St George-Hyslop told Alzforum.
“The underlying conclusion of this body of work is that altering the posttranslational state, and the action of chaperones, are two ways in which the cell physiologically regulates this assembly process,” said St George-Hyslop. “It’s going to need now a little bit of work to try to figure out exactly who the partners are—what are the methyltransferases, the demethylases, the kinases, the phosphatases, and the chaperones other than TNPO1, and most importantly, what regulates them,” he said. “Somewhere in that currently unclear mist of factors, we should be able to find something that can potentially be manipulated.”
Role of RNA Sequence
Turning to RNA, Alberti and Hyman were puzzled why proteins like FUS and TDP-43 aggregate in the cytosol, but remain soluble in the nucleus, where their concentrations are quite high. The answer, they found, was RNA. First author Shovamayee Maharana first estimated concentrations of FUS and other RBPs in the nucleus, then showed that, at that same concentration in vitro, 7.6 μM, FUS phase-separated. But when the researchers repeated the experiment in the presence of total cell RNA, they got a different result. As expected, low concentrations of RNA promoted liquid drop formation, but higher concentrations, as in the nucleus, suppressed it.
FUS-ion. Microinjecting HeLa cell nuclei with RNase causes immediate condensation of GFP-FUS (green) into liquid-like droplets. Arrowheads show where smaller FUS assemblies fuse into larger spherical bodies. The cell at lower right was not injected with RNase. [From Maharana et al., 2018. Courtesy of Science/AAAS.]
To test whether RNA prevents phase separation of RBPs in vivo, the researchers microinjected RNase into HeLa cell nuclei. Almost immediately, FUS and other RBPs condensed into liquid-like droplets (see movie). Injecting concentrated FUS had the same effect. RNA also retarded aberrant aggregation of FUS into fibrils, and FUS mutants with impaired RNA binding aggregated more readily and were more toxic to cells. The results suggest that high RNA in the nucleus buffers against aggregation of RBPs, the authors conclude. When RBPs move into the cytoplasm, as they do in times of stress, they become prone to aggregate, and anything that prolongs time spent in the cytosol will increase their chances of aggregation, say the authors. Based on their results, they predict that changes in RNA levels or the ability of proteins to bind RNA are frequent causes of age-related protein-misfolding diseases.
“It’s a neat setup, where each compartment has its own system for dealing with these proteins,” said Shorter. “The Kapβ2 TNPO1 in cytoplasm stops FUS from getting into trouble in that compartment, and when FUS is back in the nucleus, the high concentration of RNA prevents aggregation.”
RNA has another important function in dictating the individual identities of phase-separated membraneless organelles, says the final study. First author Erin Langdon and coworkers from the Gladfelter lab demonstrated that its three-dimensional structure dictates if RNA will get recruited into droplets in cells. Proteins play a role in determining the conformation of RNA, so are active participants in the process, they show. This mechanism is likely relevant for sorting of specific RNAs to stress granules, P bodies, and other individualized droplets. Gladfelter emphasized that it’s too early to speculate what this might have to do with neurodegenerative disease. “The field is still young—so much remains unknown about normal granule assembly, it’s impossible to say yet what might go wrong in disease,” she told Alzforum.—Pat McCaffrey
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