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Integrator subunit INTS12 links ribotoxic stress to transcription-coupled nucleotide excision repair

Nature Structural & Molecular Biology (2026)Cite this article

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Abstract

Cells use transcription-coupled nucleotide excision repair (TC-NER) to efficiently resolve transcription-blocking DNA lesions caused by genotoxic stress such as ultraviolet (UV) irradiation. However, UV also induces RNA damage, triggering a cytoplasmic ribotoxic stress response (RSR). Whether and how RSR affects nuclear TC-NER has remained unclear. Here we identify INTS12, a flexible, poorly characterized subunit of the Integrator complex, as a key mediator linking RSR to TC-NER. Specifically, RSR-activated ZAK signaling induces phosphorylation of INTS12, enhancing its interaction with CSB and promoting recruitment of the Integrator complex to lesion-stalled RNA polymerase II (Pol II). This facilitates Pol II clearance and enables efficient DNA repair through TC-NER. Disruption of this pathway compromises TC-NER and transcription recovery, thereby increasing cellular sensitivity to UV-induced damage. Notably, the requirement for INTS12-mediated Pol II removal is context dependent, as it is not advantageous during the transcription-coupled response to formaldehyde-induced DNA–protein crosslinks, which rely on a distinct proteasome-dependent degradation pathway. Together, these findings uncover a regulatory axis connecting RNA damage signaling to DNA repair and highlight a context-dependent role of INTS12 in maintaining genome integrity.

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Fig. 1: INTS12 is rapidly phosphorylated in response to UV-induced ribotoxic stress.
Fig. 2: Phosphorylation of INTS12 enhances the recruitment of Integrator complex to CSB.
Fig. 3: INTS12 is crucial for transcription recovery and DNA repair by TC-NER.
Fig. 4: INTS12 promotes the processing and removal of lesion-stalled Pol II following UV irradiation.
Fig. 5: A context-dependent role of INTS12 in TC-DPC repair.

Data availability

MS data were deposited to ProteomeXchange (PXD066142). ChIP-seq and RNA-seq data were uploaded to the Genome Sequence Archive of The National Genomics Data Center, the China National Center for Bioinformation/Beijing Institute of Genomics and the Chinese Academy of Sciences (HRA009292). All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank L. Zhang, J. Wang, W. Deng, Q. Zhao, C. Xu, L. Shen, J. Jin and the core facility of the Life Sciences Institute for helpful suggestions, discussions and technical assistance. This work was supported in part by Zhejiang Provincial Natural Science Foundation of China (LRG25C060001), National Natural Science Foundation of China (32541027, 32570646, 32370591, 32271343, 32070632 an 92053114), National Key R&D Program of China (2022YFA1303000), Fundamental Research Funds for the Central Universities (226-2025-00028), Innovative Research Team of High-level Local University in Shanghai (to J.H.) and Medical Science Data Center of Fudan University.

Author information

Author notes

  1. These authors contributed equally: Zhuo Li, Ran Li, Min Yang, Yanchao Huang.

Authors and Affiliations

  1. Zhejiang Key Laboratory of Molecular Cancer Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Zhuo Li, Ran Li, Min Yang, Jiaye Yang, Yangqing Shao, Huanyi Fu, Yu-Xin Xiao, Chengyu Li, Huipeng Jiao, Dong Fang, Bing Yang, Lei Li, Jun Huang, Long Zhang & Huasong Lu

  2. Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang Province, Hangzhou City University School of Medicine, Hangzhou, China

    Ran Li

  3. Shanghai Fifth People’s Hospital, Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Institutes of Biomedical Sciences, Fudan University, Shanghai, China

    Yanchao Huang, Qian Zhu & Jinchuan Hu

  4. Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China

    Weiqi Zhao & Jun Xu

  5. Tongji University Cancer Center, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, China

    Yi Lu

  6. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, State Key Laboratory of Genetic Engineering, Shanghai Key Laboratory of Medical Epigenetics, Shanghai Key Laboratory of Radiation Oncology, Human Phenome Institute, Fudan University, Shanghai, China

    Fei Xavier Chen

  7. Department of Neurology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Huasong Lu

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Contributions

Z.L., J.H. and H.L. conceptualized and design the study. H.L., J.H., Z.L., R.L., M.Y. and Y.H. developed the methodology. Z.L., M.Y., Y.H., J.Y., Q.Z., Y.S., W.Z. and C.L. acquired the data. H.L., J.H., Z.L., R.L., M.Y., Y.H., J.Y., Q.Z., Y.S. and C.L. performed data analysis. H.F., Y.-X.X., H.J., D.F., B.Y., Y.L., J.X., L.L., J.H., F.X.C. and L.Z. provided valuable discussion. Z.L., J.H. and H.L. supervised the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Jinchuan Hu or Huasong Lu.

