Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

Biomedical Research Centre Hradec Králové University Hospital 500 01 Hradec Králové Czech Republic

Cancer Mechanisms and Biomarkers Group Radiation Effects Department Centre for Radiation Chemical and Environmental Hazards Public Health England Chilton Oxfordshire OX11 0RQ UK

Department of Oncology and Radiotherapy and 4th Department of Internal Medicine Hematology University Hospital 500 05 Hradec Králové Czech Republic

Department of Radiation Dosimetry Nuclear Physics Institute of the Czech Academy of Sciences 180 00 Prague 8 Czech Republic

Department of Radiobiology Faculty of Military Health Sciences in Hradec Králové University of Defence in Brno 500 01 Hradec Králové Czech Republic

Institute of Hematology and Blood Transfusion 128 00 Praha 2 Czech Republic

Oxford Nanopore Technologies Gosling Building Edmund Halley Way Oxford OX4 4DQ UK

Zobrazit více v PubMed

Wang Y., Liu J., Huang B.O., Xu Y.M., Li J., Huang L.F., Lin J., Zhang J., Min Q.H., Yang W.M., et al. Mechanism of alternative splicing and its regulation. Biomed. Rep. 2015;3:152–158. doi: 10.3892/br.2014.407. PubMed DOI PMC

Wang B.D., Lee N.H. Aberrant RNA Splicing in Cancer and Drug Resistance. Cancers. 2018;10:458. doi: 10.3390/cancers10110458. PubMed DOI PMC

Shabalina S.A., Ogurtsov A.Y., Spiridonov N.A., Koonin E.V. Evolution at protein ends: Major contribution of alternative transcription initiation and termination to the transcriptome and proteome diversity in mammals. Nucleic Acids Res. 2014;42:7132–7144. doi: 10.1093/nar/gku342. PubMed DOI PMC

Sprung C.N., Li J., Hovan D., McKay M.J., Forrester H.B. Alternative Transcript Initiation and Splicing as a Response to DNA Damage. PLoS ONE. 2011;6:e25758. doi: 10.1371/journal.pone.0025758. PubMed DOI PMC

Barak Y., Gottlieb E., Juven-Gershon T., Oren M. Regulation of mdm2 expression by p53: Alternative promoters produce transcripts with nonidentical translation potential. Genes Dev. 1994;8:1739–1749. doi: 10.1101/gad.8.15.1739. PubMed DOI

Rossi M., Demidov O.N., Anderson C.W., Appella E., Mazur S.J. Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites. Nucleic Acids Res. 2008;36:7168–7180. doi: 10.1093/nar/gkn888. PubMed DOI PMC

Forrester H.B., Li J., Hovan D., Ivashkevich A.N., Sprung C.N. DNA Repair Genes: Alternative Transcription and Gene Expression at the Exon Level in Response to the DNA Damaging Agent, Ionizing Radiation. PLoS ONE. 2012;7:e53358. doi: 10.1371/journal.pone.0053358. PubMed DOI PMC

O’Brien G., Cruz-Garcia L., Majewski M., Grepl J., Abend M., Port M., Tichý A., Sirak I., Malkova A., Donovan E., et al. FDXR is a biomarker of radiation exposure in vivo. Sci. Rep. 2018;8:684. doi: 10.1038/s41598-017-19043-w. PubMed DOI PMC

Cruz-Garcia L., O’Brien G., Donovan E., Gothard L., Boyle S., Laval A., Testard I., Ponge L., Wozniak G., Miszczyk L., et al. Influence of Confounding Factors on Radiation Dose Estimation Using In Vivo Validated Transcriptional Biomarkers. Health Phys. 2018;115:90–101. doi: 10.1097/HP.0000000000000844. PubMed DOI PMC

Tichy A., Kabacik S., O’Brien G., Pejchal J., Sinkorova Z., Kmochova A., Sirak I., Malkova A., Beltran C.G., Gonzalez J.R., et al. The first in vivo multiparametric comparison of different radiation exposure biomarkers in human blood. PLoS ONE. 2018;13:e0193412. doi: 10.1371/journal.pone.0193412. PubMed DOI PMC

