biostudies-arrayexpressNie ChenHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15822Genomic 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 and heightened sensitivity to etoposide—a drug targeting active chromatin. In summary, we have elucidated that snRNA functions as a pivotal transcription-repair bridge, connecting transcriptional processes via spliceosomes to ATM-dependent repair mechanisms to promote accurate repair within transcriptionally active chromatin.biostudies-arrayexpressSample Collection - Each group was prepared with 2x107 cells. As for DIvA U2OS stable cells, cells were treated with IRAK1 inhibitor for 13 hours, followed by the addition of 300 nM 4-hydroxytamoxifen (4OHT) and treated for 1 hour to induce DSBs. As for iCUT HeLa stable cells, cells were treated with IRAK1 inhibitor for 17.5 hours, followed by the addition of 1 μM 4OHT and treated for 5 hours to induce DSBs.Nucleic Acid Extraction - Cells were gently scraped from the culture dishes using a cell scraper, washed twice with PBS, and fixed with 1% formaldehyde in PBS at room temperature for 10 minutes, then quenched by incubation with 0.125 M glycine for 5 minutes. Cells were washed once with PBS and lysed sequentially using Buffer 1 (50 mM HEPES, pH 7.5; 140 mM NaCl; 1 mM EDTA, pH 8.0; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100) and Buffer 2 (200 mM NaCl; 1 mM EDTA; 0.5 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA); 10 mM Tris-HCl, pH 8.0) at 4 °C with rotation for 10 minutes each. After centrifugation, pellets were treated with lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0). All buffers used contained 1 mM PMSF, 0.6-2 mg/mL aprotinin, and 1x protein phosphatase inhibitor cocktail. The lysate was sonicated with a Covaris M220 focused ultrasonicator for 8 minutes in DIvA cells or 4 minutes in iCUT cells, this generated DNA fragments in the size range of 200-500 bp. After sonication, the samples were centrifuged at 15,000 rpm for 10 minutes. The supernatants were diluted with 5x volume of NETN lysis buffer with proteinase and phosphatase inhibitors, part of each sample was taken as input. The remaining samples were incubated overnight at 4 °C with rotation after the addition of 5 µg of pNBS1 Ser343 (ab109453, Abcam), γH2AX (Millipore, 05-636) or pATM Ser1981 (ab81292, Abcam) antibody. Protein A beads (Millipore) were then added to each sample, and the samples were incubated with rotation at 4 °C for 2-3 hours. The beads were washed sequentially using the following buffers: one wash with low-salt buffer (0.1% SDS, 1% Triton X-100; 2 mM EDTA, pH 8.0; 20 mM Tris-HCl, pH 8.0; 150 mM NaCl); one wash with high-salt buffer (0.1% SDS, 1% Triton X-100; 2 mM EDTA, pH 8.0; 20 mM Tris-HCl, pH 8.0; 500 mM NaCl); one wash with LiCl buffer (10 mM Tris-HCl, pH 8.0; 1% sodium deoxycholate; 1% NP-40; 250 mM LiCl; 1 mM EDTA, pH 8.0); and two washes with TE buffer (10 mM Tris-HCl, pH 8.0; and 1 mM EDTA, pH 8.0). The beads were eluted by incubation in 200 µL of elution buffer (10 mM Tris-HCl, 1% SDS, 1 mM EDTA) at 65 °C overnight with rotation (1,200 rpm) in a Thermo Mixer-Heat. RNA was digested by incubating the samples with 10 µg/mL RNase A at 37 °C for 1 hour, followed by protein digestion with 20 µg/mL proteinase K at 55 °C for 1 hour. DNA was purified by phenol-chloroform extraction, the supernatant was added with 10 µL of glycogen (20 mg/mL), 100 µL of ammonium acetate (7.5 M), and 1 mL ethanol, then incubated at -80 °C overnight. After centrifuging with 15,000 rpm at 4 °C for 30 minutes, the pellets were washed with 75% ethanol. Finally, we resuspended the pellet in nuclease-free water to prepare the sample for DNA library building.Sequencing - Libraries were pooled and sequenced on Illumina platforms with PE150 by Novogene.Library Construction - ChIP-seq library preparation was proceeded as follows: the DNA fragments were end-repaired and A-tailed, then ligated with sequencing adaptors; the final DNA library was obtained after size selection and PCR amplification.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The BAM files were converted to bigwig files using deepTools.Sequence Alignment - Raw data was processed using Trim-Galore for quality control and adapter trimming. Subsequently, reads were aligned to the hg38 genome using HISAT2. Duplicate reads were removed using Sambamba, and the resulting alignments were sorted into a BAM format with SAMtools. For samples from replicate groups, duplicate reads were merged before further analysis.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000ChIP-seqHomo sapiensNie ChenWang JiadongfalsesnRNA Orchestrates ATM Activation to Promote Accurate Repair within Transcriptionally Active Chromatin (ChIP-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 and heightened sensitivity to etoposide—a drug targeting active chromatin. In summary, we have elucidated that snRNA functions as a pivotal transcription-repair bridge, connecting transcriptional processes via spliceosomes to ATM-dependent repair mechanisms to promote accurate repair within transcriptionally active chromatin.2026-08-31T00:00:00Z2026-08-31T01:00:55.043Z2025-10-22T15:30:54.578ZE-MTAB-15822ERP182722EFO_0002944EFO_0004170EFO_0002692EFO_0004917EFO_0005518EFO_0003816EFO_0004184