biostudies-arrayexpressNie ChenHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15793Genomic integrity within transcriptionally active regions is crucial for averting oncogenic mutations, yet the precise mechanisms governing DNA double-strand break (DSB) repair in these areas remain elusive. This study reveals that the IRAK1-spliceosome axis orchestrates small nuclear RNA (snRNA) as a central molecular hub, potently activating the ATM at DSB sites within active chromatin to enable precise repair. Mechanistically, IRAK1 phosphorylates spliceosomal SR proteins to recruit snRNA to DSBs, inducing condensation of the MRN complex near transcriptionally active regions to create an ATM activation platform. Inhibition of the IRAK1-spliceosome-snRNA axis compromises ATM activation at damaged sites in active regions, leading to increased mutations exclusively within these regions andbiostudies-arrayexpressLibrary Construction - As for RIP-seq, rRNA was removed using rRNA probes as described in RNA component separation section. Library preparation was performed using the Hieff NGS® Ultima Dual-mode RNA Library Prep Kit (Yeasen) according to the manufacturer's protocol. Notably, RNA fragmentation was not conducted during library preparation since the RNA had already been fragmented by sonication. After adapter ligation, the libraries were purified and subjected to size selection, yielding a main peak between 260-280 bp. The libraries were then PCR-amplified for 22 cycles, followed by final purification prior to sequencing.Nucleic Acid Extraction - Cells were lysed with SDS lysis buffer (1% SDS; 50 mM Tris, pH 8.0) supplemented with proteinase, phosphatase, and RNase inhibitors, then sonicated with Covaris M220 focused ultrasonicator. The lysate was centrifuged, and the supernatant was collected, 10% of the supernatant was prepared as input fraction. The remaining part was incubated at 4 °C for 2-3 hours with S-beads, followed by washing four times with IP washing buffer (50 mM HEPES, pH 7.5; 300 mM KCl; 0.05% NP-40; 0.05 mM DTT). DNase I (5 µL) and RNase inhibitor (1 µL) were added to the beads along with 100 µL NT2 buffer (50 mM HEPES, pH 7.5; 150 mM KCl; 1 mM MgCl₂; 0.05% NP-40), then incubated at 37 °C on a shaker for 30 minutes. Next, beads were washed once with 1 mL NT2 buffer, 5 µL of 10% SDS, 10 µL PSequencing - Libraries were pooled and sequenced on Illumina platforms with PE150 by Novogene.Sample Collection - 293T cells were transfected with SFB-NBS1 plasmid, treated with IRAK1 inhibitor for 1 hour, then exposed to 10 Gy IR and released for 1 hour, 2x107 cells were prepared per group. Next, Cells were subjected to UV irradiation at 254 nm (400 mJ/cm2), then collected and washed once with DEPC-PBS.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw data was quality-controlled using Fastp, the paraments were set as follows: --trim_poly_g --poly_g_min_len 6 --trim_poly_x --poly_x_min_len 6 --cut_front --cut_tail --cut_window_size 4 --qualified_quality_phred 15 --low_complexity_filter --complexity_threshold 30 --length_required 4. The cleaned data was aligned to the hg38 genome using STAR, generating BAM files that were subsequently sorted. Reads mapping to specific regions on chr21 (positions 8,100,000-8,261,000; 8,320,000-8,461,000; and 8,986,600-8,988,710) were excluded to remove rRNA reads. For samples with duplicates, reads were merged prior to downstream analysis.Data Transformation - The BAM files were converted to bigwig files using deepTools.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RIP-seqHomo sapiensNie ChenWang JiadongfalsesnRNA Orchestrates ATM Activation to Promote Accurate Repair within Transcriptionally Active Chromatin (RIP-seq)Genomic integrity within transcriptionally active regions is crucial for averting oncogenic mutations, yet the precise mechanisms governing DNA double-strand break (DSB) repair in these areas remain elusive. This study reveals that the IRAK1-spliceosome axis orchestrates small nuclear RNA (snRNA) as a central molecular hub, potently activating the ATM at DSB sites within active chromatin to enable precise repair. Mechanistically, IRAK1 phosphorylates spliceosomal SR proteins to recruit snRNA to DSBs, inducing condensation of the MRN complex near transcriptionally active regions to create an ATM activation platform. Inhibition of the IRAK1-spliceosome-snRNA axis compromises ATM activation at damaged sites in active regions, leading to increased mutations exclusively within these regions and2026-08-31T00:00:00Z2026-08-31T01:01:18.784Z2025-10-20T13:27:55.606ZE-MTAB-15793ERP182512EFO_0002944EFO_0004170EFO_0005310EFO_0004917EFO_0005518EFO_0003816EFO_0004184