Manning G., Macaeva E., Majewski M., Kriehuber R., Brzóska K., Abend M., Doucha-Senf S., Oskamp D., Strunz S., Quintens R., et al. Comparable dose estimates of blinded whole blood samples are obtained independently of culture conditions and analytical approaches. Second RENEB gene expression study. Int. J. Radiat. Biol. 2017;93:87–98. doi: 10.1080/09553002.2016.1227105. PubMed DOI

Manning G., Kabacik S., Finnon P., Bouffler S., Badie C. High and low dose responses of transcriptional biomarkers in ex vivo X-irradiated human blood. Int. J. Radiat. Biol. 2013;89:512–522. doi: 10.3109/09553002.2013.769694. PubMed DOI

Kabacik S., Mackay A., Tamber N., Manning G., Finnon P., Paillier F., Ashworth A., Bouffler S., Badie C. Gene expression following ionising radiation: Identification of biomarkers for dose estimation and prediction of individual response. Int. J. Radiat. Biol. 2011;87:115–129. doi: 10.3109/09553002.2010.519424. PubMed DOI

Port M., Ostheim P., Majewski M., Voss T., Haupt J., Lamkowski A., Abend M. Rapid High-Throughput Diagnostic Triage after a Mass Radiation Exposure Event Using Early Gene Expression Changes. Radiat. Res. 2019 doi: 10.1667/RR15360.1. PubMed DOI

Ghandhi S.A., Smilenov L.B., Elliston C.D., Chowdhury M., Amundson S.A. Radiation dose-rate effects on gene expression for human biodosimetry. BMC Med. Genom. 2015;8:22. doi: 10.1186/s12920-015-0097-x. PubMed DOI PMC

Paul S., Amundson S.A. Development of gene expression signatures for practical radiation biodosimetry. Int. J. Radiat. Oncol. Biol. Phys. 2008;71:1236–1244. doi: 10.1016/j.ijrobp.2008.03.043. PubMed DOI PMC

Paul A., Drecourt A., Petit F., Deguine D.D., Vasnier C., Oufadem M., Masson C., Bonnet C., Masmoudi S., Mosnier I., et al. FDXR Mutations Cause Sensorial Neuropathies and Expand the Spectrum of Mitochondrial Fe-S-Synthesis Diseases. Am. J. Hum. Genet. 2017;101:630–637. doi: 10.1016/j.ajhg.2017.09.007. PubMed DOI PMC

Macaeva E., Saeys Y., Tabury K., Janssen A., Michaux A., Benotmane M.A., De Vos W.H., Baatout S., Quintens R. Radiation-induced alternative transcription and splicing events and their applicability to practical biodosimetry. Sci. Rep. 2016;6:19251. doi: 10.1038/srep19251. PubMed DOI PMC

Budworth H., Snijders A.M., Marchetti F., Mannion B., Bhatnagar S., Kwoh E., Tan Y., Wang S.X., Blakely W.F., Coleman M., et al. DNA repair and cell cycle biomarkers of radiation exposure and inflammation stress in human blood. PLoS ONE. 2012;7:e48619. doi: 10.1371/journal.pone.0048619. PubMed DOI PMC

Soltani B., Ghaemi N., Sadeghizadeh M., Najafi F. Redox maintenance and concerted modulation of gene expression and signaling pathways by a nanoformulation of curcumin protects peripheral blood mononuclear cells against gamma radiation. Chem. Biol. Interact. 2016;257:81–93. doi: 10.1016/j.cbi.2016.07.021. PubMed DOI

Odkhuu E., Mendjargal A., Koide N., Naiki Y., Komatsu T., Yokochi T. Lipopolysaccharide downregulates the expression of p53 through activation of MDM2 and enhances activation of nuclear factor-kappa B. Immunobiology. 2015;220:136–141. doi: 10.1016/j.imbio.2014.08.010. PubMed DOI

Lu H., Giordano F., Ning Z. Oxford Nanopore MinION Sequencing and Genome Assembly. Genom. Proteom. Bioinform. 2016;14:265–279. doi: 10.1016/j.gpb.2016.05.004. PubMed DOI PMC

