ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader

Abstract

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability. The telomeric repeat-containing RNA (TERRA) that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop. In tumor cells, R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres (ALT) pathway. Dysregulated R-loops can cause stalled replication forks and telomere instability. However, how R-loops are recognized and regulated, particularly at telomeres, is not well understood. We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination. Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses. In addition, ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway. Using the proximity-dependent biotin identification (BioID) technology, we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases, including DHX9. Importantly, ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9. Our findings suggest that ILF3 may function as a reader of telomeric R-loops, helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

Keywords

ILF3 / RNA:DNA hybrids / telomeric R-loops / homologous recombination / telomeric DNA damage responses

References

[1] Argaud D, Boulanger MC, Chignon A et al. Enhancer-mediated enrichment of interacting JMJD3-DDX21 to ENPP2 locus prevents R-loop formation and promotes transcription. Nucleic Acids Res 2019;47:8424–38.

[2] Arora R, Lee Y, Wischnewski H et al. RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells. Nat Commun 2014;5:5220.

[3] Azzalin CM, Reichenbach P, Khoriauli L et al. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 2007;318:798–801.

[4] Bah A, Wischnewski H, Shchepachev V et al. The telomeric transcriptome of Schizosaccharomyces pombe. Nucleic Acids Res 2012;40:2995–3005.

[5] Blackburn EH. Telomeres: no end in sight. Cell 1994;77:621–3.

[6] Blackburn EH. Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett 2005;579:859–62.

[7] Boguslawski SJ, Smith DE, Michalak MA et al. Characterization of monoclonal antibody to DNARNA and its application to immunodetection of hybrids. J Immunol Methods 1986;89:123–30.

[8] Castella S, Bernard R, Corno M et al. IlF3 and NF90 functions in RNA biology. Wiley Interdiscip Rev RNA 2015;6:243–56.

[9] Cesare AJ, Kaul Z, Cohen SB et al. Spontaneous occurrence of telomeric DNA damage response in the absence of chromosome fusions. Nat Struct Mol Biol 2009;16:1244–51.

[10] Chen Y-A, Shen Y-L, Hsia H-Y et al. Extrachromosomal telomere repeat DNA is linked to ALT development via cGAS-STING DNA sensing pathway. Nat Struct Mol Biol 2017;24:1124–31.

[11] Cheung DH, Ho ST, Lau KF et al. Nucleophosmin interacts with PIN2/TERF1-interacting Telomerase Inhibitor 1 (PinX1) and Attenuates the PinX1 inhibition on telomerase activity. Sci Rep 2017;7:43650.

[12] Cho NW, Dilley RL, Lampson MA et al. Interchromosomal homology searches drive directional ALT telomere movement and synapsis. Cell 2014;159:108–21.

[13] Chu HP, Cifuentes-Rojas C, Kesner B et al. TERRA RNA antagonizes ATRX and protects telomeres. Cell 2017;170:86–101.e16.

[14] Cohen S, Puget N, Lin Y et al. Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations. Nat Commun 2018;9:533.

[15] Corthesy B, Kao PN. Purification by DNA affinity chromatography of two polypeptides that contact the NF-AT DNA binding site in the interleukin 2 promoter. J Biol Chem 1994;269:20682–90.

[16] Cristini A, Groh M, Kristiansen MS et al. RNA/DNA hybrid interactome identifies DXH₉ as a molecular player in transcriptional termination and R-loop-associated DNA damage. Cell Rep 2018;23:1891–905.

[17] Cristini A, Ricci G, Britton S et al. Dual processing of R-Loops and Topoisomerase I induces transcriptiondependent DNA double-strand breaks. Cell Rep 2019;28:3167–3181.e6.

[18] Crossley MP, Song C, Bocek MJ et al. R-loop-derived cytoplasmic RNA–DNA hybrids activate an immune response. Nature 2022;613:187–94.

[19] de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 2005;19:2100–10.

[20] Dilley RL, Verma P, Cho NW et al. Break-induced telomere synthesis underlies alternative telomere maintenance. Nature 2016;539:54–8.

[21] Feretzaki M, Lingner J. A practical qPCR approach to detect TERRA, the elusive telomeric repeat-containing RNA. Methods 2017;114:39–45.

[22] Feretzaki M, Pospisilova M, Valador Fernandes R et al. RAD51-dependent recruitment of TERRA lncRNA to telomeres through R-loops. Nature 2020;587:303–8.

[23] Graf M, Bonetti D, Lockhart A et al. Telomere length determines TERRA and R-loop regulation through the cell cycle. Cell 2017;170:72–85.e14.