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Nature Structural & Molecular Biology thanks Julian Stingele and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Characterization of INTS12 phosphorylation.

a, Lysates from HeLa cells that were either untreated or treated with UV (20 J/m2, 20 min recovery), 4-NQO (5 µM, 1 h), MNNG (100 µM, 1 h), MMS (10 mM, 30 min), Anisomycin (20 µM, 1 h), Cycloheximide (100 µg/mL, 1 h) or b, HU (4 mM, 4 h), Bleomycine (10 ug/mL, 1 h), IR (10 Gy, 30 min recovery), Calicheamicin (100 nM, 1 h) as shown in b were separated on Phos-tag SDS-PAGE and immunoblotted with the indicated antibodies. c, HeLa cells expressing the indicated Integrator complex subunit were left untreated or treated with 4-NQO (5 µM, 1 h) and then analyzed as in a. d, Lysates from HeLa cells that were either untreated or treated with 4-NQO (1 µM, 1 h) and recover for the indicated time were analyzed as in a. e, Control or ZAK KO cells were untreated or treated with UV (20 J/m2, 20 min recovery) and then analyzed as in a. f, HeLa cells expressing the indicated Flag-tagged FL or truncated INTS12 proteins were untreated or treated with 4-NQO and then analyzed as in a. g, Mass spectrum of representative INTS12 peptide containing the indicated phosphorylated residue, with both b-ions and y-ions shown in green and yellow, respectively. h, HeLa cells expressing the indicated Flag-tagged mutant INTS12 proteins were untreated or treated with UV and then analyzed as in a. i, Sequence alignments of various INTS12 orthologues in different vertebrates. Green color, Serine (S)/Threonine (T) residues. Conserved phosphorylation residues are highlighted by the red boxes. j, INTS12 protein levels in control, INTS12 KO, and INTS12 KO cells reconstituted with stably expressed WT or 3 A INTS12-Flag were analyzed by WB. k, HeLa cells were depleted of INTS12 (INTS12 KO) and reconstituted with stably expressed WT or 3 A INTS12-Flag. Lysates from cells that were either untreated or treated with 4-NQO were analyzed as in a. All Western blots are representative of three independent experiments.

Source data

Extended Data Fig. 2 The role of INTS12 in transcriptional regulation and Integrator complex formation.

a, The levels of INTS1 in control, INTS12 KO, and INTS12 KO cells reconstituted with stably expressed WT or 3 A INTS12-Flag were analyzed by WB. b, Lysates from control or INTS12 KO cells that were treated with CHX for the indicated time were analyzed by WB. c, d, Gradient centrifugation using nuclear extracts from control or INTS12 KO cells (c) or INTS12 KO cells reconstituted with stably expressed WT or 3 A INTS12 (d). The fractionated samples were analyzed using SDS-PAGE followed by WB. e, Schematic illustrating the preDRB-nRNA-seq. Cells released from DRB pretreatment were labeled with 4-SU at the indicated times. f, Heatmaps of preDRB-nRNA-seq data from the TSS into the first 60 kb of active genes larger than 60 kb in control and INTS12 KO cells, followed by the indicated times. g, Metaplots showing the average level of nascent transcription of genes defined in f in control or INTS12 KO cells. Wavefront positions are defined as the distance from the TSS where the reads density drops below an arbitrary threshold (dashed line). All Western blots are representative of three independent experiments.

