m0276s  (New England Biolabs)

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkag080

Figure Lengend Snippet: Detecting RNA damage by 5FU using AquIRE. ( A ) Schematic of the experimental protocol for AquIRE. Total RNA, containing modifications (e.g. 5FU), is polyA-tailed, then immobilized on oligodT beads. RNA is sequentially exposed to primary antibody to detect RNA elements, a biotin-tagged secondary antibody then Alexa Fluor TM -tagged streptavidin. Finally, water is used to elute a fluorescent signal. Figure in part generated in BioRender: https://BioRender.com/f11o153 . ( B ) HCT116 cells were treated with 2.5 µM 5FU for the indicated times, RNA extracted, then equal amounts of RNA analysed for RNA content. The graph shows AquIRE fluorescent measurements normalized against vehicle treatment set to 0. Bars represent the average of three biological replicates, each shown as grey circles and the error bars are SEM. Significance was calculated by analysis of variance (ANOVA) using Šídák multiple comparison testing. ( C ) HCT116 cells were treated as in panel (A) then stained for 5FU incorporation into RNA and counterstained with DAPI. Scale bar 50 µm. ( D ) Violin plots of the quantification of the 5FU incorporation in cells as shown in panel (C). Each grey circle represents one of 50 individual cells analysed per timepoint in this biological replicate. The thick orange lines are the mean and dashed lines are quartiles. Data are presented relative to vehicle treatment (0 h), which is set to 0. The black line extending from the y- axis shows the average of vehicle treatment. Significance was determined by Kruskal–Wallis analysis with Dunn’s multiple-comparison testing. Significant differences between drug-treated groups are annotated. All treatments were significantly different to 0 h. ( E ) HCT116 cells were treated with 5FU at 2.5 µM for the times shown, and analysis performed as in panel (A). Bars represent the average of at least three biological replicates for each timepoint, shown as grey circles, and the error bars are SEM. Significance was analysed by ANOVA. ( F ) HCT116 were treated with 2.5 µM 5FU for the indicated times and presented as in panel (C). Scale bar 50 µm. (G) 5FU intensity per cell from panel (F) was calculated for 50 cells per indicated timepoint. Data are presented in violin plots as described in panel (D). Significance was calculated using a Kruskal–Wallis test without multiple/individual comparisons. ( H ) RNA was analysed from HCT116, DLD1, or RKO cells were treated with vehicle or 10 µM 5FU for 72 h. Graph shows the levels of 5FU incorporation relative to the vehicle set to 0. Data are n = 3 biological replicates, with error bars showing SEM. Significance was determined by unpaired t -test. ( I ) HCT116 cells were treated with 10 µM 5FU for 24 h then their cytoplasmic fraction separated by sucrose density gradient. From these gradients, OD 254 nm polysome traces were obtained and overlaid here. Data are representative of two biological replicates. ( J ) RNA was extracted from the sub-polysome and polysome fractions of the 5FU treated sample shown in panel (I), with total RNA distribution between the fractions (left) and 5FU:RNA content determined by AquIRE (right) plotted ± SEM. * P < .05, ** P < .01, **** P < .0001.

Article Snippet: The RNA was polyadenylated using E.Coli polyA polymerase (NEB), unless otherwise stated, as per the manufacturer’s protocol recommendations.

