Ryan VH, Dignon GL, Zerze GH, Chabata CV, Silva R, Conicella AE, Amaya J, Burke KA, Mittal J, Fawzi NL. Mechanistic View of hnRNPA2 Low-Complexity Domain Structure, Interactions, and Phase Separation Altered by Mutation and Arginine Methylation. Mol Cell. 2018 Feb 1;69(3):465-479.e7. Epub 2018 Jan 18 PubMed.
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University of Modena and Reggio Emilia
The finding by Veronica H. Ryan and colleagues that wild-type and disease-linked mutated hnRNPA2 promote the phase separation of other low-complexity proteins, such as TDP-43, is important because it suggests that co-phase separation of these proteins could potentially converge, in a plethora of diseases, onto a common pathomechanism: conversion of dynamic membrane-less condensates into irreversible protein aggregates. This conversion into protein aggregates is enhanced by the defective clearance of misfolded proteins and by a general failure of the protein folding and degradation machineries, which has also been linked to aging and neurodegeneration. In agreement, Marrone and colleagues report that drugs that induce autophagy rescue FUS stress-granule pathology in iPS cell and fruit-fly models, most likely by removing misfolded proteins that would otherwise co-aggregate with stress granules. Based on these data, drugs that promote the degradation of misfolded proteins and thus prevent the conversion of stress granules into aggregates hold great promise for the development of therapeutic approaches for several types of age-related neurodegenerative diseases, with different symptoms, ranging from cognitive to motor defects.
View all comments by Serena CarraSt. Jude Children's Research Hospital
Ryan and colleagues make an important observation, and conceptually the same as we observed in our recent Neuron paper in which ALS/FTD-causing mutations were found to alter the biophysical properties of TIA-1 (Mackenzie et al., 2017). Disease-causing mutations in hnRNPA1, hnRNPA2, and TIA-1 strengthen the cohesive forces that give rise to phase transitions, and change the material properties of the liquid phase. Not addressed in the Fawzi paper is the impact on intracellular phase transitions, but the prediction is that it would have the same impact as TIA-1 mutations, which impair the dynamics of RNA granules. Indeed, in our initial paper describing disease mutations in hnRNPA1 and hnRNPA2 we reported that poorly dynamic stress granules containing these RNA-binding proteins accumulate in patient-derived cells. These same changes increase the propensity of the protein to assemble into a fibrillar “aggregate” similar to that observed in disease. It remains to be determined which is more important for driving cellular dysfunction: altered material properties (and therefore function) of the RNA granules that contain mutant hnRNP (hnRNPA1, TIA-1, hnRNPA2, TDP-43, FUS), or acquisition of a toxic property by the aggregated form of the protein. I favor the former, but time will tell. The fact that these phase transitions are regulated by posttranslational modifications is also very important, and suggests strategies for finding small molecules to restore normal RNA granule dynamics.
The Marrone et al. paper is conceptually consistent with the observation that poorly dynamic RNA granules (e.g., stress granules) that arise from mutations in hnRNPs are cleared by autophagy, otherwise known as granulophagy (Buchan et al., 2013), and underscore the potential for this process to be targeted for therapeutic benefit.
References:
Mackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, Pottier C, Annu K, Baker M, Perkerson RB, Kurti A, Matchett BJ, Mittag T, Temirov J, Hsiung GR, Krieger C, Murray ME, Kato M, Fryer JD, Petrucelli L, Zinman L, Weintraub S, Mesulam M, Keith J, Zivkovic SA, Hirsch-Reinshagen V, Roos RP, Züchner S, Graff-Radford NR, Petersen RC, Caselli RJ, Wszolek ZK, Finger E, Lippa C, Lacomis D, Stewart H, Dickson DW, Kim HJ, Rogaeva E, Bigio E, Boylan KB, Taylor JP, Rademakers R. TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics. Neuron. 2017 Aug 16;95(4):808-816.e9. PubMed.
Buchan JR, Kolaitis RM, Taylor JP, Parker R. Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell. 2013 Jun 20;153(7):1461-74. PubMed.