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Extended Data Fig. 3 INTS12 contributes to Integrator recruitment to CSB.

a, Venn diagram showing the number of proteins identified by MS from the INTS12-WT or 3 A interactome. b, Gene Ontology analysis of the MS-identified proteins that specifically interacted with INTS12-WT after UV irradiation. c, INTS12 KO cells reconstituted with stably expressed WT or 3 A INTS12-Flag were untreated or treated with 4-NQO. Chromatin fractions (Input) and anti-Flag immunoprecipitates (IPs) were analyzed by WB. d, Purified recombinant CSB and INTS12 proteins were analyzed by SDS-PAGE and visualized by Coomassie blue staining. e, Control or CSB KO cells expressing INTS12-Flag were either untreated or treated with UV as indicated and then analyzed as in c. f, g, Control or INTS12 KO cells expressing Flag-PAF1 or not (-) were untreated (g) or treated with UV as indicated (f) and then analyzed as in c. h–j, Control or INTS12 KO cells expressing INTS3-Flag (h), INTS5-Flag (i), INTS10-Flag (j) or not (-) were untreated or treated with UV as indicated and then analyzed as in c. k, l, Control or INTS12 KO expressing INTS5-Flag (k) or not (l) were treated with DMSO or 4-NQO (5 µM, 1 h) and subject to PLA using the indicated antibodies. Right: quantification of PLA foci per nucleus (n = 141). P < 0.0001. m, HeLa cells expressing INTS10-Flag together with either INTS3-Strep or INTS3 (∆CMBM)-Strep were lysed to prepare for the whole cell extracts (WCEs). WCEs (Input) and anti-strep immunoprecipitates (IPs) were analyzed by WB. n, HeLa cells expressing INTS3-Strep or INTS3 (∆CMBM)-Strep were either untreated or treated with UV as indicated and then analyzed as in c. For k and l, box plots show the minimum, quartiles and maximum. Scale bar, 10 μm. Statistical analysis was performed using two-tailed unpaired t-tests. All Western blots are representative of three independent experiments.

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Extended Data Fig. 4 INTS12 and its phosphorylation are required for efficient transcriptional recovery following UV irradiation.

a, Clonogenic survival of control, INTS12 KO and CSB KO cells treated with indicated doses of 4-NQO. Graphs represent the mean ± s.d. from n = 3 repeated experiments and were normalized to the untreated colony number, which was set at 100%. b, c, Cell survival of control and INTS12 KO cells treated with indicated doses of IR (b) or Calicheamicin (c). Graphs represent the mean ± s.d. from n = 3 repeated experiments and were normalized to the untreated cell number, which was set at 100%. d, e, INTS12 KO cells reconstituted with either INTS12-WT or INTS12-3A were treated with indicated doses of UV (d) or 4-NQO (e) and subjected to clonogenic survival assays. Graphs represent the mean ± s.d. from n = 3 repeated experiments and were normalized to the untreated colony number, which was set at 100%. f, Quantification of fluorescence intensity per cell for data shown in Fig. 3b and are representative of three independent experiment (n = 238). Red lines indicate the mean intensity in each group. g, Transcription recovery in INTS12 KO cells reconstituted with either INTS12-WT or INTS12-3A following UV irradiation was measured by 5-EU labeling of newly synthesized RNA. Right: Quantification of fluorescence intensity per cell and are representative of three independent experiment (n = 216). Red lines indicate the mean intensity in each group. Scale bar, 50 μm. h, Transcription recovery in control, ZAK KO and ZAKi-treated (Nilotinib, 1 µM, pre-1h) cells following UV irradiation was measured by 5-EU labeling of newly synthesized RNA. Bottom: Quantification of fluorescence intensity per cell and are representative of three independent experiment (n = 242). Red lines indicate the mean intensity in each group. Scale bar, 20 μm. i, Metaplots showing the average TT-seq signal levels of genes between 10-25 kb, 25-50 kb, and 50-100 kb in control and INTS12 KO cells after mock or UV treatment, followed by recovery for the indicated times.

