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

Article

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

Authors Marta M. Fay, Paul J. Anderson, Pavel Ivanov

Correspondence [email protected] (P.J.A.), [email protected] (P.I.)

In Brief Fay et al. report that GGGGCC repeat RNA (rG4C2) associated with ALS/FTD drives phase separations to promote RNA granule formation in vitro and in cells. Such separations are length- and G-quadruple-structure-dependent. Targeting rG4C2 G-quadruplex structures is a potential therapeutic option for ALS/FTD.

Highlights d

ALS/FTD GGGGCC repeat RNA (rG4C2) drives phase separations in vitro and in cells

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rG4C2-mediated phase separation is length and structure dependent

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G-quadruplex structures are required for rG4C2-mediated phase separation

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Manipulating G-quadruplex structures alters rG4C2mediated phase separation

Fay et al., 2017, Cell Reports 21, 3573–3584 December 19, 2017 ª 2017 The Author(s). https://doi.org/10.1016/j.celrep.2017.11.093

Cell Reports

Article ALS/FTD-Associated C9ORF72 Repeat RNA Promotes Phase Transitions In Vitro and in Cells Marta M. Fay,1,2 Paul J. Anderson,1,2,* and Pavel Ivanov1,2,3,4,* 1Division

of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA, USA of Medicine, Harvard Medical School, Boston, MA 02115, USA 3The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 4Lead Contact *Correspondence: [email protected] (P.J.A.), [email protected] (P.I.) https://doi.org/10.1016/j.celrep.2017.11.093 2Department

SUMMARY

Membraneless RNA granules originate via phase separation events driven by multivalent interactions. As RNA is the defining component of such granules, we examined how RNA contributes to granule assembly. Expansion of hexanucleotide GGGGCC (G4C2) repeats in the first intron of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). We describe a biophysical phenomenon whereby G4C2 RNA (rG4C2) promotes the phase separation of RNA granule proteins in vitro and in cells. The ability of rG4C2 to promote phase separation is dependent on repeat length and RNA structure because rG4C2 must assume a G-quadruplex conformation to promote granule assembly. We demonstrate a central role for RNA in promoting phase separations and implicate rG4C2 G-quadruplex structures in the pathogenesis of C9-ALS/FTD. INTRODUCTION Protein-protein interactions are proposed to play a dominant role in RNA granule dynamics (Mitrea and Kriwacki, 2016); changes affecting such interactions (e.g., mutations in selected protein factors) are linked to the formation of protein aggregates, and defects in RNA granule homeostasis are implicated in neurodegenerative disease pathogenesis (Bosco et al., 2010; Burke et al., 2015; Conicella et al., 2016; Kim et al., 2013; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015). A common feature of non-coding repeat expansion disorders is the accumulation of repeat-bearing RNA foci in the nucleus and/or cytoplasm of affected cells (La Spada and Taylor, 2010). How these RNA foci form and how they impact biological processes and contribute to disease is largely unknown. A hexanucleotide expansion GGGGCC (G4C2), in the first intron of the chromosome 9 open reading frame 72 (C9ORF72) gene, is the most common mutation associated with amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/ FTD) (DeJesus-Hernandez et al., 2011; Renton et al., 2011). G4C2 repeats exceeding a defined threshold (typically R24 repeats) show association with ALS/FTD (Van Mossevelde et al.,

2017). RNA is transcribed from these repeats in the sense (G4C2 RNA [rG4C2]) and antisense (CCCCGG: rC4G2) directions. Dipeptide repeat proteins (DRP) are translated from rG4C2 and rC4G2 repeats via repeat associated non-ATG (RAN) translation. Both rG4C2 and DRP form nuclear and cytoplasmic foci when transfected into cells and have been found in C9-ALS/FTD patient samples. These foci disrupt RNA metabolism by a variety of mechanisms (Gitler and Tsuiji, 2016). Some rG4C2 cytoplasmic foci resemble stress granules (SGs), which are membraneless, microscopically visible cytoplasmic foci induced by some stresses and containing translationally stalled mRNAs, 40S ribosomal subunits, RNA binding proteins (RBPs), and signaling molecules (Kedersha et al., 2013). Both rG4C2 and DRP are known to disrupt RNA metabolism, alter SG dynamics and may directly drive disease pathogenesis (Gitler and Tsuiji, 2016). Recent reports suggest that RNA granule formation is dependent on weak interactions between prion-like, low-complexity (including DRP), and intrinsically disordered regions in proteins that facilitate phase separation, a biophysical phenomenon that allows concentration of proteins into discrete non-membranous subcellular compartments (reviewed in Hyman et al. [2014]; Mitrea and Kriwacki [2016]). The chemical biotinylated isoxazole (b-isox) can promote phase separation in vitro by concentrating and condensing low-complexity domain- and intrinsically disordered region-containing proteins into structures that resemble RNA-granules (Han et al., 2012; Kato et al., 2012). Multiple RBPs contain distinct RNA-binding domains and intrinsically disordered regions, and these proteins can undergo phase transitions such as formation of liquid droplets, hydrogels, or, even, insoluble amyloids in vitro (Burke et al., 2015; Conicella et al., 2016; Lin et al., 2015; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015; Zhang et al., 2015). Addition of RNA to such protein-driven phase separations influences their ability to separate (Elbaum-Garfinkle et al., 2015; Lin et al., 2015; Molliex et al., 2015; Zhang et al., 2015). Additionally, repeat expansion RNA can promote an in vitro phase separation in isolation (Jain and Vale, 2017). rG4C2 forms higher order structures including G-quadruplexes and hairpins in vitro (Fratta et al., 2012; Haeusler et al., 2014; Reddy et al., 2013; Su et al., 2014). G-quadruplexes are stable structures formed by stacks of four guanosine residues hydrogen bonded via Hoogsteen base pairing and stabilized by specific metal cations (Neidle, 2012). The availability of structural data combined with the link to neurodegenerative disease

