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Figure 1. rG4s initiate aSyn sol-gel phase transition (A) Representative images of MAP2 (blue) and mouse endogenous aSyn (green) with DCP1a or p62 (magenta) in mouse cultured neurons following hPFF treatment. Scale bars, 5 mm. (B) Analyses of aSyn dispersion (left), pS129+ immunoreactivity relative to MAP2+ area (center), and co-localization of aSyn with markers of RNA granule and aggresome (right) in mouse cultured neurons after hPFF treatment. (C) aSyn FRAP assay of hPFF-treated <t>HEK293T</t> cells following transient expression of mCh-aSyn (left, center). Scale bars, 2 mm. Apparent diffusion coefficients of mCh-aSyn calculated from FRAP assay (right). n.d., not detected. (D and E) In vitro aSyn (69 mM) phase separation (D) and FRAP assay (E) dependent on PEG exposure and total RNA. Scale bars, 10 (DIC), 5 (mCh-aSyn), and 1 (FRAP) mm, respectively. DIC, differential interference contrast. (F) Schematic illustration of RNA Bind-n-Seq experiment for aSyn and the top enriched RNA motif (left). Fold enrichment of the top two 12-mers (circle marks) and two randomly chosen 12-mers (square marks) across aSyn concentrations (0, 0.69, 3.45, 6.9, 34.5, and 69 mM). (G) Schematic illustration of intramolecular parallel G4 (left). Electrophoresis mobility shift assay (EMSA) for the interaction of aSyn with RNA oligomers in the presence of 100 mM NaCl (center, right). aSyn concentrations were the same as those in the RNA Bind-n-Seq experiment (F). (H) SPR sensorgrams for the interaction of aSyn with G4tr or G4mt. RU, response unit. (I) In vitro aSyn (69 mM) phase separation (top) and FRAP assay (bottom) with G4tr or G4mt in the presence of 15% PEG. Scale bars, 5 and 1 mm, respectively. (J) Proteostat intensity within phase-separated RNA (1 mM) and aSyn (69 mM) in the presence of 15% PEG. Scale bars, 5 mm. Data are presented as mean ± SEM. **p < 0.01 by two-way (B, C [FRAP], E, G, and I) and one-way (J) ANOVA with Bonferroni’s multiple comparisons test. The color-graded scales below the FRAP images are the lookup table representing the fluorescence intensity. See also Figures S1 and S2, Video S1, related to (C), and Tables S1, related to (F) and S3, related to statistical analysis.
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Figure 1. rG4s initiate aSyn sol-gel phase transition (A) Representative images of MAP2 (blue) and mouse endogenous aSyn (green) with DCP1a or p62 (magenta) in mouse cultured neurons following hPFF treatment. Scale bars, 5 mm. (B) Analyses of aSyn dispersion (left), pS129+ immunoreactivity relative to MAP2+ area (center), and co-localization of aSyn with markers of RNA granule and aggresome (right) in mouse cultured neurons after hPFF treatment. (C) aSyn FRAP assay of hPFF-treated HEK293T cells following transient expression of mCh-aSyn (left, center). Scale bars, 2 mm. Apparent diffusion coefficients of mCh-aSyn calculated from FRAP assay (right). n.d., not detected. (D and E) In vitro aSyn (69 mM) phase separation (D) and FRAP assay (E) dependent on PEG exposure and total RNA. Scale bars, 10 (DIC), 5 (mCh-aSyn), and 1 (FRAP) mm, respectively. DIC, differential interference contrast. (F) Schematic illustration of RNA Bind-n-Seq experiment for aSyn and the top enriched RNA motif (left). Fold enrichment of the top two 12-mers (circle marks) and two randomly chosen 12-mers (square marks) across aSyn concentrations (0, 0.69, 3.45, 6.9, 34.5, and 69 mM). (G) Schematic illustration of intramolecular parallel G4 (left). Electrophoresis mobility shift assay (EMSA) for the interaction of aSyn with RNA oligomers in the presence of 100 mM NaCl (center, right). aSyn concentrations were the same as those in the RNA Bind-n-Seq experiment (F). (H) SPR sensorgrams for the interaction of aSyn with G4tr or G4mt. RU, response unit. (I) In vitro aSyn (69 mM) phase separation (top) and FRAP assay (bottom) with G4tr or G4mt in the presence of 15% PEG. Scale bars, 5 and 1 mm, respectively. (J) Proteostat intensity within phase-separated RNA (1 mM) and aSyn (69 mM) in the presence of 15% PEG. Scale bars, 5 mm. Data are presented as mean ± SEM. **p < 0.01 by two-way (B, C [FRAP], E, G, and I) and one-way (J) ANOVA with Bonferroni’s multiple comparisons test. The color-graded scales below the FRAP images are the lookup table representing the fluorescence intensity. See also Figures S1 and S2, Video S1, related to (C), and Tables S1, related to (F) and S3, related to statistical analysis.

