Journal: bioRxiv

Article Title: AquIRE reveals multiple mechanisms of clinically induced RNA damage and the conservation and dynamics of glycoRNAs

doi: 10.1101/2025.04.01.646554

Figure Lengend Snippet: A) Schematic of the AquIRE detection approach to determine covalent RNA binding protein (RBP) crosslinking to RNA. B) HCT116 cells were crosslinked with UV at 254nm at the shown intensities directly prior to lysis and RNA extraction. Fibrillarin and NPM1 crosslinking to total RNA was detected by AquIRE compared to the fluorescence of the elution buffer (water) which acts as the experimental negative control. Values are plotted as raw fluorescence values. Bars represent the mean of 3 biological replicates ±SEM. Significance was determined by unpaired t test. C) Total RNA from HCT116 cells extracted after the indicated UV crosslinking protocols was analysed for NanoOrange binding. This version of AquIRE allows total protein crosslinking to be detected. Significance was determined by unpaired t test. D) Schematic of the additional step for AquIRE to include an on-bead protein digest using proteinase K. This is added after bead immobilisation but before primary antibody incubation. E) RNA as in B was analysed with the additional protein digest step or a parallel incubation without enzyme. Detection of fibrillarin crosslinking to RNA was performed by AquIRE. Values shown are the raw fluorescence reads from 3 biological replicates ±SEM. Statistical significance compared to column 3 was determined by ANOVA using Šídák multiple comparison testing. F) Left, HCT116 cells were treated with 200mM salt for the indicated times and G3BP1 localisation determined by immunofluorescence in parallel to DAPI staining. Scale bar 50µm.Right, quantification of the percentage of cells exhibiting at least 3 stress granules, defined as being a cytoplasmic G3BP1 foci. Data represent the average of 3 biological replicates ±SEM. G) HCT116 cells were treated as in F and RNA extracted. AquIRE was used to detect three stress granule resident proteins in the absence of UV induced crosslinking. The mean fluorescence is shown relative to no salt treatment for 3 biological replicates ±SEM. For G3BP1, statistical comparison to the 0 timepoint used an ANOVA with Šídák multiple comparison testing. For TIA1 and eIF4E, unpaired t tests were used. H) The crosslinking of two cytoplasmic proteins was analysed from the same RNA as analysed in G. Data are the mean fluorescence presented relative to 0 minutes salt for three biological replicates ±SEM. Significance was analysed by unpaired t test. I) NanoOrange AquIRE was used on the same RNA as in G and H. Mean fluorescence is shown relative to 0 minutes for 3 biological replicates ±SEM. Statistical comparison to the 0 timepoint was performed using an ANOVA with Šídák multiple comparison testing. J) Representation of a cell with cytoplasmic stress granules in red. Zoomed image shows the proximity of protein and RNA in these granules, as well as the presence of ROS. K) HCT116 cells were analysed as in F for after 150 minute exposure to 200mM NaCl alone or in combination with 2mM NAC. Scale bar 50µm. L) Stress granule positive cell percentages as in F, plotted as the average of 3 technical replicates ±SEM. M) HCT116 cells were pretreated with the DCFDA detection reagent then NaCl, NAC or the positive control TBHP for 150 minutes. Mean fluorescence reads, proportional to ROS levels, are plotted for 3 biological replicates relative to NaCl treated cells set to 1. Significance was tested by ANOVA with Šídák multiple comparison testing, not including columns 1 or 5, with significant values plotted between the three experimental groups of interest. N) HCT116 cells were treated with NaCl and/or NAC as in K, then isolated total RNA analysed by AquIRE for G3BP1 crosslinking. The mean fluorescence from three biological replicates is plotted ±SEM relative to RNA from untreated cells. Statistical comparison was performed using an ANOVA with Šídák multiple comparison testing. Only significant differences are shown. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Human cell pellets were lysed by immediate incubation with Zymo RNA lysis buffer.

Techniques: RNA Binding Assay, Lysis, RNA Extraction, Fluorescence, Negative Control, Binding Assay, Incubation, Comparison, Immunofluorescence, Staining, Positive Control, Isolation