biostudies-arrayexpressNie ChenHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15831Genomic 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-arrayexpressNucleic Acid Extraction - Next, total RNA was extracted with Trizol, and the 50-500 nt RNA were separated as described in RNA component separation section. RNA sample was added T4 PNK (M0201S, NEB), followed by incubation at 37 °C for 1 hour. RppH (M0356S, NEB) was then added, and the mixture was incubated at 37 °C for 30 minutes. RNA purification was completed using the phenol-chloroform extraction method.Sample Collection - HeLa cells were treated with IRAK1 inhibitor for 1 hour, then exposed to 10 Gy IR and released for 1 hour.Library Construction - For library preparation, Hieff NGS® Ultima Dual-mode RNA Library Prep Kit (Yeasen) was used; however, RNA fragmentation step was omitted. After PCR amplification, 1x Hieff NGS® DNA Selection Beads (Yeasen) were added for purification. The selection process was performed as follows: first, 0.6x DNA selection beads were added, and the magnetic rack was used to separate beads and supernatant. The DNA bound to the beads was purified to obtain Part I library. The supernatant was transferred to a new PCR tube, and 0.2x beads were added; the DNA bound to these beads was then purified to obtain Part II library.Sequencing - Libraries were pooled and sequenced on Illumina platforms with PE150 by Novogene.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - The sequencing data for both Part I and Part II were analyzed using the following workflow. Raw data was quality-controlled using Fastp, with parameters set as described in the RIP-seq related analysis section. Cleaned data was aligned to the hg38 genome using Bowtie2, the parameters were set as follows: -D 15 -R 2 -N 1 -k 1 -L 12 -i S,1,1.15. Then BAM files were sorted, and reads mapping to rDNA regions near chr21 were removed.Data Transformation - Based on the GRCh38 GTF file, read counts for each gene were obtained using multicov function of BEDtools, which were subsequently normalized to CPM values. Subsequently, the CPM matrices for Part I and Part II were combined, with gene-level CPM value averaged, and low-expression genes removed.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of total RNAHomo sapiensNie ChenWang JiadongfalsesnRNA Orchestrates ATM Activation to Promote Accurate Repair within Transcriptionally Active Chromatin (small RNA-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 and2026-08-31T00:00:00Z2026-08-31T01:00:30.765Z2025-10-23T12:52:23.748ZE-MTAB-15831ERP182822EFO_0002944EFO_0004170EFO_0009653EFO_0004917EFO_0005518EFO_0003816EFO_0004184