Clarke J., Wu H.-C., Jayasinghe L., Patel A., Reid S., Bayley H. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 2009;4:265. doi: 10.1038/nnano.2009.12. PubMed DOI

Jain M., Koren S., Miga K.H., Quick J., Rand A.C., Sasani T.A., Tyson J.R., Beggs A.D., Dilthey A.T., Fiddes I.T., et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat. Biotechnol. 2018;36:338. doi: 10.1038/nbt.4060. PubMed DOI PMC

Rhoads A., Au K.F. PacBio Sequencing and Its Applications. Genom. Proteom. Bioinform. 2015;13:278–289. doi: 10.1016/j.gpb.2015.08.002. PubMed DOI PMC

Mantere T., Kersten S., Hoischen A. Long-Read Sequencing Emerging in Medical Genetics. Front. Genet. 2019;10:426. doi: 10.3389/fgene.2019.00426. PubMed DOI PMC

Weirather J.L., de Cesare M., Wang Y., Piazza P., Sebastiano V., Wang X.J., Buck D., Au K.F. Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis. F1000Research. 2017;6:100. doi: 10.12688/f1000research.10571.2. PubMed DOI PMC

Cruz-Garcia L., O’Brien G., Sipos B., Mayes S., Love M.I., Turner D.J., Badie C. Generation of a Transcriptional Radiation Exposure Signature in Human Blood Using Long-Read Nanopore Sequencing. Radiat. Res. 2020;193:143–154. doi: 10.1667/RR15476.1. PubMed DOI PMC

Rieger K.E., Chu G. Portrait of transcriptional responses to ultraviolet and ionizing radiation in human cells. Nucleic Acids Res. 2004;32:4786–4803. doi: 10.1093/nar/gkh783. PubMed DOI PMC

Kis E., Szatmári T., Keszei M., Farkas R., Ésik O., Lumniczky K., Falus A., Sáfrány G. Microarray analysis of radiation response genes in primary human fibroblasts. Int. J. Radiat. Oncol. Biol. Phys. 2006;66:1506–1514. doi: 10.1016/j.ijrobp.2006.08.004. PubMed DOI

Abend M., Badie C., Quintens R., Kriehuber R., Manning G., Macaeva E., Njima M., Oskamp D., Strunz S., Moertl S., et al. Examining Radiation-Induced In Vivo and In Vitro Gene Expression Changes of the Peripheral Blood in Different Laboratories for Biodosimetry Purposes: First RENEB Gene Expression Study. Radiat. Res. 2016;185:109–123. doi: 10.1667/RR14221.1. PubMed DOI

Kelemen O., Convertini P., Zhang Z., Wen Y., Shen M., Falaleeva M., Stamm S. Function of alternative splicing. Gene. 2013;514:1–30. doi: 10.1016/j.gene.2012.07.083. PubMed DOI PMC

Rinn J.L., Snyder M. Sexual dimorphism in mammalian gene expression. Trends Genet. 2005;21:298–305. doi: 10.1016/j.tig.2005.03.005. PubMed DOI

Trabzuni D., Ramasamy A., Imran S., Walker R., Smith C., Weale M.E., Hardy J., Ryten M. Widespread sex differences in gene expression and splicing in the adult human brain. Nat. Commun. 2013;4:2771. doi: 10.1038/ncomms3771. PubMed DOI PMC

Karlebach G., Veiga D.F.T., Mays A.D., Kesarwani A.K., Danis D., Kararigas G., Zhang X.A., George J., Ananda G., Steinhaus R., et al. The impact of sex on alternative splicing. BioRxiv. 2018 doi: 10.1101/490904. DOI

Liu A., Zhang H., Qin F., Wang Q., Sun Q., Xie S., Wang Q., Tang Z., Lu Z. Sex Associated Differential Expressions of the Alternatively Spliced Variants mRNA of OPRM1 in Brain Regions of C57BL/6 Mouse. Cell. Physiol. Biochem. 2018;50:1441–1459. doi: 10.1159/000494644. PubMed DOI

