ALS/FTD-Associated C9ORF72 Repeat RNA Promotes Phase Transitions In Vitro and in Cells  Marta M. Fay, Paul J. Anderson, Pavel Ivanov  Cell Reports  Volume 21, Issue 12, Pages 3573-3584 (December 2017) DOI: 10.1016/j.celrep.2017.11.093 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions

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 μm. (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 μg/mL) or cycloheximide (CHX, 10 μg/mL) for 30 min prior to fixation. Quantification of the percentage of cells with SG (cells with ≥ 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 eIF2α-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 ≥ 3 (B and E–G). >37 cells (C) and >50 foci (D) were quantified per condition across 3 biological repeats. See also Figure S1. Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 rG4C2 Promotes Foci Formation In Vitro (A) Images detecting pelleted 3′ FAM-labeled r(G4C2)4, r(C4G2)4, r(A4C2)4, or scrambled control (rScram) (0.5 μM) after incubation with U2OS lysate (upper panels) or buffer (lower panels), concentrated by centrifugation, and then assayed by fluorescence microscopy. Scale bar, 10 μm. (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 μM RNA with 75, 150, and 200 mM NaCl (labeled left to right) (C), or r(G4C2)4 (0.5 μM) with or without 2.5% Ficoll (D). Data are represented as mean ± SD, n ≥ 3. See also Figure S2. Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions

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 ≥ 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 ≥ 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 μg (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. Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions

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. Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions

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. Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions

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 μM, ++ for 10 μM) for 30 min. (G) SG quantification of U2OS cells transfected with r(G4C2)4 or r(C4G2)4 preincubated with TMPyP4 or NMM (100 μM) for 30 min prior to transfection. An asterisk denotes statistical significance with p < 0.05. Data are represented as mean ± SD, n ≥ 3. Cropped blots are indicated by white separation. See also Figure S6. Cell Reports 2017 21, 3573-3584DOI: (10.1016/j.celrep.2017.11.093) Copyright © 2017 The Author(s) Terms and Conditions