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Extended Data Fig. 5 INTS12 and its phosphorylation facilitate DNA repair by TC-NER following UV irradiation.

a, qRT-PCR analysis of the mRNA levels of XPC in the indicated cells. The error bars indicate mean ± s.d. with n = 3 biologically independent samples. b, Unscheduled DNA synthesis in mock or UV-irradiated XPC-deficient cells that were either untreated or treated by ZAKi (1 µM, pre-1h) were analyzed by EdU labeling. Right: Quantification of relative EdU levels per cell (n = 128, 128, 121, 123). Red lines indicate the mean intensity in each group. Scale bar, 50 μm. c, Metaplots of XR-seq signal across gene body regions for nonoverlapping protein-coding genes in DMSO- or ZAKi-treated cells after UV treatment. d, Relative quantification of TCR activity based on the ratio of XR-seq read counts from TSs and NTSs in expressed protein-coding genes (n = 3901, TPM > 5). P < 7.03E-298. e, Metaplots showing the average level of TCR-seq signals from the TSS until the TTS (−2 kb and +2 kb, respectively) in control or INTS12 KO cells after mock or UV treatment, followed by recovery for the indicated times. f, Genome browser tracks showing the strand-specific TCR-seq read distribution across the representative gene in mock- or UV-irradiated control and INTS12-KO cells. g, Time course analysis of the recovery index (RI) in control and INTS12-KO cells following UV irradiation to estimate TC-NER kinetics on a global scale. h, Strand-specificity index (SSI) scatterplots derived from TCR-seq signals of allexpressed genes in control or INTS12 KO cells after mock or UV treatment, followed by recovery for the indicated times. Statistical analysis was performed using two-tailed unpaired t-tests.

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Extended Data Fig. 6 INTS12-mediated removal of lesion-stalled Pol II following UV irradiation.

a, Soluble and chromatin fractions from untreated or UV-irradiated control and INTS12 KO cells were analyzed by WB. b, Control or CSA KO cells expressing INTS12-Flag or not (-) were either untreated or treated with UV irradiation (20 J/m2, 1 h recovery). Chromatin fractions (Input) and anti-Flag immunoprecipitates (IPs) were analyzed by WB. c, Lysates from parental or Halo-RPB1 U2OS cells were analyzed by WB. d, FRAP analysis of Halo-RPB1 U2OS cells that were either untreated or treated with UV (8 J/m2, 1 h recovery) or FA (0.3 mM, 1 h). The relative fluorescence intensity (RFI) of Halo-RPB1 was measured every second for 220 s and normalized to pre-bleach fluorescence intensity (set to 1.0). Right: relative immobile fractions of Halo-RPB1, which were calculated by comparing RFI after UV treatment to untreated samples. Data are the mean ± s.e.m. of n = 4 biologically independent samples for the untreated and FA-treated groups, and n = 6 for the UV-treated group. e, Chromatin fractions from control, CSB KO, or INTS12 KO cells treated with THZ and/or UV as indicated were analyzed by WB. f, Control or INTS12 KO cells were either untreated or treated with UV (20 J/m2) followed by CHX (100 µg/mL) treatment. Lysates from cells that were harvested at the indicated time points were analyzed by WB. g, Metaplots of PADD-seq signals around TSSs and TESs for active genes longer than 50 kb under the indicated conditions. Right: quantification of Pol II retention on damage sites by relative change of PADD-seq signals from 0.5 h to 2 h on active genes longer than 20 kb. P < 9.8E-159. h, R-loop levels in mock- or 4-NQO-treated control, INTS12 or CSB KO cells were detected by GFP-dRNH1. Bottom: Quantification of mean nuclear GFP-dRNH1 intensity per cell and are representative of three independent experiment (n = 231, 242, 202, 202, 241, 231). Red lines indicate the mean intensity in each group. Scale bar, 50 μm. Statistical analysis was performed using two-tailed unpaired t-tests. All Western blots are representative of three independent experiments.

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Extended Data Fig. 7 INTS12 phosphorylation and the Integrator’s endonuclease activity are required for establishing UV-induced Pol II elongation wave.

