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• W3113647834 abstract "Article22 December 2020Open Access Transparent process Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration Veronica H Ryan Veronica H Ryan Neuroscience Graduate Program, Brown University, Providence, RI, USA Search for more papers by this author Theodora M Perdikari Theodora M Perdikari Biomedical Engineering Graduate Program, Brown University, Providence, RI, USA Search for more papers by this author Mandar T Naik Mandar T Naik Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA Search for more papers by this author Camillo F Saueressig Camillo F Saueressig orcid.org/0000-0001-6372-0695 Department of Neuroscience, Brown University, Providence, RI, USA Search for more papers by this author Jeremy Lins Jeremy Lins Department of Neuroscience, Brown University, Providence, RI, USA Search for more papers by this author Gregory L Dignon Gregory L Dignon orcid.org/0000-0001-8016-8652 Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA Search for more papers by this author Jeetain Mittal Jeetain Mittal orcid.org/0000-0002-9725-6402 Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA Search for more papers by this author Anne C Hart Corresponding Author Anne C Hart [email protected] orcid.org/0000-0001-7239-4350 Department of Neuroscience, Brown University, Providence, RI, USA Search for more papers by this author Nicolas L Fawzi Corresponding Author Nicolas L Fawzi [email protected] orcid.org/0000-0001-5483-0577 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA Search for more papers by this author Veronica H Ryan Veronica H Ryan Neuroscience Graduate Program, Brown University, Providence, RI, USA Search for more papers by this author Theodora M Perdikari Theodora M Perdikari Biomedical Engineering Graduate Program, Brown University, Providence, RI, USA Search for more papers by this author Mandar T Naik Mandar T Naik Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA Search for more papers by this author Camillo F Saueressig Camillo F Saueressig orcid.org/0000-0001-6372-0695 Department of Neuroscience, Brown University, Providence, RI, USA Search for more papers by this author Jeremy Lins Jeremy Lins Department of Neuroscience, Brown University, Providence, RI, USA Search for more papers by this author Gregory L Dignon Gregory L Dignon orcid.org/0000-0001-8016-8652 Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA Search for more papers by this author Jeetain Mittal Jeetain Mittal orcid.org/0000-0002-9725-6402 Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA Search for more papers by this author Anne C Hart Corresponding Author Anne C Hart [email protected] orcid.org/0000-0001-7239-4350 Department of Neuroscience, Brown University, Providence, RI, USA Search for more papers by this author Nicolas L Fawzi Corresponding Author Nicolas L Fawzi [email protected] orcid.org/0000-0001-5483-0577 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA Search for more papers by this author Author Information Veronica H Ryan1, Theodora M Perdikari2, Mandar T Naik3, Camillo F Saueressig4, Jeremy Lins4, Gregory L Dignon5, Jeetain Mittal5, Anne C Hart *,4 and Nicolas L Fawzi *,3 1Neuroscience Graduate Program, Brown University, Providence, RI, USA 2Biomedical Engineering Graduate Program, Brown University, Providence, RI, USA 3Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA 4Department of Neuroscience, Brown University, Providence, RI, USA 5Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA *Corresponding author. Tel: +1 401 863 2822; E-mail: [email protected] *Corresponding author. Tel: +1 401 863 5232; E-mail: [email protected] The EMBO Journal (2021)40:e105001https://doi.org/10.15252/embj.2020105001 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch-TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress-induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp-1, suggesting gain-of-function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity. SYNOPSIS Specificity of contacts formed in phase-separated RNA granules is complex. We show phosphorylation alters hnRNPA2 interactions and expression of activated Fyn kinase prevents neurodegeneration, suggesting a path to therapies for aggregation diseases. hnRNPA2 granule components hnRNPF and TOG specifically partition into hnRNPA2 low complexity domain droplets. Tyrosine phosphorylation prevents this partitioning as well as aggregation of hnRNPA2 disease-associated mutants. A C. elegans model of ALS/FTD