Cell Reports 21, 3573–3584, December 19, 2017 ª 2017 The Author(s). 3573 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

makes rG4C2 an ideal candidate to study the mechanisms by which specific RNAs modulate RNA granule assembly. In analogy to SG nucleator proteins that initiate SG formation when overexpressed in the absence of stress (Kedersha et al., 2013), we hypothesized that rG4C2 G-quadruplexes work as RNA templates that create a local microenvironment that drives RNA granule formation. We show here that rG4C2 in a G-quadruplex conformation drives the assembly of RNA granules both in vitro and in cells. In vitro, rG4C2 condenses a cohort of proteins that is similar but not identical those condensed by b-isox. Importantly, rG4C2 repeat length correlates with the degree of protein condensation in vitro and in cells, mirroring the relationship between C9ORF72 repeat length and ALS/FTD pathogenesis. Our studies offer a framework for understanding how RNA participates in RNA granule assembly, and suggest ways in which targeting the rG4C2 G-quadruplex might be beneficial in the treatment of patients with ALS/FTD. RESULTS rG4C2 Promotes SGs in Cells We have previously shown that transfection of various synthetic and natural RNA species promotes SG formation (Emara et al., 2010; Ivanov et al., 2014). Interestingly, transfection of r(G4C2) 4, but not r(C4G2)4, also promotes formation of cytoplasmic foci positive for G3BP1, a standard SG marker (Figures S1A– S1C). These r(G4C2)4-induced cytoplasmic foci also contain the SG markers eIF3b, eIF4G, and poly(A) mRNA (Figures S1A and S1B). As the length of G4C2 repeats is linked to C9-ALS/ FTD pathogenesis (DeJesus-Hernandez et al., 2011; Renton et al., 2011) and some C9-ALS/FTD patient cells are positive for rG4C2 cytoplasmic foci (Donnelly et al., 2013), we tested whether variable length rG4C2 repeats differently affect granule formation. Transfection of in-vitro-transcribed rG4C2 into U2OS cells promotes G3BP1-positive granules in a repeat length dependent manner (Figures 1A and 1B). To determine whether repeat length alters SG properties, we assessed the number of SGs per cell and found that cells transfected with longer repeats showed a decrease in SGs per cell (Figure 1C). In assessing SG size, we observed no difference (Figure 1D). Since rG4C2-induced granules contain known SG markers, we asked whether these granules are in dynamic equilibrium with polysomes by treating with cycloheximide and puromycin, drugs that inhibit or promote SG assembly, respectively. rG4C2-38X (38 repeats of the G4C2) induced granules decrease when treated with cycloheximide and increase when treated with puromycin (Figure 1E), consistent with these granules being in dynamic equilibrium with polysomes (Kedersha et al., 2000). The presence of SG markers and their dynamic behavior indicates rG4C2-induced foci can be classified as bona fide SGs (Aulas et al., 2017). Fluorescence recovery after photobleaching (FRAP) was employed to assess the dynamics of both the r(G4C2)4 and mCherry-tagged G3BP1. The fluorescent signal from transiently transfected 30 FAM-labeled r(G4C2)4 is static and does not return after bleaching as indicated by only 15% recovery after photobleaching, whereas mCherry-tagged G3BP1 exhibits dynamic behavior quickly returning to the 3574 Cell Reports 21, 3573–3584, December 19, 2017

bleached region with 70% of the signal recovering (Figures 1F and S2). SGs form due to a block in translation initiation which can occur though several mechanisms. One such mechanism is via phosphorylation of eukaryotic initiation factor 2-alpha (eIF2a) on serine 51. eIF2a brings the initiator methionine tRNA to the 43S initiation complex, and phosphorylation effectively prevents initiator tRNA recruitment and, consequently, inhibits translation. To assess whether eIF2a phosphorylation is required for rG4C2mediated SG assembly, we utilized mouse embryonic fibroblasts (MEFs) with a serine 51 to alanine point mutation in eIF2a (S51A). Transfection of r(G4C2)4 promotes SG assembly in wild-type MEFs, but not in MEFs containing the eIF2a-S51A mutation (Figure 1G), indicating eIF2a phosphorylation is required for rG4C2 induced SG assembly. Together, these data indicate rG4C2 promotes SGs in a length dependent manner, and rG4C2 is a stable component of these granules. Importantly, transfection of plasmids used for in vitro transcription encoding variable-length G4C2 repeats also promotes formation of rG4C2-containing nuclear foci (Figure S1D), consistent with other investigations (DeJesus-Hernandez et al., 2011). These data also indicate that rG4C2 is sufficient to promote RNA granules both in nuclear and cytoplasmic compartments. rG4C2-Mediated Condensation Promotes In Vitro RNA Granule Assembly rG4C2 can promote SGs and nuclear foci (Figures 1 and S1). Formation of RNA granules has been mechanistically linked to phase separation, a process whereby demixing high concentrations of macromolecules in crowded microenvironments results in the assembly of non-membrane-bound entities (Hyman et al., 2014; Mitrea and Kriwacki, 2016). As b-isox assembles RNA granule-like structures in cell lysates (Kato et al., 2012), we hypothesized that rG4C2 might promote a phase separation in vitro. To test this, we developed a visual assay to assess phase separation. Antisense r(C4G2)4 oligo, an oligo substituting the purine for purine (adenosine for guanosine) r(A4C2)4, and a scrambled control (rScram), a scrambled version of r(G4C2)4 with the same guanosine content as r(G4C2)4, were used as controls. Briefly, 30 fluorescently labeled r(G4C2)4, r(C4G2)4, r(A4C2)4, or rScram were incubated with U2OS lysate, concentrated by centrifugation, spotted on a slide and then examined using fluorescence microscopy. Fluorescently labeled r(G4C2) 4 promotes microscopically visible foci in cell lysates, but not in buffer controls (Figure 2A, left panel). These foci form in a concentration-dependent manner (Figure 2B). rScram also forms microscopically visible foci in cell lysate yet to a lesser extent than r(G4C2)4 (Figures 2A and 2B). Neither r(A4C2)4 nor r(C4G2)4 form foci even at higher concentrations (Figures 2A and 2B). To assess the properties of these foci, we tested whether electrostatic interactions play a role in their assembly. Under hypotonic conditions, more r(G4C2)4 foci form, while hypertonic conditions prevent foci formation (Figure 2C). Similar trends were observed with rScram and r(C4G2)4 (Figure 2C), indicating that RNA foci formation is more prominent under hypotonic conditions yet the extent is RNA specific. We also assessed whether