Matlin A.J., Clark F., Smith C.W. Understanding alternative splicing: Towards a cellular code. Nat. Rev. Mol. Cell Biol. 2005;6:386–398. doi: 10.1038/nrm1645. PubMed DOI

McGlincy N.J., Smith C.W. Alternative splicing resulting in nonsense-mediated mRNA decay: What is the meaning of nonsense? Trends Biochem. Sci. 2008;33:385–393. doi: 10.1016/j.tibs.2008.06.001. PubMed DOI

Dhamija S., Menon M.B. Non-coding transcript variants of protein-coding genes—What are they good for? RNA Biol. 2018;15:1025–1031. doi: 10.1080/15476286.2018.1511675. PubMed DOI PMC

Cirulli E.T., Heinzen E.L., Dietrich F.S., Shianna K.V., Singh A., Maia J.M., Goedert J.J., Goldstein D.B. A whole-genome analysis of premature termination codons. Genomics. 2011;98:337–342. doi: 10.1016/j.ygeno.2011.07.001. PubMed DOI PMC

Maquat L.E. When cells stop making sense: Effects of nonsense codons on RNA metabolism in vertebrate cells. RNA. 1995;1:453–465. PubMed PMC

Salmena L., Poliseno L., Tay Y., Kats L., Pandolfi P.P. A ceRNA hypothesis: The Rosetta Stone of a hidden RNA language? Cell. 2011;146:353–358. doi: 10.1016/j.cell.2011.07.014. PubMed DOI PMC

Ezkurdia I., Rodriguez J.M., Carrillo-de Santa Pau E., Vázquez J., Valencia A., Tress M.L. Most highly expressed protein-coding genes have a single dominant isoform. J. Proteome Res. 2015;14:1880–1887. doi: 10.1021/pr501286b. PubMed DOI PMC

Consortium T.U. UniProt: A worldwide hub of protein knowledge. Nucleic Acids Res. 2018;47:D506–D515. doi: 10.1093/nar/gky1049. PubMed DOI PMC

Consortium F., Clst R.P., Forrest A.R., Kawaji H., Rehli M., Baillie J.K., de Hoon M.J., Haberle V., Lassmann T. A promoter-level mammalian expression atlas. Nature. 2014;507:462–470. doi: 10.1038/nature13182. PubMed DOI PMC

Kimura K., Wakamatsu A., Suzuki Y., Ota T., Nishikawa T., Yamashita R., Yamamoto J., Sekine M., Tsuritani K., Wakaguri H., et al. Diversification of transcriptional modulation: Large-scale identification and characterization of putative alternative promoters of human genes. Genome Res. 2006;16:55–65. doi: 10.1101/gr.4039406. PubMed DOI PMC

Davuluri R.V., Suzuki Y., Sugano S., Plass C., Huang T.H. The functional consequences of alternative promoter use in mammalian genomes. Trends Genet. 2008;24:167–177. doi: 10.1016/j.tig.2008.01.008. PubMed DOI

Arce L., Yokoyama N.N., Waterman M.L. Diversity of LEF/TCF action in development and disease. Oncogene. 2006;25:7492–7504. doi: 10.1038/sj.onc.1210056. PubMed DOI

Xu C., Park J.-K., Zhang J. Evidence that alternative transcriptional initiation is largely nonadaptive. PLoS Biol. 2019;17:e3000197. doi: 10.1371/journal.pbio.3000197. PubMed DOI PMC

Christmann M., Kaina B. Transcriptional regulation of human DNA repair genes following genotoxic stress: Trigger mechanisms, inducible responses and genotoxic adaptation. Nucleic Acids Res. 2013;41:8403–8420. doi: 10.1093/nar/gkt635. PubMed DOI PMC

Liu G., Chen X. The ferredoxin reductase gene is regulated by the p53 family and sensitizes cells to oxidative stress-induced apoptosis. Oncogene. 2002;21:7195–7204. doi: 10.1038/sj.onc.1205862. PubMed DOI