associated with hnRNPA2-D290V shows glutamatergic neurodegeneration. Deletion of tdp-1 (TDP-43 ortholog) or expression of activated Fyn (tyrosine kinase that phosphorylates hnRNPA2) reduces neurodegeneration. Introduction Cells, particularly highly polarized cells like neurons, organize their cytosol using both membrane-bound organelles and membraneless organelles (MLOs), which are condensates of RNA and proteins (Banani et al, 2017). Neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) have been linked to disruption of the components and properties of MLOs, possibly through MLO stabilization by mutations in proteins capable of liquid–liquid phase separation (LLPS), a phenomenon where proteins and nucleic acids demix from the surrounding solution (Nedelsky & Taylor, 2019; Ryan & Fawzi, 2019). The relationship between stress granules, MLOs observed in cells after exposure to exogenous stress, and these ALS/FTD-associated proteins, including FUS, TDP-43, hnRNPA1, and hnRNPA2, has been a primary focus of work relating MLOs to neurodegeneration (Kim et al, 2013; Burke et al, 2015; Molliex et al, 2015; Patel et al, 2015; Conicella et al, 2016; Monahan et al, 2017; Ryan et al, 2018; Wang et al, 2018a). However, many of these disease-associated proteins are also found in physiological MLOs observed in the absence of stress, notably transport granules. Transport granules, called neuronal granules when found in neurons, move mRNAs from the perinuclear space to sites of local translation. Importantly, many ALS/FTD-associated RNA-binding proteins that have essential nuclear functions including roles in transcription and splicing (Tollervey et al, 2011; Rogelj et al, 2012; Martinez et al, 2016) are also found in cytoplasmic RNA transport granules (Yasuda et al, 2013; Alami et al, 2014). Local translation is important in many cell types but is critical for myelination by oligodendrocytes and neuronal functions, including synaptic plasticity and axon guidance. Different kinds of mRNA transport granules likely exist, with distinct mRNA cargos, including neuronal β-actin mRNA transport granules containing zip-code-binding protein/IGF2BP1 (Zhang et al, 2001; Elvira et al, 2006; Kiebler & Bassell, 2006) and hnRNPA2-containing granules transporting myelin basic protein mRNA in oligodendrocytes or Arc, neurogranin, and αCamKII mRNAs in neurons (Ainger et al, 1993; Brumwell et al, 2002; Shan et al, 2003; Gao et al, 2008). Several other protein components of hnRNPA2-containing transport granules have been identified, including hnRNPF, hnRNPAB, hnRNPE1, hnRNPK, and the microtubule-associated protein ch-TOG (CKAP5) (Kosturko et al, 2005; Kosturko et al, 2006; Francone et al, 2007; Laursen et al, 2011; Raju et al, 2011; White et al, 2012; Torvund-Jensen et al, 2014). Importantly, hnRNPA2 transport granules appear to contain a specific set of proteins and exclude related proteins associated with other cytoplasmic granules (e.g., other RNA-binding proteins found in stress granules), yet the mechanisms of granule component specificity remain unclear. Some of these protein interactions with hnRNPA2 are RNA-dependent (Laursen et al, 2011; Raju et al, 2011; Torvund-Jensen et al, 2014) but hnRNPF and ch-TOG both directly bind to hnRNPA2 (Kosturko et al, 2005; White et al, 2012; Falkenberg et al, 2017). Interactions within the granules are regulated; mRNA is released for local translation in processes when hnRNPA2 and hnRNPF are locally phosphorylated by the tyrosine kinase Fyn (White et al, 2008; White et al, 2012). Yet, the molecular basis for both hnRNPA2-hnRNPF and hnRNPA2-TOG interactions and their disruption by phosphorylation remains unknown. hnRNPA2 mutations cause multisystem proteinopathy (MSP), a degenerative disease with clinical features of ALS/FTD, inclusion body myopathy, and Paget’s disease of bone (PDB) (mutation: D290V) (Kim et al, 2013) as well as PDB alone in a separate family (mutation: P298L) (Qi et al, 2017). These disease mutations drive aggregation of the protein in vitro (Kim et al, 2013; Ryan et al, 2018). The D290V mutation also enhances hnRNPA2 stress granule localization (Martinez et al, 2016) even in the absence of stress (Kim et al, 2013), induces aggregation in the cytoplasm (Kim et al, 2013) and nucleus (Martinez et al, 2016), and results in abnormal splicing changes and decreased survival in neuronal culture (Martinez et al, 2016). Therefore, the mechanistic interactions leading to hnRNPA2 function and dysfunction provide an interesting model for a large class