Figure 1. rG4C2 Promotes SG Formation (A) U2OS cells were transiently transfected with equimolar in-vitro-transcribed rG4C2 of indicated length or vehicle control (Ctrl) for 6 hr, fixed, permeabilized, assayed by immunofluorescence-detecting G3BP1 (green) and eIF3b (red), and counterstained with Hoechst (blue) to visualize nuclei. Scale bar, 40 mm. (B–D) Quantification of the percentage of cells with SGs (B), number of SGs per cell (C), and SG size (D). (E) U2OS cells transiently transfected with rG4C2-38x and then treated with puromycin (puro, 5 mg/mL) or cycloheximide (CHX, 10 mg/mL) for 30 min prior to fixation. Quantification of the percentage of cells with SG (cells with R 2 G3BP1 positive foci). (F) FRAP analysis of rG4C2-induced SGs in U2OS cells stably expressing mCherry-G3BP1 and transiently transfected with 3‘ FAM-labeled r(G4C2)4. Graphs represent relative fluorescence of mCherry-G3BP1 (red) and 3‘ FAM-labeled r(G4C2)4 (green) before (first 3 data points) and after photobleaching (last 10 data points). (G) Quantification of the percentage of SG positive wild-type (black bars) or eIF2a-S51A mutant (white with black dots) MEFs transfected with r(G4C2)4. An asterisk denotes statistical significance, p < 0.05. Data are represented as mean ± SD, n R 3 (B and E–G). >37 cells (C) and >50 foci (D) were quantified per condition across 3 biological repeats. See also Figure S1.

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Figure 2. rG4C2 Promotes Foci Formation In Vitro

(A) Images detecting pelleted 30 FAM-labeled r(G4C2)4, r(C4G2)4, r(A4C2)4, or scrambled control (rScram) (0.5 mM) after incubation with U2OS lysate (upper panels) or buffer (lower panels), concentrated by centrifugation, and then assayed by fluorescence microscopy. Scale bar, 10 mm. (B–D) Quantification of assay described in (A) using r(G4C2)4 (green), r(C4G2)4 (red), r(A4C2)4 (yellow), or rScram (gray) at indicated RNA concentration (B), 0.25 mM RNA with 75, 150, and 200 mM NaCl (labeled left to right) (C), or r(G4C2)4 (0.5 mM) with or without 2.5% Ficoll (D). Data are represented as mean ± SD, n R 3. See also Figure S2.

molecular crowding affects r(G4C2)4 foci formation, and found that molecular crowding using Ficoll promotes r(G4C2)4 foci formation (Figure 2D). Together, these data indicate that molecular crowding and electrostatic interactions drive RNA foci formation. At high concentrations, poly-guanosine, but not other poly-nucleosides, can assume a gel-like state (Bang, 1910). Visual inspection of the r(G4C2)4 condensed material revealed a transparent pellet, in contrast to b-isox, which forms a white opaque pellet of similar size. In contrast to b-isox, r(G4C2)4 requires cell lysate in order to assume a sedimentable state because it does not sediment in buffer alone (Figure S3), suggesting that cellular components are required for in vitro r(G4C2)4 foci formation. Material condensed by r(G4C2)4 appears to be specific, as shown by differences in condensed versus total banding pattern identified on a silver-stained SDS-polyacrylamide gel (Figure 3A, compare lanes 1 and 6). Proteins condensed by b-isox and r(G4C2)4 were extracted and assayed by mass spectrometry. Using a cutoff of R10 peptides, 333 proteins were condensed 3576 Cell Reports 21, 3573–3584, December 19, 2017

by r(G4C2)4 as compared with 196 condensed by b-isox (Figure 3B). 19 of the top 25 r(G4C2)4 identified proteins/ protein complexes have been previously identified as RNA granule components (Figure 3C) (Kato et al., 2012; Lessel et al., 2017; Wen et al., 2010). Gene ontology term analysis of r(G4C2)4 condensed proteins indicates an enrichment in RNA processing, (Figure 3D). Together, this supports that we are observing an in vitro formed granule promoted by RNA. Western blotting was used to confirm the mass spectrometry identified proteins. In common with b-isox, r(G4C2)4 condenses the large and small ribosomal subunits, select RBPs including YB-1 and HuR, some translation initiation factors, and components that regulate SG assembly including G3BP1, Caprin1, and USP10 (Figure 3E). Consistent with the mass spectroscopy results, and in contrast to b-isox, RNA helicases including DDX3 and DDX6, the RNA binding protein TIAR, and the ALS-associated proteins TDP-43 and Ataxin-2 are largely not condensed by r(G4C2)4 (Figure 3E). These data indicate that r(G4C2)4 and b-isox condense both overlapping and unique subsets of proteins.

Figure 3. Selective Condensation of RNA Granule-Related Proteins by rG4C2 (A) U2OS lysate incubated with indicated RNAs, b-isox, or vehicle controls (H2O and DMSO), pelleted, resuspended, resolved by 4%–20% SDS-PAGE, and then silver stained. (B) Venn diagram of proteins identified by mass spectroscopy that were condensed by r(G4C2)4 and b-isox. Number of proteins identified to be unique for rG4C2 (green), b-isox (red), or overlapping (yellow) is shown. Cutoffs set at R 10 total peptides. (C) Top r(G4C2)4 condensed targets identified by mass spectroscopy. Proteins previously identified as components of RNA granules are highlighted in yellow. (D) Gene ontology (GO) analysis of proteins identified by mass spectroscopy that were pelleted by r(G4C2)4. Cutoffs were set at R 10 total peptides. Top hits sorted by p value with a fold enrichment of > 3. (E) Proteins pelleted by indicated RNAs, b-isox, or vehicle controls (H2O, DMSO) after incubation with U2OS lysate for 60 min at 4 C, washed twice, and resolved on 4%–20% SDS-PAGE gels. Western blots probed for indicated antibodies. (F–H) Western blots probed for G3BP1, eIF3b, and TIAR. Same reaction as (E), except using indicated RNAs (F), equimolar amounts of in-vitro-transcribed RNAs of indicated length (G), and equal mg (lanes 1–3) compared to equimolar (lanes 4–6) amounts of in-vitro-transcribed RNAs of indicated length (H). Cropped blots are indicated by white separation. See also Figure S2 and Table S4.

