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Maintenance of R-loop structures by phosphorylated hTERT preserves genome integrity

Abstract

As aberrant accumulation of RNA–DNA hybrids (R-loops) causes DNA damage and genome instability, cells express regulators of R-loop structures. Here we report that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates R-loop formation. We found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles both in telomerase-positive cells and telomerase-negative cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT did not associate with telomerase RNA component in nuclear speckles but, instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT gene in ALT cells ablated RdRP activity and impaired tumour growth. Using a genome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures and DNA damage. These findings indicate that RdRP activity of p-hTERT guards against genome instability by removing R-loop structures.

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Fig. 1: Expression of phosphorylated hTERT proteins in ALT cells.
Fig. 2: Enzymatic activity of hTERT in ALT cells.
Fig. 3: p-hTERT localizes in NSs in ALT cells.
Fig. 4: Involvement of RdRP in R-loop resolution on TERRA sequences.
Fig. 5: CRISPR-Cas9 editing of TERT gene locus in U2OS cells.
Fig. 6: Synthetic lethality between inhibition of RdRP and Fanconi anaemia/BRCA.
Fig. 7: Genome instability triggered by suppression of hTERT.

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Data availability

All sequencing datasets have been deposited at the Gene Expression Omnibus with accession codes GSE226966 and GSE226994. All the data supporting the findings of this study are available within the article and from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by the Japan Society for the Promotion of Science KAKENHI (22K15550 to M.M., 20J00036 and 20H03438 to A.Y.), the Japan Science and Technology Agency (JPMJMS2022 to A.Y.) and several grants from the Japan Agency for Medical Research and Development: the P-CREATE grant (JP21cm0106584 and JP23ama221523 to M.M. and JP21cm0106115 to K.M.), the Practical Research for Innovation Cancer Control (JP20ck0106403 to K.M.), Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS; JP22ama121008 to Y.K.) and the Translational Research programme (JP24ym0126804 to M.M.). T.T. was supported by the Uehara Memorial Foundation and Tokai University Tokuda Memorial Cancer/Genome Basic Research Grant. This work was supported by the Center for Promotion of Translational Research, National Cancer Center Japan.

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Authors and Affiliations

Authors

Contributions

M.M., A.N., M.Y., S.U. and K.-I.F. performed biochemical and cellular experiments. T.Y. and S.K. performed animal experiments. T.U. and H.M. conducted bioinformatics analyses of CRISPR screening. Y.K. generated a phospho-specific monoclonal antibody against hTERT. T.T. provided FANC/BRCA mutant cell clones. N.S. supported purification of NSs. M.M., A.N., A.Y., Y.T., M.W. and K.M. designed the experiments and discussed the interpretation of the results. M.M., A.N. and K.M. wrote the paper.

Corresponding author

Correspondence to Kenkichi Masutomi.

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Extended data

Extended Data Fig. 1 Detection of phosphorylated hTERT proteins and hTERT mRNAs in ALT cells.

a, IIF imaging of p-hTERT. Telomerase-positive cell lines (293T, HeLa, and Huh-7 cells) and ALT cell lines (U2OS, VA-13, and Saos-2 cells) were immunostained with an anti-p-hTERT mouse mAb (TpMab-35). Scale bar, 10 μm. b, c, IIF imaging of p-hTERT (TpMab-35) in HeLa and U2OS cells after treatment with hTERT siRNAs (b) and a CDK1 inhibitor (RO-3306) (c). Scale bar, 10 μm. d, e, Comparison between reverse transcriptases for RT-PCR analysis (n = 4 independent experiments per group). cDNA was synthesized from total RNAs with PrimeScript (PS), Superscript IV (SSIV), or Superscript IV VILO. The relative expressions are noted below the panel (for b). f, Schematic diagram of hTERT mRNA. Arrows depict locations of primers used for quantitative RT-PCR analysis. g, Determination of hTERT mRNA levels by quantitative RT-PCR analysis using LT5/6 primer pairs or new primer pairs targeting the exon 15 and 16 junction of hTERT mRNA (n = 3 independent experiments per group). h, i, Detection of p-hTERT (TpMab-35) in U2OS and HeLa cells synchronized in mitosis with nocodazole. Data are presented as mean ± SD (for e, g). Experiments were repeated three times (for a, c) and twice (for b, d, h, i) with similar results. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 Telomerase and RdRP activity in ALT cells.

