biostudies-arrayexpressNie ChenHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15815Genomic 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-arrayexpressSequencing - 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.Sample Collection - DIvA cells were seeded in 15-cm dishes and treated with an IRAK1 inhibitor for 19 hours, followed by treatment with 300 nM 4OHT for 1 hour. Cells were washed once with PBS, then incubated at room temperature with gentle shaking in 7 mL of 2 mM Disuccinimidyl glutarate (DSG) solution in PBS for 45 minutes. After washing with PBS, cells were treated with 7 mL of 3% formaldehyde in PBS and incubated at room temperature for 10 minutes. The crosslinking reaction was quenched by adding glycine to a final concentration of 500 mM for 5 minutes at room temperature. Following fixation, cells were washed twice with cold PBS, collected, and counted. A total of 2x107 cells were prepared per group for subsequent steps.Nucleic Acid Extraction - Cells were lysed with 1 mL pre-chilled Cell Lysis Buffer (10 mM HEPES, pH 7.5; 20 mM KCl; 1.5 mM MgCl2; 0.5 mM EDTA; 1 mM tris(2-carboxyethyl) phosphine [TCEP]; 0.5 mM PMSF; 0.1% NP-40). The lysate was incubated on ice for 10 minutes, followed by centrifugation. The nuclear pellet was treated with Nuclear Lysis Buffer (20 mM HEPES, pH 7.5; 50 mM KCl; 1.5 mM MnCl2; 1% NP-40; 0.4% sodium deoxycholate; 0.1% N-lauroylsarcosine; 1 mM TCEP; 0.5 mM PMSF). The cells were resuspended to a concentration of 5x106 cells/mL and incubated on ice for 10 minutes. The sample was then subjected to sonication for 6 minutes by Covaris M220 focused ultrasonicator. Following sonication, 100 µL of TURBO DNase (Invitrogen) was added to the sample, then incubated at 37 °C for 10 minutes. The reaction was terminated by adding 24 µL DNase Stop Solution (250 mM EDTA, 125 mM EGTA). Subsequently, 1.5 mL of 1.4x GuSCN Hybridization Buffer (1x buffer was prepared as follows: 20 mM Tris-HCl, pH 7.5; 7 mM EDTA; 3 mM EGTA; 150 mM LiCl; 1% NP-40; 0.2% N-lauroylsarcosine; 0.1% sodium deoxycyolate; 3 M guanidine thiocyanate; 2.5 mM TCEP) was added to each 600 µL of the sample. The mixture was centrifuged, and the supernatant was rapidly frozen in liquid nitrogen before being stored at -80 °C or used directly in subsequent steps. 100 µL of MyONE Streptavidin C1 magnetic beads (Invitrogen) was washed twice using 1x GuSCN Hybridization Buffer. These beads were then added to the lysate derived from 5x106 cells, then incubated at 37 °C for 30 minutes with shaking. Subsequently, the mixture was subjected to magnetic separation, and 1/10 of the supernatant was reserved as an input component. Simultaneously, 250 pmol of ssDNA probe pools were heated at 85 °C for 3 minutes to denature them, followed by immediate placement on ice. These probes were then added to the sample, which was incubated at 37 °C for 2-3 hours while shaking at 1,200 rpm. 500 µL of Streptavidin C1 beads were washed twice using GuSCN Hybridization Buffer and subsequently added to the sample. The mixture was incubated at 37 °C for 15-30 minutes while shaking. Following this, magnetic separation was performed to isolate the beads, which were then washed six times with 500 µL GuSCN Wash Buffer (20 mM Tris-HCl, pH 7.5; 10 mM EDTA; 1% NP-40; 0.2% N-lauroylsarcosine; 0.1% sodium deoxycyolate; and 3 M guanidine thiocyanate; with 2.5 mM TCEP). Each wash involved incubation at 45 °C for 10 minutes before magnetic separation. After washing, the beads were treated twice more with RNase H Elution Buffer (50 mM Tris-HCl, pH 7.5; 75 mM NaCl; 3 mM MgCl2; 0.125% N-lauroylsarcosine; 0.025% sodium deoxycholate; and 2.5 mM TCEP). The beads were then subjected to elution by the addition of 55 µL RNase Elution Buffer and 7.5 µL RNase H (M0297S, NEB), followed by mixing and incubation at 37 °C for 30 minutes while shaking. The resulting supernatant was separated using a magnetic rack and stored, while the beads were treated with 62.5 µL GuSCN Hybridization Buffer at 37 °C for 5 minutes. After once more magnetic separation, the supernatant was combined with the previously collected supernatant. Finally, 312.5 µL of NLS Digestion Buffer (20 mM Tris-HCl, pH 7.5; 10 mM EDTA; 2% N-lauroylsarcosine; and 2.5 mM TCEP), 50 µL of 5 M NaCl, and 12.5 µL of Proteinase K were added to the supernatant or input group, then incubated overnight at 60 °C to remove proteins and reverse crosslinking. The DNA sample was purified for DNA using SILANE beads.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. The cleaned reads were then mapped to the hg38 genome using STAR, the parameters were set as follows: --outFilterMismatchNmax 10 --outFilterMatchNmin 10 --alignEndsType Local --seedSearchLmax 10 --outFilterScoreMinOverLread 0.1 --outFilterMatchNminOverLread 0.1. Duplicate reads were removed and the BAM files were sorted using Sambamba.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000ChIP-seqHomo sapiensNie ChenWang JiadongfalsesnRNA Orchestrates ATM Activation to Promote Accurate Repair within Transcriptionally Active Chromatin (RAP-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.2026-08-31T00:00:00Z2026-08-31T01:01:01.039Z2025-10-22T12:16:39.304ZE-MTAB-15815ERP182701EFO_0002944EFO_0004170EFO_0002692EFO_0004917EFO_0005518EFO_0003816EFO_0004184