As r(G4C2)4 forms RNA granules in vitro, we sought to confirm this phenomenon is universal and not cell type specific. r(G4C2) 4-induced condensation of eIF3b, G3BP1, and/or HuR occurred in lysates generated from several different cell lines, as well as from mouse brain (Figure S4). SG components reliably condense G3BP1 and eIF3b with r(G4C2)4 and b-isox, whereas TIAR reliably condenses with b-isox and under some specific conditions with r(G4C2)4 (discussed below). We therefore tracked these proteins for the remainder of our studies. To further validate

our condensation assay, we assessed r(A4C2)4 and rScram controls. We found r(A4C2)4 does not promote condensation while rScram partially promotes condensation yet not to the same degree as r(G4C2)4 (Figure 3F). G4C2 repeat length is a defining feature of C9-ALS/FTD pathogenesis (DeJesus-Hernandez et al., 2011; Renton et al., 2011). To determine whether rG4C2-mediated in vitro condensation demonstrates repeat length-dependent variations, a range of G4C2 repeat lengths were in vitro transcribed and used in equal molar Cell Reports 21, 3573–3584, December 19, 2017 3577

Figure 4. rG4C2 Promotes In Vitro Phase Separations (A–E) Western blots detecting G3BP1, eIF3b, and TIAR from material condensed from U2OS lysate by r(G4C2)4, r(C4G2)4, or b-isox. Condensation was varied by temperature of wash steps (A), concentrations of RNA or b-isox (B), incubation time (C), NaCl concentration in the buffer (for lysis and the reaction) (D), and addition of the crowding agent Ficoll (E). Cropped blots are indicated by white separation. See also Figure S3.

amounts in our condensation assay (Figure 3G). Generally, longer rG4C2 repeats promote more efficient condensation as judged by G3BP1 and eIF3b. Importantly, longer (but not shorter) repeats recruit TIAR, an important SG nucleator (Gilks et al., 2004), suggesting that repeat length also influences the repertoire of proteins condensed. Importantly, when this assay is performed with equal grams of G4C2 11X, 28X, and 75X in-vitro-transcribed RNA, similar amounts of condensation are observed (Figure 3H). This experiment shows that the number of repeats is important yet these repeats can be in cis or in trans to promote this effect. rG4C2-Mediated Phase Separations Phase transitions are reversible and influenced by temperature and concentration. Increased temperature causes an increase in free energy that promotes mixing and prevents phase separation. Conversely, increased concentration of initiating factors enhances the transition and thus condensation (Hyman et al., 2014; Mitrea and Kriwacki, 2016). To assess whether temperature affects rG4C2-mediated condensation, r(G4C2)4 condensation reactions were pelleted then washed at 4 C, room temperature (20 C), or 37 C. Increased temperature prevents both r(G4C2) 4 and b-isox-mediated condensation (Figure 4A, compare lanes 1 and 2 to 3 and 4 to 5 and 6). Conversely, increasing rG4C2 or b-isox concentration causes increased condensation of G3BP1, eIF3b, and eventually condenses TIAR at higher concentrations of r(G4C2)4 (Figure 4B). These data are consistent with the visual assay that shows that assembly of r(G4C2)4-induced foci is concentration dependent (Figure 2B). Kinetically, G3BP1 and eIF3b are quickly condensed by r(G4C2)4 and are unchanged over 1 hr (Figure 4C). This is in contrast to b-isox which shows increased protein condensation over a 1 hr time course (Figure 4C, compare lanes 8 and 9). RNA granule formation is attributed to phase separation, resulting from multiple low-affinity electrostatic and other weak non-co3578 Cell Reports 21, 3573–3584, December 19, 2017

valent interactions between different molecules, which are enhanced by molecular crowding. Thus ionic conditions play a critical role in phase separation (Mitrea and Kriwacki, 2016). At physiologic salt concentration (150 mM NaCl), rG4C2 and b-isox selectively condense specific proteins including G3BP1 and eIF3b (Figure 4D, lanes 2, 5, 8, 11, and 14). At hypotonic salt concentration (75 mM NaCl), G3BP1 and eIF3b condense irrespective of whether RNAs, b-isox, or vehicle controls is added (Figure 4D, lanes 1, 4, 7, 10, and 13). Under these conditions, TIAR remains specific as it only condenses with b-isox (Figure 4D, lanes 1, 4, 7, 10, and 13, bottom panel). At hypertonic salt concentration (200 mM NaCl) rG4C2- and b-isox-mediated condensation is greatly reduced (Figure 4D, lanes 3, 6, 9, 12, and 15). 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 (Figure 4E, lanes 2, 6, 10, 13, and 17). Higher concentrations of Ficoll induce G3BP1 and eIF3b condensation independent of adding RNA, b-isox, or vehicle control, whereas TIAR condensation is specific to high concentrations of b-isox (Figure 4E). Thus electrostatic interactions and molecular crowding regulate rG4C2-mediated condensation, and such condensation occurs at physiologic salt concentrations and is promoted by molecular crowding. Cellular RNA Is Essential for rG4C2-Mediated Condensation To assess whether cellular RNA is necessary for rG4C2-mediated condensation, RNase A was added during the incubation or wash steps of the condensation reaction. Addition of RNase A efficiently degrades any RNA (data not shown) and as a result