of RNA-binding proteins mutated in disease (King et al, 2012). hnRNPA2 contains two RNA recognition motifs (RRMs) and a glycine-rich low complexity (LC) domain, which is necessary and sufficient for LLPS (Ryan et al, 2018). Importantly, both mutations are located in the aggregation-prone “prion-like” LC domain (named for resemblance in sequence composition to the polar-residue-rich domains found in yeast prion proteins) (King et al, 2012). Given that transport granule proteins are thought to interact directly with hnRNPA2 LC (Falkenberg et al, 2017) and that the LC gives hnRNPA2 the ability to undergo LLPS, we set out to determine the molecular basis for interactions of hnRNPA2 LC with hnRNPF prion-like domain (PLD) and TOG domain 1 (D1) by evaluating their ability to specifically co-phase separate into in vitro models of reconstituted multicomponent hnRNPA2 granules. We chose these domains to probe the molecular interactions between the disordered LC or PLD domains and a single domain of TOG, as the disordered domains are thought to be protein–protein interaction domains and contribute to LLPS and granule formation. Furthermore, as post-translational modifications (PTMs) alter phase separation and aggregation of RNA-binding proteins (Nott et al, 2015; Monahan et al, 2017; Ryan et al, 2018; Wang et al, 2018a), we tested the hypothesis that tyrosine phosphorylation disrupts the interactions between granule components as a possible mechanism for granule dissociation and prevents protein aggregation in vitro. We also hypothesized that hnRNPA2 mutation could induce neurodegeneration in an animal model and that promoting tyrosine phosphorylation could prevent toxicity. Here, we used nuclear magnetic resonance (NMR) spectroscopy, molecular simulation, in vitro phase separation assays, and a novel C. elegans model to map the protein–protein interactions between components of hnRNPA2-containing transport granule assembly, probe a potential mechanism of physiological disassembly, and assess strategies to prevent hnRNPA2-associated neurodegeneration. Results hnRNPA2 arginine residues are required for in vitro interaction with transport granule component hnRNPF hnRNPA2 was previously shown to undergo LLPS (Ryan et al, 2018) and interact with other protein components of myelin basic protein mRNA transport granules, including hnRNPF (White et al, 2012). Importantly, hnRNPF interacts directly with hnRNPA2 protein in transport granules (White et al, 2012), but the biophysical details of this interaction between these two prion-like domain containing proteins remain unclear. Here, we sought to reconstitute and structurally characterize this interaction using recombinant full-length proteins and their isolated domains in order to probe the unexplored interactions between these proteins that occur inside granules. First, we tested which domains mediate the hnRNPA2-hnRNPF interaction. Using purified recombinant hnRNPA2 low complexity domain (LC, residues 190–341) and full length (FL) with hnRNPF prion-like domain (PLD, resides 365–415), and FL (Appendix Fig S1A), we asked if fluorescently tagged hnRNPF partitions into hnRNPA2 droplets. hnRNPF (PLD or FL) did not undergo LLPS alone in the conditions and concentrations required for LLPS of hnRNPA2 LC or FL (Fig 1A, Appendix Fig S1B and C). However, hnRNPF PLD partitioned into hnRNPA2 LC (Fig 1A) and hnRNPA2 FL (Appendix Fig S1B) droplets and was equally distributed throughout the droplets. hnRNPF FL partitioned into both hnRNPA2 LC and hnRNPA2 FL droplets (Appendix Fig S1B and C). The PLD of hnRNPF was not essential for partitioning into hnRNPA2 droplets, as hnRNPF lacking the PLD (hnRNPF ∆PLD, residues 1–364) partitioned into hnRNPA2 FL droplets (Appendix Fig S1B). We attempted to test whether the hnRNPF PLD is necessary for partitioning into hnRNPA2 LC droplets and while some co-localization was observed (Appendix Fig S1C), hnRNPF ∆PLD seemed to aggregate, possibly because the hnRNPA2 LC LLPS assay conditions required crossing the hnRNPF ∆PLD predicted isoelectric point (pI), where proteins are likely to self-assemble. Taken together, these data suggest that the PLD of hnRNPF avidly partitions into hnRNPA2 droplets. Figure 1. Arginine in hnRNPA2 LC is required for the interaction with granule component hnRNPF PLD. See also Appendix Figs S1–S3 hnRNPA2 LC (AlexaFluor 488-tagged, green) undergoes LLPS, while hnRNPF PLD (AlexaFluor 555-tagged, red) does not. However, hnRNPF PLD partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. At 