a, Detection of endogenous hTERT immunoprecipitated from the indicated cells with nocodazole treatment. b, Direct telomerase assay using hTERT proteins immunoprecipitated with an anti-hTERT mouse mAb (10E9-2) from the indicated mitotic cells treated with nocodazole. 293T cells are positive controls. c, quantitative RT-PCR analysis for hTERC associated with hTERT and p-hTERT isolated with the 10E9-2 and the TpMab-3 antibodies from HeLa, U2OS, VA-13, and Saos-2 cells (n = 3 independent experiments per group). Data are presented as mean ± SD. d, RdRP activity of endogenous hTERT immunoprecipitated from HeLa cells and a recombinant hTERT (rhTERT) protein. Experiments were repeated three times (for a, b, d) with similar results. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 RdRP activity of hTERT alternative splicing variants.

a, Schematic diagram of hTERT alternative splicing (AS) variants. Arrows depict locations of primers used for quantitative RT-PCR analysis. b, Determination of hTERT AS variant levels by quantitative RT-PCR analysis (n = 3 independent experiments per group). Data are presented as mean ± SD. N.D., not detected. c, Detection of recombinant proteins of AS variants purified by a baculovirus expression system. * indicates migration of full-length hTERT or AS variants. d, IP-RdRP assay using recombinant AS variant proteins. Experiments were repeated three times (for c, d) with similar results. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 hTERT undergoing liquid-liquid phase separation in ALT cells.

a, IIF imaging of hTERT (2E4-2) and p-hTERT (TpMab-3) in HeLa and U2OS cells. Scale bar, 10 μm. b, c, Multicolor immunofluorescence imaging of p-hTERT and PML (b) or TRF2 (c) in U2OS cells. TEN, telomerase essential N-terminal domain; RBD, RNA-binding domain; RT, reverse transcriptase domain; CTE, C-terminal extension. Scale bar, 10 μm. d-g, Prediction of disorder regions on hTERT protein using the IUPred2A web interface (d) and the PONDR predictor VL-XT (e), VL3-BS (f), or VSL2 (g). The bold lines show putative disorder regions. h, IIF imaging of p-hTERT in HeLa, Huh-7, and U2OS cells disrupting LLPS with 10% 1,6-HD for 5 min. Scale bar, 10 μm. i, j, Detection of endogenous hTERT proteins (i) and RdRP activity (j) in the cells inhibiting LLPS with 1,6-HD. The 10E9-2 and 2E4-2 clones are antibodies for immunoprecipitation and immunoblotting of hTERT, respectively. k, Detection of endogenous hTERT protein under conditions for inhibiting LLPS (1,6-HD treatment and lysis with 150 or 1000 mM NaCl). Experiments were repeated three times (for a, h-k) and twice (for b, c) with similar results. Source unprocessed blots are available in source data.

Source data

Extended Data Fig. 5 Specificity of an anti-p-hTERT antibody (TpMab-3).

a, b, IIF imaging of p-hTERT (TpMab-3) and phospho-SR proteins (1H4) in HeLa cells treated with siRNAs specific for hTERT (a) and a CLK1 inhibitor (KH-CB19) (b). Experiments were repeated twice with similar results. Scale bar, 10 μm.

Extended Data Fig. 6 Association between hTERT and TERRA RNAs and RdRP activity of p-hTERT against TERRA RNAs.

a, Scheme of hTERT RIP-seq, which analyzed RNAs co-immunoprecipitated with hTERT using an anti-hTERT antibody (10E9-2) from nuclear speckle (NS)-enriched fraction. b, RIP-seq data of hTERT purified from nuclear speckles. Arrows show reads mapped to subtelomeric regions. c, Quantitative RT-PCR analysis of TERRA RNAs from different subtelomeric regions in hTERT knockdown HeLa and U2OS cells (n = 4 independent experiments per group). Data are presented as mean ± SD. One-way ANOVA method with Dunnett correction for multiple comparisons between control (siCtrl) and other groups. d, RdRP activity of hTERT purified from nuclear speckles against a synthetic TERRA RNA template. e, IP-RdRP assay using hTERT proteins immunoprecipitated with an anti-hTERT antibody (10E9-2) from the HeLa cells. A mixture of two RNA templates, control DN3AS and 5ʹ-(CCUAAC)8-3ʹ for TERRA, was used for the IP-RdRP assay. f, g, In vitro R-loop degradation assay using hTERT (f) and p-hTERT (g) complex immunoprecipitated from the NS-enriched fraction and 5′-32P-labeled TERRA RNA:DNA hybrids. RNase H is a positive control to digest RNA:DNA hybrids. Experiments were repeated three times (for d, f) and twice (for e, g) with similar results. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 7 Establishment of hTERT-CRISPR clones.