[24] Greider CW, Blackburn EH. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 1989;337:331–7.

[25] Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458–60.

[26] Henson JD, Hannay JA, McCarthy SW et al. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin Cancer Res 2005;11:217–25.

[27] Henson JD, Cao Y, Huschtscha LI et al. DNA C-circles are specific and quantifiable markers of alternativelengthening-of-telomeres activity. Nat Biotechnol 2009;27:1181–5.

[28] Herdy B, Mayer C, Varshney D et al. Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts. Nucleic Acids Res 2018;46:11592–604.

[29] Hondele M, Sachdev R, Heinrich S et al. DEAD-box ATPases are global regulators of phase-separated organelles. Nature 2019;573:144–8.

[30] Jiao X, Sherman BT, Huang da W et al. DAVID-WS: a stateful web service to facilitate gene/protein list analysis. Bioinformatics 2012;28:1805–6.

[31] Kao PN, Chen L, Brock G et al. Cloning and expression of cyclosporin A-and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90. J Biol Chem 1994;269:20691–9.

[32] Kappei D, Scheibe M, Paszkowski-Rogacz M et al. Phylointeractomics reconstructs functional evolution of protein binding. Nat Commun 2017;8:14334.

[33] Kim NW, Piatyszek MA, Prowse KR et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–5.

[34] Kim H, Li F, He Q et al. Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/CaS9 knockout cells. Cell Discov 2017;3:17034.

[35] Lago S, Tosoni E, Nadai M et al. The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids. Biochim Biophys Acta Gen Subj 2017;1861:1371–81.

[36] Lee YW, Arora R, Wischnewski H et al. TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops. Nat Struct Mol Biol 2018;25:147–53.

[37] Lessel D, Schob C, Kury S et al; DDD study. De Novo Missense Mutations in DHX30 impair global translation and cause a neurodevelopmental disorder. Am J Hum Genet 2017;101:716–24.

[38] Li X, Liu CX, Xue W et al. Coordinated circRNA biogenesis and function with NF90/NF110 in viral infection. Mol Cell 2017;67:214–227.e7.

[39] Li K, Wu JL, Qin B et al. ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer. Cell Res 2020a;30:163–78.

[40] Li Y, Song Y, Xu W et al. R-loops coordinate with SOX2 in regulating reprogramming to pluripotency. Sci Adv 2020b;6:eaba0777.

[41] Liu D, O’Connor MS, Qin J et al. Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins. J Biol Chem 2004;279:51338–42.

[42] Liu Y, Liu F, Cao Y et al. Shwachman-Diamond Syndrome Protein SBDS maintains human telomeres by regulating telomerase recruitment. Cell Rep 2018;22:1849–60.

[43] Liu S, Hua Y, Wang J et al. RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination. Cell 2021;184:1314–1329 e10.

[44] Liu J, Liu ZX, Li JJ et al. The Macrophage-Associated LncRNA MALR facilitates ILF₃ liquid-liquid phase separation to promote HIF1α signaling in esophageal cancer. Cancer Res 2023;83:1476–89.

[45] Londono-Vallejo JA, Der-Sarkissian H, Cazes L et al. Alternative lengthening of telomeres is characterized by high rates of telomeric exchange. Cancer Res 2004;64:2324–7.

[46] Ma J, Chen T, Wu S et al. iProX: an integrated proteome resource. Nucleic Acids Res 2019;47:D1211–7.

[47] Maicher A, Kastner L, Dees M et al. Deregulated telomere transcription causes replication-dependent telomere shortening and promotes cellular senescence. Nucleic Acids Res 2012;40:6649–59.

[48] Montero JJ, Lopez-Silanes I, Megias D et al. TERRA recruitment of polycomb to telomeres is essential for histone trymethylation marks at telomeric heterochromatin. Nat Commun 2018;9:1548.

[49] Mosler T, Conte F, Longo GMC et al. R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability. Nat Commun 2021;12:7314.

[50] Nazitto R, Amon LM, Mast FD et al. ILF3 is a negative transcriptional regulator of innate immune responses and myeloid dendritic cell maturation. J Immunol 2021;206:2949–65.

[51] Nie X, Xiao D, Ge Y et al. TRF2 recruits nucleolar protein TCOF1 to coordinate telomere transcription and replication. Cell Death Differ 2020;28:1062–1075.

[52] Ouyang J, Yadav T, Zhang JM et al. RNA transcripts stimulate homologous recombination by forming DR-loops. Nature 2021;594:283–8.

[53] Parrott AM, Mathews MB. Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution. Nucleic Acids Res 2007;35:6249–58.