View all comments by J. Paul TaylorBrown University
Marrone et al. describe the creation of interesting cell reporters expressing GFP-tagged forms of wild-type and disease-associated mutants of FUS. Especially interesting is the creation of closely related lines that link GFP (a large modification) with two different length linkers to the C-terminal end of FUS. Noticing that the short link alters stress granule phenotype compared to the long linker, the authors take advantage of both linker lines for designing assays optimized for sensitivity. They use these cells to show that stimulating autophagy decreases FUS accumulation in stress granules and identifies currently FDA-approved compounds for modulating.
The results will be exciting to test in recent and upcoming cell and animal models of ALS. Of further interest is the examination of a FUS variant that disturbs the canonical FUS binding site for nuclear import protein karyopherin beta2 yet does not entirely disrupt nuclear localization and redistribution after stress. These findings suggests that work on the roles of transportin/karyopherin FUS interactions and on SGs is of keen interest.
View all comments by Nicolas FawziJGU Mainz
Ryan et al. used NMR spectroscopy, molecular simulations, and biochemical LLPS assays to characterize the structural details and phase-separation properties of the hnRNP-A2 low-complexity (LC) domain. A particularly interesting finding of this work is that genetic mutations in the hnRNP-A2 LC domain that are linked to neurodegenerative disease (D290V and P298L) enhance self-interaction of the LC domain and thus promote a liquid-to-solid-state transition (i.e. aggregation) of the hnRNP-A2 LC domain. Similar findings have been reported previously for disease-linked mutations in the LC domain of other RNA-binding proteins, e.g. FUS (Patel et al., 2015; Murakami et al., 2015) or hnRNP-A1 (Molliex et al., 2015), hence this seems to be a common theme in neurodegenerative diseases. The detrimental downstream changes in cells elicited by such aberrantly self-interacting and aggregating mutant RNA-binding proteins remain to be clarified. Do they promote co-phase separation and aggregation of other LC domain-containing RNA-binding proteins, such as TDP-43? Is alternative splicing of hnRNP-A2 target altered due to enhanced self-interactions of hnRNP-A2 genes (Martinez el al., 2016)? These are interesting questions for follow-up studies sparked by the present study by Fawzi and colleagues.
Another interesting finding reported by Ryan et al. is that arginine methylation on four sites reduces LLPS of the hnRNP-A2 LC domain. This highlights that posttranslational modifications in LC domains are crucial factors influencing LLPS, as previously reported for other RNA-binding proteins (Nott et al., 2015; Monahan et al., 2017), so they are promising targets for modulating LLPS/aggregation of disease-linked LC domain-containing RNA-binding proteins. Arginine methylation seems to be a particularly interesting modification, since it is often mis-regulated in disease (Thandapani et al., 2015) and arginine methylation defects have been identified in FUS-associated neurodegeneration (Dormann et al., 2012; Suárez-Calvet et al., 2016). Thus, arginine methylation could indeed have a wider role in preventing LLPS and aggregation of LC domain-containing RNA-binding proteins.
References:
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Molliex A, Temirov J, Lee J, Coughlin M, Kanagaraj AP, Kim HJ, Mittag T, Taylor JP. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell. 2015 Sep 24;163(1):123-33. PubMed.
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Monahan Z, Ryan VH, Janke AM, Burke KA, Rhoads SN, Zerze GH, O'Meally R, Dignon GL, Conicella AE, Zheng W, Best RB, Cole RN, Mittal J, Shewmaker F, Fawzi NL. Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J. 2017 Oct 16;36(20):2951-2967. Epub 2017 Aug 8 PubMed.
Thandapani P, O'Connor TR, Bailey TL, Richard S. Defining the RGG/RG motif. Mol Cell. 2013 Jun 6;50(5):613-23. PubMed.
Dormann D, Madl T, Valori CF, Bentmann E, Tahirovic S, Abou-Ajram C, Kremmer E, Ansorge O, Mackenzie IR, Neumann M, Haass C. Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. EMBO J. 2012 Sep 11; PubMed.
Suárez-Calvet M, Neumann M, Arzberger T, Abou-Ajram C, Funk E, Hartmann H, Edbauer D, Kremmer E, Göbl C, Resch M, Bourgeois B, Madl T, Reber S, Jutzi D, Ruepp MD, Mackenzie IR, Ansorge O, Dormann D, Haass C. Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS. Acta Neuropathol. 2016 Apr;131(4):587-604. Epub 2016 Feb 19 PubMed.
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