a, b, Sanger sequencing for the hTERT-CRISPR clones #1 (a) and #2 (b). Black lines and red arrowheads indicate the gRNA sequence and deletion regions, respectively. c, Determination of hTERT mRNA levels in hTERT-CRISPR clones by quantitative RT-PCR analysis (n = 3 independent experiments per group). Data are presented as mean ± SD. One-way ANOVA method with Dunnett correction for multiple comparisons between control (U2OS) and other groups. d, Clustering and GO analyses of genes differentially expressed in hTERT-CRISPR clones. The p-values were calculated by MBCluster.Seq package. Source numerical data are available in source data.

Source data

Extended Data Fig. 8 Effects of FANC/BRCA knockdown and PARP1 inhibition in hTERT-CRISPR clones.

a, Knockdown efficiencies of siRNAs specific for FANC/BRCA genes in hTERT-CRISPR clones (n = 3 independent experiments per group). b, The representative images of colony formation assay using hTERT-CRISPR clones transfected with siRNAs specific for FANC/BRCA genes. c, The MLE beta essentiality scores for FANC/BRCA and PARP1 genes generated from MAGeCK algorithm. d, e, The representative images (d) and colony counting (e) of colony formation assay using hTERT-CRISPR clones with FANC/BRCA and PARP1 knockdowns (n = 3 independent experiments per group). f, g, Detection of hTERT proteins (f) and the representative images of colony formation assay (g) using hTERT-CRISPR clones overexpressing a wild-type hTERT or a T249A mutant. Data are presented as mean ± SD (for a, e). One-way ANOVA method with Dunnett correction for multiple comparisons between control and other groups (e). Experiments were repeated twice with similar results (for f). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 9 Effects of hTERT knockdown in cells lacking FANC/BRCA genes.

a, Knockdown efficiencies of siRNAs specific for hTERT in Capan-1 and TOV-21G cell clones corrected for BRCA2 and FANCF deficiencies (n = 4 independent experiments per group). b, Direct telomerase assay using hTERT proteins immunoprecipitated from the indicated cells. c-g, The representative images of colony formation assay using these revertant clones. h, Schematic diagram of a strand-specific RT-PCR. DRIP RNAs were subjected to quantitative RT-PCR (qRT-PCR) analysis with strand-specific primers with a tag sequence. i, qRT-PCR analysis of DRIP RNAs using tagged primers specific for sense or antisense strand TERRA RNAs (n = 3 independent experiments per group). Strand-specific qPCR primers only recognize each strand-specific cDNA. Data are presented as mean ± SD (for a, i). Experiments were repeated twice with similar results (for b). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 10 Maintenance of R-loop structures by p-hTERT and FANC/BRCA proteins.

a, Immunoblotting for BRCA2 in proteins co-immunoprecipitated with hTERT from the indicated biochemical fractions. Experiments were repeated three times with similar results. b, Model of maintenance of R-loop structures by p-hTERT and FANC/BRCA proteins. c, Model of different roles of hTERT in regulation of genome stability. hTERT elongates telomeres as the catalytic enzyme of telomerase. The 3′ telomeric overhangs invades into the telomere duplex and forms a small loop at the chromosome ends, called T-loop structure, which protects the end of linear chromosomes from degradation, fusion, and recombination. On the other hand, hTERT binds to TERRA sequences of the nascent RNA transcripts and synthesizes antisense RNAs through RdRP activity. The RdRP reaction converts RNA:DNA hybrids of R-loops to dsRNAs, preventing R-loop formation. hTERT guarantees genome stability by the two enzymatic activities. Source unprocessed blots are available in source data.

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Machitani, M., Nomura, A., Yamashita, T. et al. Maintenance of R-loop structures by phosphorylated hTERT preserves genome integrity. Nat Cell Biol 26, 932–945 (2024). https://doi.org/10.1038/s41556-024-01427-6

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