Journal: Nature Communications

Article Title: Meta-unstable mRNAs in activated CD8 + T cells are defined by interlinked AU-rich elements and m 6 A mRNA methylation

doi: 10.1038/s41467-025-67762-w

Figure Lengend Snippet: A The miCLIP workflow for identification of high-confidence crosslinks: (1) primary human CD8 + T cells subjected to in vitro activation with aCD3/aCD28 beads; (2) RNA enrichment and experimental controls, including m 6 A immunoprecipitation without UV irradiation (noUV) and input RNA samples; (3) SDS‒PAGE purification of RNA fragments crosslinked to the anti-m 6 A antibody followed by RNA isolation; (4) library preparation; (5) iCLIP analysis for determination of uniquely mapped crosslinks; (6) determination of peaks, i.e., regions with high density of crosslinks; (7) ‘positionally enriched k-mer analysis’ (PEKA) to extract sequences in high-confidence (‘thresholded’) miCLIP crosslinks. B Hierarchical clustering of 5-mers based on standardised PEKA scores at ‘thresholded sites’; here, crosslinks and Clippy peaks were used for PEKA. The median PEKA score of each miCLIP or Input group (noAct, Day1 or Day5) was calculated for all 5-mers, and the unique sets of the 20 most enriched and variant 5-mers were plotted on the heatmap. C Metagene distribution of GLORI-determined m 6 A counts at miCLIP sites (top) and number-matched control sites randomly selected from the 3’UTR (bottom), centred on RRACH, WWAWW and CRACH pentamers; GLORI counts show the effect of the METTL3 inhibitor at each position, and represent the average across two CD8 + T cell replicates. D Metagene distribution of GLORI-determined m 6 A counts centred on miCLIP-ARE sites that contain nearby RRACH motifs (left) or lack RRACH motifs (right), showing the effect of the METTL3 inhibitor. Stratification by distance highlights the spatial relationship between m 6 A deposition and RRACH proximity. The WWAWW + RRACH sites refer to miCLIP-ARE sites containing at least one RRACH motif within ≤ 50nt. E Density plot showing the motif-specific distribution of PEKA crosslinks in the miCLIP and Input samples around scaled mRNA regions. For all panels, CD8 + T cells from healthy donors were isolated; not activated (noAct; n = 4), Day1 after activation (Day1; n = 5), Day5 after activation (Day5; n = 4), Mock ( n = 12), noUV ( n = 3). Panel ( A ) was created in BioRender. Foskolou, I. (2026) https://BioRender.com/ h18sinc. Source data are provided as a Source Data file.

Article Snippet: Non-activated and Day1 activated cells were treated for 24 h with DMSO or 4 μM METTL3 inhibitor (MedChemExpress, cat. no. HY-134836), while Day5 activated T cells were treated with the same inhibitor for 5 days.

Techniques: In Vitro, Activation Assay, Immunoprecipitation, Irradiation, Purification, Isolation, Variant Assay, Control

Journal: Nature Communications

Article Title: Meta-unstable mRNAs in activated CD8 + T cells are defined by interlinked AU-rich elements and m 6 A mRNA methylation

doi: 10.1038/s41467-025-67762-w

Figure Lengend Snippet: A Actinomycin D experiments showing % of remaining mRNA in CD8⁺ T cells treated with DMSO or METTL3inhibitor. Mean ± SD; n = 3 (one donor provided material for 0,2 h). Mixed-effects model (REML), p -values indicate treatment effects. Donors were processed in parallel. Representative results from three independent experiments ( IL7R , TNF ). B mRNA levels IL7R ( n = 5), CCL4 ( n = 5), CD69 ( n = 4), CD28 ( n = 4), TCF7 ( n = 3), ZFP36L1 ( n = 3), TNF ( n = 4), IFNG ( n = 4) in CD8⁺ T cells treated with METTL3inhibitor. Fold change relative to DMSO (grey line). Reference gene 18S. Mean ± SD; paired two-tailed t tests on ΔCt values. For TNF , one Grubbs-identified outlier is displayed but excluded from statistics. C , D Representative flow cytometry plots illustrating IL7Rα gating ( C ) and quantification of IL7Rα⁺CD8⁺ T cells ( D ) ( n = 10). Paired two-tailed t tests. E , F Representative histogram ( E ) and quantification ( F ) of IL7Rα median fluorescence intensity (MFI) in IL7Rα⁺ cells treated with METTL3inhibitor relative to DMSO (grey line) ( n = 10). Mean ± SD; one-sample two-tailed t test. G GFP MFI in T cells transduced with GFP-3’UTR constructs (S3H) after treatment with FTO inhibitor followed by PMA/ionomycin activation relative to DMSO ( n = 3). One-way ordinary ANOVA. H , I IFNγ (H) and TNFα (I) protein levels measured by ELISA in DMSO (0 μM) or FTOinhibitor treated cells ( n = 3). RM one-way ANOVA. J , K Representative amplification curves (ΔRn) and Ct quantification for reference ( J ) and miCLIP-ARE ( K ) amplicons in control (grey) and METTL3-KO (pink) samples ( n = 3). Mean ± SD. L Fold-change expression (ΔΔCt) for the miCLIP-ARE site in METTL3-KO and FTO-KO cells relative to control (grey line). Mean ± SEM; unpaired two-tailed t test comparing METTL3-KO and FTO-KO; paired t test for control vs METTL3-KO on ΔCt values; ( n = 3). M – P mRNA levels ( M ) and GFP protein levels ( N – P ) in CD8 + T cells transduced with GFP-3′UTR constructs (S3O). mRNA levels normalised to 3′UTR- TNF_ WT (grey line) ( n = 10); one-way ANOVA with Holm–Šídák correction ( M ). Representative plots of resting or PMA/ionomycin-activated cells ( N ). GFP MFI quantification in activated cells ( O , P ) ( n = 15); paired two-tailed t tests. Box-and-whisker plots (min–max, median, 25th–75th percentiles). All n values reflect distinct donors. Source data are provided as a Source Data file.

Article Snippet: Non-activated and Day1 activated cells were treated for 24 h with DMSO or 4 μM METTL3 inhibitor (MedChemExpress, cat. no. HY-134836), while Day5 activated T cells were treated with the same inhibitor for 5 days.

Techniques: Two Tailed Test, Flow Cytometry, Fluorescence, Transduction, Construct, Activation Assay, Enzyme-linked Immunosorbent Assay, Amplification, Control, Expressing, Whisker Assay