Terwilliger T., Abdul-Hay M. Acute lymphoblastic leukemia: A comprehensive review and 2017 update. Blood Cancer J. 2017;7:e577. doi: 10.1038/bcj.2017.53. PubMed DOI PMC

Chiaretti S., Li X., Gentleman R., Vitale A., Vignetti M., Mandelli F., Ritz J., Foa R. Gene expression profile of adult T-cell acute lymphocytic leukemia identifies distinct subsets of patients with different response to therapy and survival. J. Blood. 2004;103:2771–2778. doi: 10.1182/blood-2003-09-3243. PubMed DOI

Wong A.C.H., Rasko J.E.J., Wong J.J. We skip to work: Alternative splicing in normal and malignant myelopoiesis. Leukemia. 2018;32:1081–1093. doi: 10.1038/s41375-018-0021-4. PubMed DOI

Manning G., Tichý A., Sirák I., Badie C. Radiotherapy-Associated Long-term Modification of Expression of the Inflammatory Biomarker Genes ARG1, BCL2L1, and MYC. Front. Immunol. 2017;8:412. doi: 10.3389/fimmu.2017.00412. PubMed DOI PMC

Lammering G., Valerie K., Lin P.S., Hewit T.H., Schmidt-Ullrich R.K. Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance. J. Eur. Soc. Ther. Radiol. Oncol. 2004;72:267–273. doi: 10.1016/j.radonc.2004.07.004. PubMed DOI

Sheng J., Zhao Q., Zhao J., Zhang W., Sun Y., Qin P., Lv Y., Bai L., Yang Q., Chen L., et al. SRSF1 modulates PTPMT1 alternative splicing to regulate lung cancer cell radioresistance. EBioMedicine. 2018;38:113–126. doi: 10.1016/j.ebiom.2018.11.007. PubMed DOI PMC

Abbaszadeh F., Clingen P.H., Arlett C.F., Plowman P.N., Bourton E.C., Themis M., Makarov E.M., Newbold R.F., Green M.H.L., Parris C.N. A novel splice variant of the DNA-PKcs gene is associated with clinical and cellular radiosensitivity in a patient with xeroderma pigmentosum. J. Med. Genet. 2010;47:176. doi: 10.1136/jmg.2009.068866. PubMed DOI

West S., Kumar S., Batra S.K., Ali H., Ghersi D. Uncovering and characterizing splice variants associated with survival in lung cancer patients. PLoS Comput. Biol. 2019;15:e1007469. doi: 10.1371/journal.pcbi.1007469. PubMed DOI PMC

Vitting-Seerup K., Sandelin A. The Landscape of Isoform Switches in Human Cancers. Mol. Cancer Res. 2017;15:1206. doi: 10.1158/1541-7786.MCR-16-0459. PubMed DOI

Badie C., Iliakis G., Foray N., Alsbeih G., Cedervall B., Chavaudra N., Pantelias G., Arlett C., Malaise E.P. Induction and Rejoining of DNA Double-Strand Breaks and Interphase Chromosome Breaks after Exposure to X Rays in One Normal and Two Hypersensitive Human Fibroblast Cell Lines. Radiat. Res. 1995;144:26–35. doi: 10.2307/3579232. PubMed DOI

Badie C., Iliakis G., Foray N., Alsbeih G., Pantellias G.E., Okayasu R., Cheong N., Russell N.S., Begg A.C., Arlett C.F., et al. Defective repair of DNA double-strand breaks and chromosome damage in fibroblasts from a radiosensitive leukemia patient. Cancer Res. 1995;55:1232–1234. PubMed

Love M.I., Soneson C., Patro R. Swimming downstream: Statistical analysis of differential transcript usage following Salmon quantification. F1000Research. 2018;7:952. doi: 10.12688/f1000research.15398.1. PubMed DOI PMC

Li H. Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–3100. doi: 10.1093/bioinformatics/bty191. PubMed DOI PMC

Patro R., Duggal G., Love M.I., Irizarry R.A., Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods. 2017;14:417. doi: 10.1038/nmeth.4197. PubMed DOI PMC

Transcriptional Dynamics of DNA Damage Responsive Genes in Circulating Leukocytes during Radiotherapy