Plasmids expressing 60 CGG repeats (referred to as 60x CGG) or 100 CGG repeats within the human FMR1 sequence, fused to GFP (referred to as 5’UTR FMR1 (CGG)100x GFP) have been described previously 33,42. For cell viability assays, a bidirectional plasmid was created by inserting GFP with a CMV promoter and terminator sequence (amplified from pEGFP) into a 5’UTR FMR1 (CGG)100x GFP plasmid with the ACG start codon deleted (referred to as Δ5’UTR FMR1 (CGG)100x GFP) 33) using BglII and EcoRI restriction sites. To ensure the stability of the expanded CGG repeats, all CGG plasmids were transformed into NEB® Stable Competent E. coli (New England Biolabs, UK) and grown at 30ºC according to manufacturer’s instructions.
Cell Culture And Transient Transfections
HGrC1 43 and COV434 44 cells were cultured in DMEM/F-12 (Gibco™, ThermoFisher Scientific, UK) supplemented with 10% fetal bovine serum (Gibco™) and maintained at 37°C in 5% CO2. For transient transfections to express 60x CGG, 5’UTR FMR1 (CGG)100x GFP or Δ5’UTR FMR1 (CGG)100x GFP plasmids or to overexpress GFP- or HA-tagged proteins of interest, cells were seeded at density of 80,000 cells per well of a 4-well chamber slide (Nunc, ThermoFisher Scientific). Single and double transfection experiments were carried out using Lipofectamine 3000 (Invitrogen, ThermoFisher Scientific) according to manufacturer’s instructions.
RNA fluorescence in situ hybridisation (FISH) combined with immunocytochemistry
Chamber slides with transfected cells were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. Cells were permeablised with 0.5% Triton X-100/PBS for 5 min, and washed in PBS before pre-hybridisation in 40% DMSO (Sigma-Aldrich, UK), 40% formamide (Sigma-Aldrich), 10% BSA (10mg/mL) and 2x saline-sodium citrate (SSC) for 30 min in a humidified hybridisation oven set to 60ºC. Chamber slides were hybridised for 2h in 40% formamide, 10% DMSO, 2x SSC, 2mM vanadyl ribonucleotide (Sigma-Aldrich), 60mg/mL yeast RNA (ThermoFisher Scientific), 30mg/mL BSA plus 0.75µg (CCG)8x-Cy3 DNA oligonucleotide probe (Integrated DNA Technologies, UK). Following hybridisation, the chamber slides were washed twice successively at 55ºC in 2x SSC/50% formamide and 2x SSC, and counterstained with 4,6-diamidino-2-phenylidole (DAPI) before mounting in Permafluor Aqueous Mounting Medium (Perkin-Elmer, ThermoFisher Scientific). To confirm the RNA composition of CGG aggregates, treatment with RNAase A (Roche Diagnostics, UK) was carried out according to manufacturer’s instructions prior to permeablisation. If immunocytochemistry was carried out immediately following in situ hybridisation, instead of counterstaining, chamber slides were washed three times in PBS before incubation with primary antibody (diluted in PBS) overnight at 4ºC. Antibody dilutions were: anti-GFP at 1:400 (ab6556, Abcam), anti-HA at 1:500 (clone 16B12, #MMS-101P, Covance), anti-FUS at 1:400 (AMAb90549, Atlas Antibodies), anti-PA2G4 at 1:50 (15348-1-AP, Proteintech) and anti-TRA2β at 1:800 (ab31353, Abcam). The next day, chamber slides were washed in PBS before incubation with an Alexa Fluor 488-conjugated secondary antibody (Molecular Probes) for 60 min at room temperature. Chamber slides were then counterstained with DAPI before mounting. Slides were imaged using either a Zeiss LSM 780 confocal microscope (Carl Zeiss, Oberkochen, Germany) or an Axioscan slide scanner (Carl Zeiss). For colocalisation analyses, Z-stack images were acquired with a Zeiss LSM 780 confocal microscope using the correct Nyquist sample rate and deconvolved using Huygens Essential. The 3D ImageJ Suite in FIJI was used to carry out segmentation and calculate percentage colocalisation using the middle slice of the Z stack.
RNA pulldown SILAC high-throughput mass spectrometry (RP-SMS) and Western blotting
RNA pulldown coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry was carried out as described previously 45. Briefly, HGrC1 cells were cultured in SILAC media (DC Biosciences, Dundee, UK), ‘heavy’ or ‘light’, supplemented with dialysed calf serum (DC Biosciences) to incorporate cells with heavy or light isotopes. Cell extracts were prepared and incubated with CGG(30x) RNA (Dharmacon, Cambridge, UK) coupled to agarose beads. Following a series of washes to remove unbound protein, proteins were electrophoresed into an SDS-PAGE gel (Bio-Rad, Watford, UK), and submitted for LC-MS/MS analysis performed using an Orbitrap™ mass spectrometer (ThermoFisher Scientific). Data was analysed using the MaxQuant software 46 to determine the ratio of heavy-labelled peptides to light-labelled peptides, and identify proteins specifically bound to CGG RNA. Pulldown experiments followed by Western blotting were used to validate mass spectrometry data. Pulldown was carried out as described above, and proteins were separated on an SDS-PAGE gel (Bio-Rad). Proteins were transferred onto Immobilon FL membrane (Millipore, Dorset, UK), which was blocked using Intercept blocking buffer (LI-COR Biosciences, Cambridge, UK). Western blotting was undertaken with anti-FUS (AMAb90549), anti-PA2G4 (15348-1-AP) and anti-TRA2β (ab31353) antibodies, at a dilution of 1:1000, overnight at 4ºC. Alexa Fluor 680- and 800-conjugated secondary antibodies (Molecular Probes) were used for detection (at 1:10,000) and blots were imaged on a LI-COR FC Odyessy.
Flow Cytometry Cell Viability Assay
HGrC1 and COV434 cells were seeded at a density of 300,000 cells per well of a 6 well plate. and transfected with either an empty pEGFP plasmid, Δ5’UTR FMR1 (CGG)100x GFP or 5’UTR FMR1 (CGG)100x GFP as described above. At 48h post transfection, cells were trypsinised, neutralised, and 6 wells per condition were pooled in order to have enough cells for flow cytometry analysis. Just before analysis, cells were incubated with DAPI (1:1000) for 3 min as a cell viability marker. Flow cytometry was carried out on a BD LSRFortessa™ and data analysed using BD FACSDiva™ software (version 8.0). Single cells were analysed for GFP expression (to identify positively transfected cells) and DAPI staining, with DAPI positive cells indicative of a compromised cell membrane.
All data are shown as mean ± standard error of the mean and were analysed using GraphPad Prism 9 software (GraphPad Software, Inc., San Diego, CA). Mann-Whitney and Freidman test statistics were carried out as appropriate. A p value of < 0.05 was considered statistically significant.