300 µM, FUS LC (AlexaFluor488-tagged, green) undergoes LLPS, but at 200 µM hnRNPF PLD (AlexaFluor555-tagged, red) still does not undergo LLPS. When mixed at 300 µM FUS LC and 200 µM hnRNPF PLD, hnRNPF PLD does not partition into FUS LC droplets. Conditions: 300 µM FUS and 200 µM hnRNPF PLD (~1% fluorescently tagged), 20 mM MES pH 5.5, 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. While FUS LC and hnRNPF PLD both have a small negative predicted net charge at neutral pH, hnRNPA2 LC has a predicted + 4 net positive charge, due to the 9 positively charged residues (8 arginine, 1 lysine) and 5 negatively charged residues. Removal of the charged residues from hnRNPA2 LC (hnRNPA2 LCCD) prevents partitioning of hnRNPF PLD into the hnRNPA2 LC phase. Addition of hnRNPA2 LC-like charged residue patterning to FUS LC (FUS LCCE) allows the partitioning of hnRNPF PLD at 40 µM. Conditions: protein concentration indicated next to image (20 µM hnRNPA2 LC and hnRNPA2 LCCD, 40 µM FUS LCCE, hnRNPF PLD concentration matches other protein in mixture (either 20 or 40 µM)) (all ~ 1% fluorescently tagged), 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCR→K droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LCR→K does not phase separate much as hnRNPA2 LC at these conditions, see Appendix Fig S1J for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. Quantification of phase separation of hnRNPA2 LC constructs used to determine the residue types important for hnRNPF PLD partitioning. hnRNPA2 LCN→S (purple) has similar phase separation to hnRNPA2 LC. hnRNPA2 LCCD (red) is consistently phase separated with ~ 5 µM protein remaining in the supernatant at all salt conditions tested. Adding back arginines to hnRNPA2 LC no charge (hnRNPA2 LCCD,R, green) brings phase separation as a function of salt to similar levels as hnRNPA2 LC. Changing all the arginine residues to lysine (removing the π-character but maintaining positive charge, hnRNPA2 LCR→K) also removes the salt dependence of phase separation but has reduced phase separation overall. Conditions: 20 µM of each protein, pH 5.5 MES, NaCl concentration as indicated, 25° C. Error bars are standard deviation of three replicates. Download figure Download PowerPoint Next, we focused on delineating the molecular basis for the interaction between hnRNPA2 LC and hnRNPF PLD. To visualize hnRNPF PLD with residue-by-residue resolution, we performed multidimensional NMR spectroscopy to observe each backbone amide position. From the narrow chemical shift dispersion and secondary shifts, we conclude that hnRNPF PLD is primarily disordered (Appendix Fig S2A and B). Then, we attempted to localize the sites of interactions between hnRNPA2 LC and hnRNPF PLD using NMR titrations. These experiments were performed at low salt conditions where hnRNPA2 LC alone does not phase separate (mimicking monomeric interactions) as NMR signal is attenuated due to LLPS into viscous, sedimenting droplets when hnRNPA2 LC is placed in the presence of salt. We found that addition of hnRNPF PLD reduces the signal intensity of hnRNPA2 LC; the converse is also true: Addition of hnRNPA2 LC reduces the signal intensity of hnRNPF PLD (Appendix Fig S2C and D). These findings are reminiscent of how Fyn-SH3, a folded domain that does not phase separate on its own, reduces hnRNPA2 LC signal intensity by inducing LLPS of hnRNPA2 LC (Amaya et al, 2018). However, we performed microscopy at the NMR conditions and found that hnRNPF PLD aggregates in these low salt conditions and these aggregates remain in the presence of hnRNPA2 LC (Appendix Fig S2E). Therefore, the decrease in hnRNPA2 LC NMR signal intensity in the presence of hnRNPF PLD is likely attributable to co-aggregation or interaction of hnRNPA2 LC with hnRNPF PLD, either of which are consistent with interaction between these domains. The NMR titrations showed signal attenuation across the entire length of hnRNPF PLD and hnRNPA2 LC, providing no evidence for specific residues or residue types in hnRNPA2 LC interactions with hnRNPF PLD (Appendix Fig S2C and D), consistent with the repetitive, low complexity sequence of these domains. An important question in the field of MLOs is the origin of specificity of granule partitioning. Although no specific sites of interaction were observed by NMR, we wondered if the sequences of hnRNPA2 LC and hnRNPF PLD could encode any specificity in partitioning. Therefore, we examined specificity by testing if hnRNPF PLD could co-phase separate with the low complexity domain of the