Figure 5. Cellular RNA Is Required for rG4C2-Mediated Phase Separation (A) Western blots detecting G3BP1, eIF3b, and TIAR from material condensed after r(G4C2)4 (lanes 1–3) was incubated with U2OS lysate (lanes 1 and 3), lysate supplemented with RNase A (lane 2), or washed with buffer with RNase A (lanes 2 and 3). (B) Western blots detecting G3BP1, eIF3b, and TIAR from material condensed after r(G4C2)4, r(C4G2)4, or b-isox were incubated with untreated (lanes 1–3 and 10), mock treated (lanes 4–6 and 11), or MNase treated (lanes 7–9 and 12) U2OS lysate. Cropped blots are indicated by white separation. See also Figure S5.

little to no G3BP1 or eIF3b is pelleted. This indicates that RNA (including r(G4C2)4) is necessary both for the initial condensation (Figure 5A, lane 2) and for maintaining the phase separation as the condensed material is partially eliminated by addition of RNase A during the washes (Figure 5A, lane 3). To determine whether cellular mRNAs are required for rG4C2-mediated condensation, lysates were treated with micrococcal nuclease (MNase) prior to the condensation assay. MNase is commonly used to prepare in vitro translation-competent lysates where endogenous mRNAs are eliminated. MNase treatment prevents rG4C2-mediated condensation and only mildly affects b-isoxmediated condensation (Figure 5B) (Han et al., 2012). Analysis of RNA that was harvested prior to centrifugation but after incubation with r(G4C2)4 shows neither major cellular RNA species (e.g., 18S/28S/5S rRNA and tRNAs), nor r(G4C2)4 or r(C4G2)4 are degraded under these conditions (Figure S5), consistent with MNase only affecting mRNAs. Together, these data indicate that cellular RNAs are necessary, but not sufficient, to mediate rG4C2-induced condensation. G-Quadruplexes Promote rG4C2-Mediated Condensation rG4C2 has been reported to assume hairpin and G-quadruplex structures (Fratta et al., 2012; Haeusler et al., 2014; Reddy et al., 2013; Su et al., 2014). Expanded repeats found in other neurological diseases including Huntington’s disease (CAGn) and myotonic dystrophy types 1 and 2 (CUGn, CCUGn) can also adopt hairpin conformations (Cleary and Ranum, 2013; Krzyzosiak et al., 2012). We compared the ability of several G-quadruplex-containing oligos, other neurological disease repeat RNAs that assume hairpin structures, and unstructured control RNAs to condense G3BP1 and eIF3b from cell lysates. Whereas G-quadruplex-containing oligos (rG4C2, rG3U3, 50 Ala tiRNA, rUUAGGG) condense G3BP1 and eIF3b, other neurological disease-associated RNA repeats do not (Figure 6A), suggesting G-quadruplex structures may promote this phase separation event. G-quadruplexes are composed of stacked G-tetrads, four guanosine residues hydrogen bonded by Hoogsteen base pairs, coordinated by a central monovalent cation (Figure 6B). The size of the cation is critical, as potassium ions support G-quadruplex assembly whereas smaller lithium ions do not (Neidle, 2012). Potassium equilibration reduces the mobility of r(G4C2)4 in native gels, a phenomenon that is not observed with lithium equilibration (Figure S6A), consistent with rG4C2 forming a G-quadruplex (Fratta et al., 2012; Haeusler et al., 2014; Reddy et al., 2013; Su

et al., 2014). These slower-migrating species are also observed under denaturing gel conditions (urea within the gel and formamide in the loading dye) (Figure S6B), indicative of the remarkable stability of G-quadruplexes. Similar shifts in mobility were also observed with longer in-vitro-transcribed rG4C2 repeats (Figure S6C). Additionally, r(G4C2)4 in the G-quadruplex conformation shows specificity in binding partners as lithium-equilibrated and potassium-equilibrated 3‘biotinylated r(G4C2)4 have different binding partners from cell lysate (Figures S6E and S6F, compare lanes 1 and 2). Together these data are consistent with rG4C2 forming G-quadruplex structures that have a specific subset of binding partners. Transfection of potassium-equilibrated rG4C2, including both r(G4C2)4 and in-vitro-transcribed r(G4C2) 11X, 28X, and 75X RNA into U2OS cells promotes more SG assembly, whereas lithium-equilibrated RNAs promote fewer SGs (Figures 6C and 6D). r(C4G2)4 equilibrated with either potassium or lithium, or potassium or lithium alone do not induce SG assembly (Figures 6C and 6D). rScram is able to promote visual foci and to condense G3BP1 and eIF3b (Figures 2A, 2B, and 3F). Because of its high G-content, this control RNA may form G-quadruplex structures (intraor inter-molecularly) that partially mimic rG4C2. We assessed lithium and potassium equilibrated rScram by gel electrophoresis and found rScram forms higher molecular weight species that are sensitive to potassium concentrations, suggesting rScram can form G-quadruplex-like structures (Figure S6D). Substitution of guanosine for its analog 7-deazaguanosine maintains Watson-Crick base pairing but does not support Hoogsteen hydrogen bonding, thereby destabilizing G-quadruplex structures (Figure 6B) (Murchie and Lilley, 1992). Introduction of two 7-deazaguanosines into r(G4C2)4 (referred to as r(G*4C2)4) affects its mobility on native and denaturing gels (Figures S6A and S6B), decreases SG formation compared to r(G4C2)4 (Figure 6C), and impairs condensation of G3BP1 and eIF3b (Figure 6E, compare lanes 1 and 2). Moreover, compounds that disrupt G-quadruplexes tetra-(N-methyl-3-pyridyl)porphyrin (TMPyP3) and tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4)) (Han et al., 2001) inhibit r(G4C2)4-mediated condensation of G3BP1 or eIF3b, whereas N-methylmorpholine (NMM), a G-quadruplex stabilizer (Nicoludis et al., 2012), slightly enhances condensation of G3BP1 and eIF3b (Figure 6F). r(G4C2)4-induced SG assembly is significantly decreased when pretreated with TMPyP4, yet no change is observed with NMM (Figure 6G). These data are consistent with a model whereby rG4C2 G-quadruplexes phase Cell Reports 21, 3573–3584, December 19, 2017 3579