Techniques: Generated, Comparison, Staining

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkag080

Figure Lengend Snippet: AquIRE sensitively quantifies the epitranscriptome across species. ( A ) Schematic of the detection of m6A using the AquIRE protocol. ( B ) Different amounts of IVT RNA with 50% m6ATP were analysed by AquIRE. Mean fluorescence reads ± SEM from three technical replicates were plotted against the quantity of m6A. Purple line plots a simple linear regression, dashed lines are the 95% confidence interval. Details of the linear regression fit are inset. ( C ) RNA was isolated from five CRC cell lines and analysed for m6A content. Values are expressed as the mean of three biological replicates ± SEM relative to the HCT116 cell line. Significance was tested by ANOVA. ( D ) AquIRE determined m6A levels in RNA samples from four tissues from three mice. Colour coded bars represent a tissue with the numbers in the annotation indicating the same animal. Values are presented as the raw fluorescence read from one technical replicate per tissue per animal. ( E ) RNA was extracted from Drosophila embryos at the indicated timepoints or adult ovary tissue then m6A levels quantified by AquIRE. The graph plots the fold fluorescence change compared to the 0–1 h timepoints from at least three biological replicates. Significance compared to 2–4 h timepoint was tested using an ANOVA with Šídák multiple comparison testing. ( F ) RNA was extracted from HCT116 cells following treatment with STM2457 for 24 h at the indicated concentrations. The graphs plot relative fluorescence compared to vehicle for total RNA, left, or polyA RNA, right. Data are from three biological replicates and show the mean ± SEM. Significance compared to vehicle was tested using an ANOVA with Šídák multiple comparison testing. ( G ) Schematic of the detection of pseudouridine (Ψ) with the AquIRE protocol. ( H ) IVT RNAs made with the indicated percentage of pseudoUTP were analysed. Mean fluorescence reads ± SEM from three technical replicates were plotted against the quantity of Ψ. Purple line plots a simple linear regression, dashed lines are the 95% confidence interval. Details of the linear regression fit are inset. ( I ) RNA was isolated from five CRC cell lines and analysed for Ψ content. Values are expressed as the mean of three biological replicates ± SEM relative fluorescence compared to HCT116. Significance was tested by ANOVA. ( J ) AquIRE was used to determine Ψ levels in RNA samples from four tissues from three mice. Data are represented as in panel (D) above. ( K ) Schematic indicating that 5FUridine cannot be converted to pseudouridine. The modified part of the Ψ base is shown in green and the hindering fluorine atom in 5FUridine in red. ( L ) RNA was extracted from HCT116 or DLD1 cells following treatment with 5FU at 10 µM for 72 h. The graphs show AquIRE data plotted as relative fluorescence compared to vehicle treatment. Data are from three biological replicates and show the mean ± SEM. For both cell lines, significance was tested using an unpaired t test. * P < .05, ** P < .01, *** P < .001, **** P < .001.

Article Snippet: The RNA was polyadenylated using E.Coli polyA polymerase (NEB), unless otherwise stated, as per the manufacturer’s protocol recommendations.

Techniques: Fluorescence, Isolation, Comparison, Modification

Journal: Nucleic Acids Research

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

doi: 10.1093/nar/gkag080

Figure Lengend Snippet: GlycoRNAs can be cell-free and are found in all domains of life. ( A ) RNA was extracted from the indicated tissues from three different mice (each a different symbol) and analysed by AquIRE for glycoRNA PNA . Graph shows the mean fluorescence per µg of RNA for each tissue, which was normalized against IVT RNA set to 0 (also shown). Significance was determined by ANOVA analysis. ( B ) RNA was extracted from Arabidopsis thaliana Col-0 ecotype leaves that had been grown in the absence of light for the indicated times. AquIRE detected glycoRNA PNA content in biological triplicates of these RNA samples compared to an equivalent mass of IVT RNA. The fold change in raw fluorescence reads is plotted compared to the 0-day timepoint. Significance was determined by ANOVA with Šídák multiple comparison testing. ( C ) RNA was extracted from Drosophila ( y 1 w 67c23 ) embryos at the indicated timepoints or adult ovary tissue and glycoRNA PNA content determined by AquIRE. Graph plots the mean fluorescence per µg for each sample from at least three biological replicates. Within the embryo samples, significance compared to 2–4 h timepoint was tested using an ANOVA with Šídák multiple comparison testing. ( D ) RNA from E. coli (TOP10) cells and growth media was isolated and quantified, then the distribution of RNA plotted for four biological replicates (left). The glycoRNA PNA , glycoRNA DBA , and glycoRNA MALI+II levels were quantified, then the distribution of signal normalized against RNA content and plotted as a percentage. Significance was determined for each glycoRNA type by two-way ANOVA with Šídák multiple comparison testing. ( E ) RNA was extracted from the media of LS174T cells, mouse plasma and serum from goat and cow. The grey bars plot the mean glycoRNA PNA fluorescent signal per µg of RNA, while the blue bars plot the RNA content of each liquid sample in ng/ml. ( F ) Ionomycin secretagogue protocol for isolation of whole organism and cell-free Xenopus laevis RNA. Embryos were treated for 10 min with ionomycin, then RNA extracted from pooled whole embryos or their growth media. ( G ) RNA samples as in panel (C) were analysed for glycoRNA PNA content by AquIRE. Graph plots the mean fluorescence per µg of RNA from four pooled embryo samples and two cell-free samples. Multiple figure panels were generated in BioRender: https://BioRender.com/t94t009 . Significance was determined by unpaired t test. * P < .05, *** P < .001, **** P < .001.

Article Snippet: The RNA was polyadenylated using E.Coli polyA polymerase (NEB), unless otherwise stated, as per the manufacturer’s protocol recommendations.

Techniques: Fluorescence, Comparison, Isolation, Clinical Proteomics, Generated