RNA-binding protein FUS. FUS is also found in stress granules but has not been found in hnRNPA2-myelin basic protein mRNA granules. Importantly, FUS LC has different amino acid composition than that of hnRNPA2. However, we found that FUS LC and FUS LC 12E, a phosphomimetic form (incorporating 12 serine to glutamate (E) substitutions) of FUS that does not undergo of FUS that does not undergo LLPS (Monahan et al, 2017), are both capable of partitioning into hnRNPA2 LC (Appendix Fig S3A). Interestingly, when hnRNPF PLD was mixed with FUS LC, it did not partition into FUS LC droplets (Fig 1B), demonstrating specificity of the hnRNPA2-hnRNPF partitioning. We hypothesized that charged residues might underlie this specificity, as both FUS LC and hnRNPF PLD are free of positively charged resides and are slightly negatively charged, while hnRNPA2 LC has a net positive charge (Fig 1C). To test the role of charged residues in hnRNPA2 LC specifying the interaction with hnRNPF, we changed almost all the charged residues from hnRNPA2 LC to serine or glutamine resulting in a “FUS LC-like” charged residue pattern (which we termed hnRNPA2 LCCD, for “charge depleted”) or changed serine/glutamine residues in FUS to charged residues to give it “hnRNPA2 LC-like” charged residue pattern including net charge, identity of charged residues, and approximate spacing between charged residues (FUS LCCE, for “charge enhanced”), and examined hnRNPF PLD partitioning into these charged residue variants. hnRNPF PLD did not partition into hnRNPA2 LCCD but did partition into FUS LCCE (Fig 1D, Appendix Fig S3B), consistent with our hypothesis. Given that arginine contacts with aromatic residues are important for phase separation in hnRNPA2 (Ryan et al, 2018) and other proteins (Vernon et al, 2018; Wang et al, 2018b), we hypothesized that partitioning of hnRNPF PLD into hnRNPA2 LC droplets required arginine in hnRNPA2 LC and tyrosine in hnRNPF PLD. Therefore, we changed hnRNPF PLD tyrosine to serine (hnRNPF PLDY→S), hnRNPA2 LC arginine to lysine (hnRNPA2 LCR→K), and also tested a form of hnRNPA2 LC with arginine residues retained but otherwise “FUS LC-like” charged residue depleted patterning (hnRNPA2 LCCD,R). First, we found that hnRNPF PLDY→S still partitioned into hnRNPA2 LC droplets (Appendix Fig S3C), implying that tyrosine in hnRNPF PLD is not the only residue type contributing to the interaction between hnRNPA2 LC and hnRNPF PLD. Second, we found that replacing arginine with lysine in hnRNPA2 LC to maintain net charge and charge patterning (hnRNPA2 LCR→K) prevented partitioning of hnRNPF PLD or hnRNPF PLDY→S, suggesting that arginine-specific contacts and not just positive charge determine partitioning (Fig 1E, Appendix Fig S3C). Third, we found that adding back arginine to hnRNPA2 LCCD allowed partitioning of hnRNPF PLD and hnRNPF PLDY→S (Fig 1E, Appendix Fig S3C). Inconveniently, FUS LCCE,R→K and FUS LCR did not undergo LLPS at the same conditions as hnRNPA2 LC, so we could not perform the complementary experiments testing hnRNPF PLD partitioning into modified FUS constructs (Appendix Fig S3C). We also tested the role of asparagine residues in hnRNPA2 LC by changing asparagine to serine (hnRNPA2 LCN→S) and the effect of serine residues in hnRNPF PLD by changing serine to alanine (hnRNPF PLDS→A), but these changes did not qualitatively alter partitioning (Appendix Fig S3C). Interestingly, both the hnRNPA2 LCCD and hnRNPA2 LCR→K substantially altered LLPS of hnRNPA2 LC, while hnRNPA2 LCN→S and hnRNPA2 LCCD,R had similar LLPS to hnRNPA2 LC (Fig 1F). Finally, we also tested the ability of hnRNPF PLD and FL to partition into FUS FL droplets. We found that both hnRNPF PLD and FL are able to partition into FUS FL droplets (Appendix Fig S3D), which is expected as FUS FL has three arginine-rich domains outside of the N-terminal LC. Therefore, though these results suggest some specificity in the interaction of disordered domains arises directly from their sequence, additional factors such as RNA-binding specificity (Helder et al, 2016) mediated by the RNA-binding domains in the full-length proteins likely contribute to granule component sorting in vivo. We conclude that arginine residues in hnRNPA2 LC are critical to specify co-phase separation with hnRNPF PLD. hnRNPA2 LC interacts with TOG D1 weakly in vitro through its disordered loops and helical face Similar to hnRNPF, ch-TOG was previously shown to interact with hnRNPA2 in myelin basic protein transport granules via contacts with hnRNPA2 LC (Kosturko