Figure 6. RNA G-Quadruplexes Promote Phase Transitions In Vitro and in Cells (A) Western blot analysis of pelleted material after indicated RNAs were incubated with U2OS cell lysate (Ai). SYBR Gold stained 15% TBE-urea gel detecting RNA loaded prior to lysate addition (Aii). (B) Four guanosine residues form a G-tetrad via hydrogen bonding using the Watson-Crick and Hoogsteen interfaces. Substitution of 7-deazaguanosine (denoted as G*) for guanosine prevents a Hoogsteen base pair (yellow ovals). The G-tetrad is coordinated with a metal ion (denoted M+) in the central channel. (C and D) SG quantification after transfection with r(G4C2)4, r(G*4C2)4, or r(C4G2)4 (C) or in-vitro-transcribed r(G4C2)-11X, 28X, or 75X equilibrated with Li+ or K+ (D). (E and F) Western blots detecting G3BP1, eIF3b, and TIAR condensed from U2OS lysate by (E) r(G4C2)4, r(G*4C2)4, or r(C4G2)4 or (F) r(G4C2)4 preincubated with TMPyP3, TMPyP4, or NMM (+ for 2 mM, ++ for 10 mM) for 30 min. (G) SG quantification of U2OS cells transfected with r(G4C2)4 or r(C4G2)4 preincubated with TMPyP4 or NMM (100 mM) for 30 min prior to transfection. An asterisk denotes statistical significance with p < 0.05. Data are represented as mean ± SD, n R 3. Cropped blots are indicated by white separation. See also Figure S6.

separate in vitro by condensing RNA granule components including G3BP1 and eIF3b, which in cells promotes SG formation. DISCUSSION The mechanisms of RNA granule assembly are extremely complex due to overlapping contributions from multiple protein:pro3580 Cell Reports 21, 3573–3584, December 19, 2017

tein and protein:RNA interactions. Recent findings highlight fundamental roles for protein-protein interactions and phase transitions in granule formation (Mitrea and Kriwacki, 2016). Paradoxically, although RNA granules are RNA-containing entities by definition, the role(s) of RNA in this process is largely unknown. Recent work from the Vale lab indicates that RNA associated with repeat expansions can phase separate in vitro and in cells (Jain and Vale, 2017). Using rG4C2 as a model RNA, here,

we demonstrated that RNA G-quadruplexes nucleate RNA granule assembly in cells and promote phase transitions in vitro. Our study is unique as the in vitro phase separation uses lysate under physiological ionic conditions, better recapitulating the cellular environment. We found rG4C2 promotes condensation of a specific subset of proteins from cell lysates (Figure 3). The majority of these proteins are also condensed by b-isox, a chemical that induces phase transitions in vitro. rG4C2 and b-isox condensed proteins are enriched for low complexity, prion-like and intrinsically disordered domains that contribute to RNA granule assembly both in vitro and in cells. However, the modes of rG4C2 and b-isox-mediated condensation differ. In the absence of lysate, b-isox forms a white pellet (data not shown), while rG4C2 fails to condense (Figures 2 and S3). B-isox forms a structural lattice, independent of other factors, that then templates low complexity domains into a specific state (Kato et al., 2012). In contrast, rG4C2-mediated condensation requires factors within the lysate, including cellular RNA (Figures 2, 5, and S3). Importantly, rG4C2 promotes RNA granule formation in cells, both cytoplasmic SGs and nuclear RNA foci (Figures 1 and S1), mirroring its ability to promote RNA-granule-like condensation in vitro. In our analysis, we tracked G3BP1, eIF3b, and TIAR due to their importance in RNA metabolism and RNA granule assembly. Whereas G3BP1 and eIF3b reliably condense with both rG4C2 and b-isox, TIAR consistently condensed with b-isox and only under some conditions with rG4C2. Interestingly, TIAR condenses with longer rG4C2 repeats or under high concentrations of short repeats (Figures 3G and 4B). These results suggest that condensation of some protein factors requires rG4C2 to reach a certain threshold, either by increasing the length of repeats or by increasing the concentration of shorter repeats. Differential condensation of protein factors may be relevant to ALS/FTD where longer repeat-containing rG4C2 transcripts form visible foci that recruit RNA binding proteins and cause their mislocalization (Gitler and Tsuiji, 2016). Like b-isox induced condensation, rG4C2-mediated condensation is reversible, fast, concentration- and temperaturedependent, and sensitive to molecular crowding. It is highly responsive to ionic conditions, which both modulate electrostatic interactions between rG4C2 and other molecules during condensation and directly contribute to stabilization of G-quadruplexes. Additionally our experiments were conducted under physiological ionic conditions. The rG4C2 G-quadruplex structure is required for granule formation (Figure 6), and our model is that rG4C2 G-quadruplexes seed granule formation. rG4C2 appears to take on a solid-like state due to its lack of dynamic properties (Figures 1F and S2), consistent with rG4C2 being a core granule component (Jain et al., 2016; Kedersha et al., 2005; Wheeler et al., 2016). Increasing the number of rG4C2 G-quadruplex units by increasing rG4C2 repeat length or rG4C2 concentration increases nucleation of microscopically visible granules and RNA-mediated condensation in vitro. In this manner, RNA structure drives granule formation. Most, if not all, RNA within the cell is thought to be coated with proteins. It is likely that a critical concentration of free RNA or disruption of RNA-protein interactions is required to allow selected RNAs to assemble G-quadruplex structures. Currently, it remains

