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The X-Linked Trichothiodystrophy-Causing Gene RNF113A Links the Spliceosome to Cell Survival Upon DNA Damage

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The X-Linked Trichothiodystrophy-Causing Gene RNF113A Links the Spliceosome to Cell Survival Upon DNA Damage ARTICLE https://doi.org/10.1038/s41467-020-15003-7 OPEN The X-linked trichothiodystrophy-causing gene RNF113A links the spliceosome to cell survival upon DNA damage Kateryna Shostak1,2,11, Zheshen Jiang1,2,11, Benoit Charloteaux1,3,10,11, Alice Mayer1,3, Yvette Habraken1,4, Lars Tharun5, Sebastian Klein5, Xinyi Xu1,2, Hong Quan Duong1,2,6, Andrii Vislovukh1,2, Pierre Close 1,7,8, ✉ Alexandra Florin5, Florian Rambow9, Jean-Christophe Marine9, Reinhard Büttner5 & Alain Chariot1,2,8 1234567890():,; Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as a therapeutic approach to treat tumors showing acquired resistance to specific drugs due to MCL-1 stabilization. 1 Interdisciplinary Cluster for Applied Genoproteomics (GIGA), University of Liege, CHU, Sart-Tilman, Liège, Belgium. 2 Laboratory of Medical Chemistry, University of Liege, CHU, Sart-Tilman, Liège, Belgium. 3 GIGA Genomics Platform, University of Liege, CHU, Sart-Tilman, Liège, Belgium. 4 Laboratory of Gene Expression and cancer, University of Liege, CHU, Sart-Tilman, Liège, Belgium. 5 Institute for Pathology-University Hospital, Cologne, Germany. 6 Center for Molecular Biology, Institute of Research and Development, Duy Tan University, 03 Quang Trung, Danang, Vietnam. 7 Laboratory of Cancer Signaling, University of Liege, CHU, Sart-Tilman, Liège, Belgium. 8 Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Wavres, Belgium. 9 Laboratory for Molecular Cancer Biology, VIB Center for Cancer Biology and KULeuven, Department of Oncology, 3000 Leuven, Belgium. 10Present address: Department of Human Genetics, CHU of Liege, University of Liege, Sart-Tilman, 4000 Liège, Belgium. 11These authors contributed equally: Kateryna Shostak, Zheshen Jiang, ✉ Benoit Charloteaux. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:1270 | https://doi.org/10.1038/s41467-020-15003-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15003-7 pliceosomes are dynamic Ribonucleoprotein (RNP) com- (TTD) and enhances sensitivity to Interstrand DNA Crosslinking Splexes required for pre-mRNA splicing, i.e., the removal of agents in C.elegans32,33, is aberrantly expressed in pulmonary non-coding intervening sequences (introns) from pre- adenocarcinomas. RNF113A promotes cell survival in Cisplatin- mRNAs and for the ligation of coding sequences (exons) to treated lung cancer-derived cells as a subunit of the spliceosome. generate mature mRNAs1–3. Intron excision occurs thanks to We characterized all transcripts whose splicing relies on RNF113A short sequence motifs in the pre-mRNA, at boundaries between and also established a link between RNF113A and MCL-1 stabili- the upstream exon and intron (the 5′ splice site) and the intron zation, with important consequences for the treatment of lung and downstream exon (the 3′splice site)1,2. RNA-protein, RNA- cancer cells showing some acquired resistance to BCL-2 inhibitors. RNA and protein–protein interactions are critical for the proper Therefore, our data define RNF113A as a promising therapeutic recognition of splice sites. These interactions are dynamic during target. the splicing cycle and are critical for the formation, rearrange- 4 ment and dissociations of the spliceosomal complexes . Results Alternate splicing of mRNA precursors allows more than 95% RNF113A is increased in pulmonary adenocarcinomas.Ina of human genes to generate a variety of RNA species and distinct search for E3 ligases overexpressed in cancer, we got interested in proteins from a single gene. Protein isoforms are selected by RNF113A which is detected in all human cases of haematological cancer cells to sustain survival and to promote tumor develop- or solid tumors (http://www.proteinatlas.org/ENSG00000125352 ment and resistance to therapies5. Cancer cells exhibit global –RNF113A/cancer). RNF113A expression was higher in our splicing deregulations due to mutations within mRNA sequences clinical cases of pulmonary adenocarcinomas than in tumor-free (“cis-acting mutations”), and in splicing factors (“trans-acting lung parenchymas, independently of the K-RAS or EGFR muta- mutations”) or to changes in expression levels of splicing tional status (Fig. 1a and Supplementary Data 1). A majority of factors6,7. As a result, the spliceosome has been defined as a lung malignancies showed a mostly nuclear staining of RNF113A promising therapeutic targets, as least for aggressive Myc-driven (Fig. 1b). A weak and almost exclusively nuclear staining of tumors and for malignant glioma8,9. Yet, transcripts whose spli- RNF113A was detected in normal lung epithelium cells (Sup- cing is deregulated in cancer cells only start to be identified10. plementary Fig. 1a). Importantly, patients with high levels of The DNA damage response (DDR) helps the body to face RNF113A showed a shorter survival rate (Fig. 1c). RNF113A thousands of DNA lesions11,12. These lesions, if not repaired, lead expression increased at both mRNA and protein levels in to mutations or genomic aberrations that threaten viability. DNA Cisplatin-treated A549 and BZR-T33 adenocarcinoma-derived damage is induced by multiple sources ranging from byproducts cells (Fig. 1d, e). Consistently, RNF113A expression was barely of cell metabolism and oxidative damage to ionizing radiation detectable in untreated A549 cells but was detected in Cisplatin- and chemotherapeutic agents. Nucleotide excision repair (NER) treated cells by immunofluorescence analyses (Fig. 1f). Camp- repairs single-stranded DNA damage by removing helix- tothecin, a topoisomerase I inhibitor, also increased RNF113A distorting DNA lesions induced by UV-light, ROS-induced expression in both A549 and BZR-T33 cells while Etoposide, a cyclopurines and intrastrand crosslinks (ISCs) generated by topoisomerase II inhibitor, did not (Fig. 1g, h and Supplementary chemotherapeutic drugs such as Cisplatin13. These ISCs activate Fig. 1b, respectively). RNF113A expression was also weakly several signal transduction pathways/kinases such as ATR14. ATR induced by γ-irradiation (Supplementary Fig. 1c). RNF113A targets substrates including Chk1 in order to help tumor cells expression was also induced by Cisplatin in normal human der- survive the DNA damage. mal fibroblasts (Supplementary Fig. 1d). Therefore, RNF113A The double-strand breaks (DSBs) do not occur as frequently as expression is induced by some DNA-damaging signals and is DNA single-strand breaks (SSBs) but are more difficult to repair increased in lung cancer. and therefore extremely toxic. The DDR includes the sensing of CCAAT/enhancer-binding protein beta (C/EBPβ) expression is the broken DNA molecule, the activation of specific signaling induced by DNA-damaging agents34. Therefore, we investigated pathways and finally, the repair of the DNA lesion11. DSB repair whether C/EBPβ is required for the induction of RNF113A involves two main pathways, the homologous recombination expression by Cisplatin. C/EBPβ deficiency interfered with (HR) and the non-homologous end-joining (NHEJ)15,16.In Cisplatin-dependent induction of RNF113A in A549 cells (Fig. 1i). NHEJ, DSBs are sensed by the Ku70/80 heterodimer that recruits Consistently, we found C/EBPβ binding sites on the RNF113A the DNA dependent protein kinase catalytic subunit (DNA- promoter using the TFbind software (http://tfbind.hgc.jp/) PKcs), Paralog of XRCC4 and XLF (PAXX) and end-processing (Fig. 1j). C/EBPβ was recruited on site 1 in unstimulated A549 enzymes leading to repair by the DNA ligase IV/DNA ligase IV- cells and on sites 1 to 4 in Cisplatin-treated cells (Fig. 1j). p53 was X-ray cross complementing protein 4 (XRCC4)/XRCC4-like dispensable for RNF113A expression as the incubation of A549 factor (XLF, also referred to as Cernunnos) complex11,17,18. The cells with Nutlin, which disrupts the interaction of the E3 ligase recruitment of DNA-PKcs to DSBs occurs within seconds fol- MDM2 with p53, or with JNJ26854165, a MDM2 inhibitor35, lowing DNA damage19. DSBs trigger DNA-PKcs autopho- did not impact on RNF113A expression (Fig. 1k). Therefore, sphorylation on the PQR cluster that includes Ser2056 and on the Cisplatin induces the expression of RNF113A through a C/EBPβ- ABCDE cluster of 6 amino acid residues between Thr2609 and dependent but p53-independent pathway. Thr2647, which is also targeted by the kinase ATM upon DNA damage20–22. These phosphorylations alter the affinity of DNA- PKcs for DNA and inactivate its kinase activity to promote RNF113A protects from Cisplatin-dependent cell death.We NHEJ23–25. next explored whether RNF113A is involved in the DDR. Cancer cells show
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