a, Heatmaps of Pol II ChIP-Rx signals across gene body regions in control cells after mock or different doses of UV treatment, followed by 1 h recovery. Genes were ranked by decreasing Pol II occupancy under mock treatment conditions. FC, fold change. b, Heatmaps of Pol II ChIP-seq signals across gene body regions in CSB KO cells after mock or UV treatment, followed by recovery for the indicated times. Genes were ranked by decreasing Pol II occupancy under mock treatment conditions. FC, fold change. c, Metaplots of Pol II ChIP-seq signals across gene body regions in CSB KO cells after mock or UV treatment, followed by recovery for the indicated times. d, Schematic illustrating the preDRB-nRNA-seq. Cells released from DRB pretreatment were labeled with 4-SU at the indicated times after recovery from UV irradiation. e, Heatmaps of the Pol II ChIP-Rx signals across gene body regions in INTS12 KO cells reconstituted with stably expressed WT INTS12-Flag after mock treatment or UV treatment, followed by recovery for the indicated times. Genes were ranked by decreasing Pol II occupancy under mock treatment conditions. FC, fold change. f, Metaplots of Pol II ChIP-Rx signals across gene body regions in INTS12-WT cells after mock or UV treatment, followed by recovery for the indicated times. g, Heatmaps of the Pol II ChIP-Rx signals across gene body regions in INTS12 KO cells reconstituted with stably expressed WT INTS12-Flag after mock treatment or UV treatment, followed by recovery for the indicated times. Genes were ranked by decreasing Pol II occupancy under mock treatment conditions. FC, fold change. h, Metaplots of Pol II ChIP-Rx signals across gene body regions in INTS12-3A cells after mock or UV treatment, followed by recovery for the indicated times. i, Cumulative distribution of PSI in INTS12 KO cells reconstituted with stably expressed WT or 3 A INTS12-Flag. j, Lysates from parental or INTS11-dTAG DLD1 cells treated with indicated chemicals were analyzed by WB. k, l, Heatmaps of Pol II ChIP-Rx signals across gene body regions in DMSO (k) or dTAG (l) treated INTS11-dTAG DLD1 cells after mock or UV treatment, followed by recovery for the indicated times. Genes were ranked by decreasing Pol II occupancy under mock treatment conditions. FC, fold change. m, n, Metaplots of Pol II ChIP-Rx signals across gene body regions in DMSO (m) or dTAG (n) treated INTS11-dTAG DLD1 cells after mock or UV treatment, followed by recovery for the indicated times.

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Extended Data Fig. 8 INTS12-coordinated Pol II removal is dispensable for TC-DPC repair.

a, INTS12 KO cells reconstituted with stably expressed WT or 3 A INTS12-Flag were either untreated or treated with FA (1 mM, 1 h). Chromatin fractions (Input) and anti-Flag immunoprecipitates (IPs) were analyzed by WB. b, Control or INTS12 KO cells expressing INTS10-Flag or not (-) were untreated or treated with FA (1 mM, 1 h) and analyzed as in a. c, HeLa cells were untreated or treated with Camptothecin (CPT, 1 µM, 30 min, left) or 5-Aza-2’-deoxycytidine (5-aza-dc, 100 µM, 30 min, right) as indicated. Lysates were separated on Phos-tag SDS-PAGE and immunoblotted with the indicated antibodies. d, Lysates from HeLa cells that were untreated or treated with 4ST (4 d) and UVA (recover 30 min) were analyzed as in c. e, Schematic illustrating the potential role of INTS12 and its coordinated Pol II removal in TC-DPC repair. f, Box plots showing the DPC-seq coverage per gene in control, INTS12 KO, or CSB KO cells with or without 4 h recovery after FA treatment (n = 9288). Control, P < 2.2E-16; INTS12 KO, P < 2.2E-16; CSB KO, P = 1.7E-15. g, Heatmaps of DPC-seq data in control, INTS12 KO, or CSB KO cells after FA treatment followed by recovery for the indicated times. Genes were grouped into non, low, medium and high transcriptional activity gene sets as determined by TT-seq. h, i, Metaplots showing the average DPC-seq signal levels of all expressed (h) or non-expressed (i) genes in control, INTS12 KO, or CSB KO cells with or without 4 h recovery after FA treatment. j, Genome browser tracks showing the DPC-seq read distribution across the representative inactive gene in control, INTS12 KO, or CSB KO cells after FA treatment followed by recovery for the indicated times. For f, box plots show the minimum, quartiles and maximum. Statistical analysis was performed using two-tailed unpaired t-tests. All Western blots are representative of three independent experiments.

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Supplementary Table 1: Detailed information regarding the plasmids and oligonucleotides used in the study. Supplementary Table 2: Light microscopy reporting table.

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Li, Z., Li, R., Yang, M. et al. Integrator subunit INTS12 links ribotoxic stress to transcription-coupled nucleotide excision repair. Nat Struct Mol Biol (2026). https://doi.org/10.1038/s41594-026-01766-y

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