key: cord-012654-m8nlsutd authors: Song, Zhiquan; Xie, Ying; Guo, Zongpei; Han, Yang; Guan, Hua; Liu, Xiaodan; Ma, Teng; Zhou, Ping-kun title: Genome-wide identification of DNA-PKcs-associated RNAs by RIP-Seq date: 2019-07-05 journal: Signal Transduct Target Ther DOI: 10.1038/s41392-019-0057-6 sha: doc_id: 12654 cord_uid: m8nlsutd nan highest binding potential. Motif analysis showed that DNA-PKcs preferentially binds the AGGA sequence, which was in accordance with previous findings (Fig. 1c) . 7 Then, the docking between DNA-PKcs RNA-binding sites deduced from the web server PRIdictor and the RNA motif AGGA was performed on the FRODOCK webserver, and the docking structure was analyzed using PyMOL software (Fig. 1c) . After analysis with a stringent cutoff,~500 RNAs were precipitated by DNA-PKcs. To categorize the RNAs bound by DNA-PKcs, we performed KEGG analysis. This analysis showed a number of signatures involved in the Focal adhesion and Receptor-ECM interaction pathways (Fig. 1d) . Then, the ITGA3, ITGA5, ITGAV, SDC4, and CD44 RNAs were selected for validation of the RIP-Seq results. The RIP-qPCR results showed different fold enrichment values for these five RNAs, which are involved in cell adhesion (Fig. 1e) . Regulation of RNA alternative splicing is a crucial process in RNA-binding proteins function, and aberrant splicing is often associated with various human diseases including cancers; 8 therefore, to discern how DNA-PKcs modulates bound RNAs, we sought to determine whether DNA-PKcs could affect CD44 alternative splicing. Specific primers to amplify the CD44 standard sequence and variants were designed, and qPCR was performed to examine the expression of different variants after U2OS cells were treated with NU7441 and NU7026, which target DNA-PKcs. The results showed that V4, V9, and V10 increased. Next, western blotting further verified that after DNA-PK inhibition, expression of CD44 variants increased (Fig. 1f) . In summary, our findings strongly support a model wherein the DNA-PKcs protein controls a variety of biological processes, including alternative splicing, through its RNA-binding activity. Further work will elucidate the accessory factors of DNA-PKcs in regulating alternative splicing and how alternative splicing may contribute to the DNA damage response mediated by DNA-PKcs. Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks Beyond DNA repair: DNA-PK function in cancer Human Ku70/80 interacts directly with hTR, the RNA component of human telomerase The human telomerase RNA component, hTR, activates the DNA-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein A1 Long noncoding RNA LINP1 regulates repair of DNA double-strand breaks in triple-negative breast cancer DNA-dependent protein kinase (DNA-PK) phosphorylates nuclear DNA helicase II/RNA helicase A and hnRNP proteins in an RNA-dependent manner Therapeutic targeting of splicing in cancer Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.