unknown whether and to what extent RNA G-quadruplexes fold in vivo. Algorithms predict that a high percentage of the transcriptome is capable of forming G-quadruplexes (Huppert and Balasubramanian, 2005; Todd et al., 2005), yet recent genome-wide sequencing methodologies suggest G-quadruplexes are not formed in cells (Guo and Bartel, 2016; Kwok et al., 2016), indicating a clear need for further investigation. Our data indicate that when folded into a G-quadruplex rG4C2 has differential binding partners (Figures S6E and S6F) and nucleates RNA granules (Figure 6), and that this occurs to a greater extent with more RNA repeats. We observed no change in SG formation when we transfected r(G4C2)4 and NMM treated r(G4C2)4 (Figure 6G). This suggests that we cannot further promote G-quadruplex folding with NMM suggesting that RNA G-quadruplexes can exist in cells. For C9-ALS/FTD, we hypothesize that by increasing the length of rG4C2, more repeats allow more templating, increasing the recruitment of proteins/RNA to allow RNA granule assembly. In turn, nucleation of granules may affect RNA metabolism in neurons and trigger pathological cascades culminating in neuron loss. These events may be more detrimental in neurons due to the necessity of localized translation, which requires transport of RNAs and translational machinery down long axonal processes. We propose that other RNA structures may also template or affect RNA granules in cells. Consistent with this hypothesis, SGs form in response to stress that causes translating polysomes to disassemble producing a sudden excess of free mRNAs in the cytoplasm. It is hypothesized that this mRNA pool nucleates the formation of RNA granules by binding a large set of RBPs (Bounedjah et al., 2014; Wheeler et al., 2016). It will be important to determine whether other mRNA structures regulate SG formation. Proteins participating in physiological phase transitions are prone to assemble protein aggregates when they possess disease-associated mutations (Bosco et al., 2010; Burke et al., 2015; Conicella et al., 2016; Kim et al., 2013; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015). Similarly, various RNAs acquire repeat expansion mutations that increase the structural complexity and/or sequence units (Cleary and Ranum, 2014; Krzyzosiak et al., 2012). We propose that RNA plays an active role in RNA granule assembly and that RNA-mediated perturbations in granule dynamics may underline cellular toxicity and contribute to disease. EXPERIMENTAL PROCEDURES Cell Culture and Cell Treatments Cell lines (U2OS, U251, LN229, and MEFs) were maintained in 5%–7% CO2 and cultured in DMEM supplemented with 20 mM HEPES, 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Stable U2OS cells expressing mCherry-tagged G3BP1 were previously described (Kedersha et al., 2016). Reagents Synthetic RNA oligos were purchased from Integrated DNA Technologies (Table S1), and reagents and antibodies are indicated in Tables S2 and S3. RNA and b-isox Precipitation 3 3 106 U2OS cells or comparable density for other cells were plated on 10-cm dishes. The next day, cells were washed with Hank’s balanced salt solution

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(HBSS) and scraped with 500 mL condensation buffer (CB: 50 mM HEPES, 10% glycerol, 150 mM NaCl, 5 mM MgCl2, 0.5% NP40, Halt Protease and phosphatase inhibitors, 1 mM DTT, and RNaseIN). For mouse brain, the brain was harvested, flash frozen, and stored at 80 C. Samples were dounce homogenized and centrifuged at 14,000 3 g for 10 min at 4 C, and supernatant was used as lysate. RNA (1 mM, unless otherwise indicated), b-isox (100 mM), or vehicle controls (H2O or DMSO) were added to lysate and incubated for 1 hr at 4 C. Samples were centrifuged at 14,000 3 g for 15 min at 4 C, supernatant was discarded, and the resultant pellet was washed twice with CB. The pellet was resuspended in 13 SDS loading dye for western blot or silver stain analysis, trichloroacetic acid (TCA) for mass spectrometry, or Trizol for RNA analysis. Modifications to the procedure are indicated. For MNase reactions, lysates were made as above, treated with MNase (0.06 U/mL), supplemented with 1 mM CaCl2 for 10 min at 37 C, and quenched with 2 mM EGTA. Mock treatment included all steps but lacked MNase. For RNase A experiments, RNase A (1 mg/mL) was added to the reaction or wash steps. For salt-adjusted experiments, cells were lysed and washed in CB with indicated NaCl. For Ficoll addition, Ficoll was supplemented to lysate prior to reaction and to wash buffer. Western Blot and Silver Stain Samples were generated as described above, and input controls were diluted in 13 SDS sample buffer, heated at 95 C for 5 min, and run on Mini-Protean TGX gels. Proteins were transferred to nitrocellulose using Trans-Blot Turbo (Bio-Rad) and blocked in 5% non-fat milk in tris-buffered solution with tween 20 (TBST) for R20 min. Primary antibodies were diluted in 5% normal horse serum (NHS) and incubated overnight at 4 C or 1 hr at 20 C. Blots were washed R3 times for 10 min at 20 C with TBST. Secondary antibodies were incubated for R30 min at 20 C. Blots were washed; ECL was applied; and signals were detected using film or Chemidoc Imaging System (Bio-Rad). For silver stain analysis, samples were run on 4%–20% Tris-Glycine gels and then stained using the Pierce Silver Stain Kit in accordance with the manufacturer’s recommendations. Visualization and Quantification of In Vitro Precipitates Lysates were generated as described above and then mixed with RNA as indicated. Samples were centrifuged at 14,000 3 g for 15 min, and pellets were resuspended in 10 mL CB. 4 mL was placed on a glass slide with a 22 3 22 mm coverslip on top. 10 min post-mounting, 3–5 random fields were imaged with a 603 oil objective on the Nikon microscope described elsewhere. Images were quantified using ImageJ by Thresholding with the Triangle algorithm and then the ‘‘Analyze Particles.’’ function (particle = 5 to N pixels2). Quantification of SGs ImageJ was used to quantify the number of SGs per cell and the size of the SGs. 8-bit grayscale G3BP1 images were thresholded; the cell was outlined via freehand selection; and ‘‘Analyze Particles’’ assessed both size and number of particles per cell. Data were exported to Excel, and graphs were generated with Prism 5.0. To assess SG size, approximately the same minimum threshold was used and the size noted is in pixels, where 3.2 pixels = 1 mm. In Vitro Transcription Gel-purified pEF6-(G4C2) repeat plasmids (May et al., 2014; Mori et al., 2013) cleaved with XbaI were used in standard reaction HiScribe T7 polymerase RNA synthesis or T7-Riboprobe System in accordance with the manufacturer’s recommendations. After 3 hr incubation, 1 mL DNase I was added and incubated for 30 min. RNA was extracted with Trizol. RNA Gel Analysis 500 mL HBSS and 700 mL Trizol were added to the pellet of the condensation reaction and then RNA was extracted. For denaturing gels, RNA was heated in denaturing loading dye, cooled, and analyzed on 15% tris base, boric acid, EDTA (TBE)-urea gel. For native gels, RNA was diluted in 13 sample buffer and run on 10% TBE gel (10% acrylamide:bis-acrylamide [19:1], 0.53 TBE, 0.01% ammonium persulfate [APS], and 0.1% tetramethylethylenediamine [TEMED]). To visualize RNA, gels were stained with SYBR Gold (1:10,000 in 0.53 TBE) for 10 min and visualized by UV light.