et al, 2005; Francone et al, 2007; Falkenberg et al, 2017). Given our results with hnRNPF, we hypothesized that one domain of ch-TOG, TOG D1 (residues 1–250) (Appendix Fig S4A), would also partition into hnRNPA2 droplets. We used a single domain of ch-TOG as the individual domains are highly similar in sequence and structure and each isolated domain was previously shown to interact similarly with hnRNPA2 at nanomolar affinity (Falkenberg et al, 2017). As expected for a single globular domain, recombinant TOG D1 did not undergo LLPS in the micromolar concentration range or buffers tested, but fluorescently tagged TOG D1 partitioned into hnRNPA2 LC and FL droplets (Fig 2A, Appendix Fig S4B). To map the interactions between hnRNPA2 LC and TOG D1, we performed NMR resonance assignment experiments on TOG D1 and confirmed its primarily α-helical secondary structure (Appendix Fig S4C and D). Surprisingly, when we performed NMR titrations with hnRNPA2 LC and TOG D1, we found no significant interaction between hnRNPA2 LC and TOG D1 in the dispersed (not phase-separated) state using this technique (Appendix Fig S4E and F), counter to our expectations. The lack of evidence for tight binding suggests that the dissociation constant for the interaction between these two proteins is likely in the micromolar to millimolar range, weaker than previously reported (which we attribute to possible artifacts arising from LLPS or aggregation, see Discussion). Figure 2. Transport granule component TOG D1 interacts weakly with hnRNPA2 LC. See also Appendix Figs S4–S6 A. AlexaFluor 488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor 555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Fig 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B. Similar to hnRNPF PLD, AlexaFluor 555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. C, D. TOG D1 homology structure with Γ2 values from PRE experiments for hnRNPA2 LC (C) S285C and (D) S329C. Amino acids are colored based on Γ2 value: Red corresponds to Γ2 > 2, orange to 2 > Γ2 > 1, yellow to 1 > Γ2 > 0.5. E. AlexaFluor 555-tagged hnRNPF PLD or FL and AlexaFluor 405-tagged TOG D1 partition simultaneously into AlexaFluor 488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm. Download figure Download PowerPoint Similar to our results with hnRNPF, we observed that the co-phase separation of hnRNPA2 LC and TOG D1 is specific; TOG D1 partitioned into hnRNPA2 LC but not FUS LC droplets (Fig 2B). However, as with hnRNPF, TOG D1 is able to partition into FUS FL droplets (Appendix Fig S5A). We hypothesized that because TOG D1 has a charged surface (Appendix Fig S5B), the charge variants of hnRNPA2 LC and FUS LC would also alter TOG D1 partitioning. Indeed, TOG D1 partitioned into FUS LCCE droplets, yet it also partitioned into hnRNPA2 LCCD droplets (Appendix Fig S5C). This result is different than what we observed for hnRNPF PLD and indicates that charged residues contribute to the specificity of partitioning, but other interactions contribute as well. To begin to elucidate these interactions, we turned to NMR techniques that provide position-specific information on weak, transient interactions. High resolution transverse relaxation optimized (TROSY-based) paramagnetic relaxation enhancement (PRE) experiments on mixtures of NMR invisible hnRNPA2 LC tagged with a small (~120 Da) paramagnetic label at a single engineered cysteine site (either S285C or S329C) (Ryan et al, 2018) and NMR-visible (2H 15N) TOG D1 revealed weak interactions, consistent with a dissociation constant weaker than 100 µM, across several sites on TOG D1 (Appendix Fig S5D). These interactions suggest that the region of hnRNPA2 LC bearing the paramagnetic tag comes in close proximity with particular parts of the TOG D1 surface. We then mapped the PRE values on a homology model of TOG D1 and found that they localized to disordered loops and helix faces of TOG D1 (Fig 2C and D). Upon sorting PRE values ≥ 0.5 s−1 by residue type, enhancement is most often observed at non-polar positions (28/76 residues with PREs) and charged residues (27/76). However, non-polar amino acids are the most prevalent typ" @default.
• W3113647834 created "2021-01-05" @default.
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• W3113647834 title "Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration" @default.
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• W3113647834 doi "https://doi.org/10.15252/embj.2020105001" @default.
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