[54] Patel RC, Vestal DJ, Xu Z et al. DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR. J Biol Chem 1999;274:20432–7.

[55] Petti E, Buemi V, Zappone A et al. SFPQ and NONO suppress RNA:DNA-hybrid-related telomere instability. Nat Commun 2019;10:1001.

[56] Pickett HA, Reddel RR. Molecular mechanisms of activity and derepression of alternative lengthening of telomeres. Nat Struct Mol Biol 2015;22:875–80.

[57] Prendergast L, McClurg UL, Hristova R et al. Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability. Nat Commun 2020;11:4534.

[58] Roux KJ, Kim DI, Raida M et al. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 2012;196:801–10.

[59] Roy D, Zhang Z, Lu Z et al. Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site. Mol Cell Biol 2010;30:146–59.

[60] Salas-Armenteros I, Perez-Calero C, Bayona-Feliu A et al. Human THO-SiN3A interaction reveals new mechanisms to prevent R-loops that cause genome instability. EMBO J 2017;36:3532–47.

[61] Satoh M, Shaheen VM, Kao PN et al. Autoantibodies define a family of proteins with conserved double-stranded RNA-binding domains as well as DNA binding activity. J Biol Chem 1999;274:34598–604.

[62] Schoeftner S, Blasco MA. Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nat Cell Biol 2008;10:228–36.

[63] Schwanhausser B, Busse D, Li N et al. Global quantification of mammalian gene expression control. Nature 2011;473:337–42.

[64] Shay JW, Wright WE. Telomeres and telomerase: three decades of progress. Nat Rev Genet 2019;20:299–309.

[65] Shi G, Hu Y, Zhu X et al. A critical role of telomere chromatin compaction in ALT tumor cell growth. Nucleic Acids Res 2020;48:6019–31.

[66] Takai H, Smogorzewska A, de Lange T. DNA damage foci at dysfunctional telomeres. Curr Biol 2003;13:1549–56.

[67] Tan J, Duan M, Yadav T et al. An R-loop-initiated CSB-RAD52-POLD3 pathway suppresses ROS-induced telomeric DNA breaks. Nucleic Acids Res 2020;48:1285–300.

[68] Tominaga-Yamanaka K, Abdelmohsen K, Martindale JL et al. NF90 coordinately represses the senescenceassociated secretory phenotype. Aging (Albany NY) 2012;4:695–708.

[69] Tran P, Pohl T, Chen C et al. PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nat Commun 2017;8:15025.

[70] Tyanova S, Temu T, Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 2016;11:2301–19.

[71] Vrbsky J, Akimcheva S, Watson JM et al. siRNA-mediated methylation of Arabidopsis telomeres. PLoS Genet 2010;6:e1000986.

[72] Vumbaca F, Phoenix KN, Rodriguez-Pinto D et al. Double-stranded RNA-binding protein regulates vascular endothelial growth factor mRNA stability, translation, and breast cancer angiogenesis. Mol Cell Biol 2008;28:772–83.

[73] Wang IX, Grunseich C, Fox J et al. Human proteins that interact with RNA/DNA hybrids. Genome Res 2018;28:1405–14.

[74] Xu Z, Duc KD, Holcman D et al. The length of the shortest telomere as the major determinant of the onset of replicative senescence. Genetics 2013;194:847–57.

[75] Yan G, Yang J, Li W et al. Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC₁-dependent amino acid sensing. Nat Cell Biol 2023;25:754–64.

[76] Yang SF, Sun AA, Shi Y et al. Structural and functional characterization of the RBBP4-ZNF827 interaction and its role in NuRD recruitment to telomeres. Biochem J 2018;475:2667–79.

[77] Yasuhara T, Kato R, Hagiwara Y et al. Human Rad52 promotes XPG-mediated R-loop processing to initiate transcription-associated homologous recombination repair. Cell 2018;175:558–570.e11.

[78] Yeager TR, Neumann AA, Englezou A et al. Telomerasenegative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res 1999;59:4175–9.

[79] Yu TY, Kao YW, Lin JJ. Telomeric transcripts stimulate telomere recombination to suppress senescence in cells lacking telomerase. Proc Natl Acad Sci U S A 2014;111:3377–82.

[80] Yuan W, Al-Hadid Q, Wang Z et al. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution. Nucleic Acids Res 2021;49:8573–91.

[81] Zhang C, Chen L, Peng D et al. METTL3 and N6-methyladenosine promote homologous recombinationmediated repair of DSBs by modulating DNA-RNA hybrid accumulation. Mol Cell 2020;79:425–442.e7.

RIGHTS & PERMISSIONS

2023 The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.