3582 Cell Reports 21, 3573–3584, December 19, 2017

Immunofluorescence 1 3 105 U2OS cells were plated on 12-mm glass coverslips. The following day, for RNA transfections, cells were transfected with indicated RNAs using Lipofectamine 2000. Cycloheximide (100 mg/mL) and puromycin (5 mg/mL) treatment was carried out 30 min prior to fixation as indicated. 6–8 hr post-transfection, cells were washed with PBS, fixed with 4% paraformaldehyde at 20 C for 15 min, permeabilized with methanol (20 C) for 5 min, and blocked (5% NHS) for R20 min. Primary and secondary antibody incubations were carried out for R1 hr at 20 C or overnight at 4 C. Between antibody incubations coverslips were washed 3 times for 5 min with PBS. Hoechst 33258 was added during secondary antibody incubation to visualize nuclei. Coverslips were mounted with Vinol and visualized using a Nikon Eclipse E800 microscope with a mercury lamp and standard filters (UV-2A 360/40; 420/LP), Cy2 (FITC HQ 480/40; 535/50), Cy3 (Cy 3 HQ 545/30; 610/75), and Cy5 (Cy 5 HQ 620/60; 700/75) equipped with a SPOT Pursuit digital Camera (Diagnostics Instruments) and the manufacturer’s software. Cells were counted as positive for SGs if R2 G3BP1 foci were present, and nuclei were counted for the total number of cells. Graphs were generated with GraphPad Prism 5 and represent compiled data from 3–5 (203 or 403 magnification) images per experiment across R 3 independent experiments. FRAP U2OS cells expressing mCherry-tagged G3BP1 were plated onto 35-mm FluoroDishes. The following day, cells were transfected with 30 FAM-labeled r(G4C2)4 using Lipofectamine 2000. Cells were imaged on a Nikon Eclipse TE2000U Inverted Microscope using an Eclipse EZ-C1 system (v.3.90; Nikon) and a Plan Apo 603 Pan Apo (NA 1.40) objective lens or the Zeiss LSM 800 with Airyscan confocal system on a Zeiss Axio Observer Z1 Inverted Microscope. FAM fluorescence was excited with the 488-nm line from a Melles Griot 488 Ion Laser and detected with a 515/30 emission filter; mCherry fluorescence was obtained using Melles Griot 543 laser excitation and a 590/50 emission filter or the best settings as selected by the Zeiss software. Three images were collected prior to photo bleaching a selected region (SG) using 100% (5% for the Zeiss system) laser power, and ten images were collected post-photobleaching. Data points include >5 SGs per experiment across 3 experiments. Graphs were generated using GraphPad Prism (v.5). Mass Spectrometry For mass spectrometry, b-isox and RNA precipitation assays were scaled up and completed as above, and pellets were resuspended in 20% TCA. Samples were incubated on ice for 20 min and spun down in at 14,000 3 g for 20 min at 4 C. The resultant pellet was washed with 10% TCA and then with ice-cold acetone. The resultant pellet was air-dried for 5 min and stored at 80 C until analyzed by the Taplin Mass Spectrometry Facility (Harvard Medical School, Boston, MA) and can be found in Table S4. DATA AND SOFTWARE AVAILABILITY The mass spectrometry data reported in this paper can be found on Mendeley: 10.17632/xdm865xf2c.3. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, six figures, and four tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2017.11.093 ACKNOWLEDGMENTS We thank the Anderson and the Ivanov lab members for their discussion; Victoria Ivanova for technical support; Christian Haass, Dieter Edbauer, and Kohji Mori (Ludwig-Maximilians University, Munich, Germany) for contributing the pEF6G4C2-repeat constructs; and Katia Urso from the Antonios Aliprantis lab (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA) for the mice used to harvest brain tissue. This work was supported by grants from

the NIH (GM111700 and GM121410 to P.J.A., NS094918 to P.I., and T32AI007306 to M.M.F.). AUTHOR CONTRIBUTIONS Investigation, M.M.F.; Conceptualization, M.M.F., P.I.; Supervision, P.I. and P.J.A.; Writing-Original Draft, M.M.F.; Writing-Review and Editing, M.M.F., P.J.A., and P.I.; Funding Acquisition, P.I. and P.A. DECLARATION OF INTERESTS

Emara, M.M., Ivanov, P., Hickman, T., Dawra, N., Tisdale, S., Kedersha, N., Hu, G.F., and Anderson, P. (2010). Angiogenin-induced tRNA-derived stressinduced RNAs promote stress-induced stress granule assembly. J. Biol. Chem. 285, 10959–10968. Fratta, P., Mizielinska, S., Nicoll, A.J., Zloh, M., Fisher, E.M., Parkinson, G., and Isaacs, A.M. (2012). C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes. Sci. Rep. 2, 1016. Gilks, N., Kedersha, N., Ayodele, M., Shen, L., Stoecklin, G., Dember, L.M., and Anderson, P. (2004). Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol. Biol. Cell 15, 5383–5398.

The authors declare no competing interests.

Gitler, A.D., and Tsuiji, H. (2016). There has been an awakening: emerging mechanisms of C9orf72 mutations in FTD/ALS. Brain Res. 1647, 19–29.

Received: May 8, 2017 Revised: October 27, 2017 Accepted: November 28, 2017 Published: December 19, 2017

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