Journal: Cell

Article Title: RNA G-quadruplexes form scaffolds that promote neuropathological α-synuclein aggregation

doi: 10.1016/j.cell.2024.09.037

Figure Lengend Snippet: Figure 1. rG4s initiate aSyn sol-gel phase transition (A) Representative images of MAP2 (blue) and mouse endogenous aSyn (green) with DCP1a or p62 (magenta) in mouse cultured neurons following hPFF treatment. Scale bars, 5 mm. (B) Analyses of aSyn dispersion (left), pS129+ immunoreactivity relative to MAP2+ area (center), and co-localization of aSyn with markers of RNA granule and aggresome (right) in mouse cultured neurons after hPFF treatment. (C) aSyn FRAP assay of hPFF-treated HEK293T cells following transient expression of mCh-aSyn (left, center). Scale bars, 2 mm. Apparent diffusion coefficients of mCh-aSyn calculated from FRAP assay (right). n.d., not detected. (D and E) In vitro aSyn (69 mM) phase separation (D) and FRAP assay (E) dependent on PEG exposure and total RNA. Scale bars, 10 (DIC), 5 (mCh-aSyn), and 1 (FRAP) mm, respectively. DIC, differential interference contrast. (F) Schematic illustration of RNA Bind-n-Seq experiment for aSyn and the top enriched RNA motif (left). Fold enrichment of the top two 12-mers (circle marks) and two randomly chosen 12-mers (square marks) across aSyn concentrations (0, 0.69, 3.45, 6.9, 34.5, and 69 mM). (G) Schematic illustration of intramolecular parallel G4 (left). Electrophoresis mobility shift assay (EMSA) for the interaction of aSyn with RNA oligomers in the presence of 100 mM NaCl (center, right). aSyn concentrations were the same as those in the RNA Bind-n-Seq experiment (F). (H) SPR sensorgrams for the interaction of aSyn with G4tr or G4mt. RU, response unit. (I) In vitro aSyn (69 mM) phase separation (top) and FRAP assay (bottom) with G4tr or G4mt in the presence of 15% PEG. Scale bars, 5 and 1 mm, respectively. (J) Proteostat intensity within phase-separated RNA (1 mM) and aSyn (69 mM) in the presence of 15% PEG. Scale bars, 5 mm. Data are presented as mean ± SEM. **p < 0.01 by two-way (B, C [FRAP], E, G, and I) and one-way (J) ANOVA with Bonferroni’s multiple comparisons test. The color-graded scales below the FRAP images are the lookup table representing the fluorescence intensity. See also Figures S1 and S2, Video S1, related to (C), and Tables S1, related to (F) and S3, related to statistical analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER RIP-Assay Kit for microRNA MBL Cat#RN1005 RNeasy Mini Kit Qiagen Cat#74104 Deposited data Raw sequencing data: RNA Bind-n-Seq This paper GEO: GSE235418 Raw sequencing data: RIP-Seq This paper GEO: GSE234238 Raw sequencing data: RNA-Seq This paper GEO: GSE267382 Experimental models: Cell lines AAVpro 293T Takara Bio Cat#632273 HEK293T ATCC Cat#CRL-3216; RRID: CVCL_0063 Neuro-2a ATCC Cat#CCL-131; RRID: CVCL_0470 Experimental models: Organisms/strains Mouse: C57BL/6J Japan SLC N/A Mouse: C57BL/6N-Sncatm1Mjff/J The Jackson Laboratory Cat#016123; RRID: IMSR_JAX:016123 Oligonucleotides RNA oligonucleotides for LLPS, CD, EMSA, and SPR experiments; see Table S6 This paper N/A A primer for RNA-Bind-n-Seq; see Table S6 This paper N/A Primers for RT-qPCR; see Table S6 This paper N/A FISH probes; see Table S6 This paper N/A Recombinant DNA pET-hSNCA Izawa et al.56 N/A pET-mCh-hSNCA This paper N/A pET-hSNCA DNterm This paper N/A pET-mCh-hSNCA DNterm This paper N/A pET-hSNCA DNAC This paper N/A pET-hSNCA DCterm This paper N/A pET-mSNCA This paper N/A pHTC HaloTag CMV-neo Vector Promega Cat#G7711 pCAG-neo Fujifilm Cat#163-25601 pHR-FUSN-mCh-Cry2WT Addgene RRID: Addgene_101223 MCP-YFP Addgene RRID: Addgene_101160 pHR-Tre3G-293GGGGCC-123MS2 Addgene RRID: Addgene_99149 pCMV-mCh-hSNCA This paper N/A pCAG-hSNCA This paper N/A pCAG-MCP-mCh-Cry2 This paper N/A pCAG-G4tr-MS2 This paper N/A pCAG-G4mt-MS2 This paper N/A pHelper Stratagene Cat#240071 pAAV2/9n Addgene RRID: Addgene_112865 pAAV-hSyn-DIO{ChETA-mRuby2}onW3SL Addgene RRID: Addgene_111389 pAAV-TH-iCre This paper N/A pAAV-hSyn-DIO-MCP-Cry2 This paper N/A pAAV-hSyn-G4tr-MS2 This paper N/A pAAV-hSyn-G4mt-MS2 This paper N/A Software and algorithms FIJI (1.53) NIH RRID: SCR_002285 GraphPad Prism (7.05) GraphPad Software RRID: SCR_002798 (Continued on next page) ll OPEN ACCESS Cell 187, 1–14.e1–e10, November 27, 2024 e3 Please cite this article in press as: Matsuo et al., RNA G-quadruplexes form scaffolds that promote neuropathological a-synuclein aggregation, Cell (2024), https://doi.org/10.1016/j.cell.2024.09.037 Article

Techniques: Sublimation, Cell Culture, Dispersion, FRAP Assay, Expressing, Diffusion-based Assay, In Vitro, Electrophoresis, Mobility Shift