Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells Da-Peng Daia,1, Wei Gana,1, Hiroshi Hayakawab,1, Jia-Lou Zhuc,1, Xiu-Qing Zhangc,1, Guo-Xin Hud, Tao Xud, Zhe-Li Jiangd, Li-Qun Zhanga, Xue-Da Hue, Ben Nied, Yue Zhouf, Jin Lia,g, Xiao-Yang Zhoua, Jian Lia, Tie-Mei Zhanga, Qing Heh, Dong-Ge Liui, Hai-Bo Chenj, Nan Yangk, Ping-Ping Zuok, Zhi-Xin Zhangl, Huan-Ming Yangm, Yao Wanga, Samuel H. Wilsonn, Yi-Xin Zenga, Jian-Ye Wanga, Mutsuo Sekiguchib,2, and Jian-Ping Caia,2 aMOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, 100730 Beijing, People’s Republic of China; bFrontier Research Center, Fukuoka Dental College, 8140193 Fukuoka, Japan; cBeijing Genomics Institute-Shenzhen, 518083 Shenzhen, People’s Republic of China; dDepartment of Pharmacology, Wenzhou Medical College, Wenzhou, 325035 Zhejiang, People’s Republic of China; eCancer Institute and Hospital, Chinese Academy of Medical Sciences, 100021 Beijing, People’s Republic of China; fThermo Fisher Scientific Demonstration Laboratory, 201206 Shanghai, People’s Republic of China; gPeking University Fifth School of Clinical Medicine, Beijing Hospital, 100730 Beijing, People’s Republic of China; hDepartment of Cardiology, Beijing Hospital, 100730 Beijing, People’s Republic of China; iDepartment of Pathology, Beijing Hospital, 100730 Beijing, People’s Republic of China; jDepartment of Neurology, Beijing Hospital, 100730 Beijing, People’s Republic of China; kInstitute of Basic Medical Sciences, Chinese Academy of Medical Sciences, 100021 Beijing, People’s Republic of China; lBeijing Red Cross Blood Center, 100088 Beijing, People’s Republic of China; mJames D. Watson Institute of Genome Sciences, 310058 Hangzhou, People’s Republic of China; and nGenome Integrity and Structural Biology Laboratory, National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved March 16, 2018 (received for review November 9, 2017) Reactive oxygen species formed within the mammalian cell can misincorporation of 8-oxoG into mRNA (9). Nevertheless, an produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can influence of 8-oxoG in mRNA in mammalian cells is still unclear. cause base mispairing during gene expression. Here we found that In the present study, we developed a luciferase reporter system administration of 8-oxoGTP in MTH1-knockdown cells results in for the detection of sequence-altered abnormal proteins, and we increased 8-oxoG content in mRNA. Under this condition, an amber also applied a deep-sequencing method to measure ultra-rare mutation of the reporter luciferase is suppressed. Using second- mutations in oxidized mRNA to investigate the role of MTH1 in generation sequencing techniques, we found that U-to-G changes 8-oxoG–induced RNA mutagenesis in human cells. We found at preassigned sites of the luciferase transcript increased when 8- that the incorporation of 8-oxoG into the mRNA strand can oxoGTP was supplied. In addition, an increased level of 8-oxoG β induce translation errors, thus accelerating the production of the content in RNA induced the accumulation of aggregable amyloid pathogenic amyloid β peptides in mammalian cells. peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein syn- Results thesis in mammalian cells. Further work is required to assess the sig- Introduction of 8-oxoG into RNA of Living Cells. Since HeLa cells nificance of these findings under normal physiological conditions. contain a relatively large amount of MTH1, we reduced the MTH1 content of cells using shRNA treatment. HeLa cells were 8-oxoG | MTH1 | RNA oxidization | amyloid β peptides Significance n aerobically growing cells, reactive oxygen and nitrogen spe- Icies (ROS) are generated as byproducts of ATP production and oxygen utilization. Although most of these radicals are We had previously shown that 8-oxoG:A mismatch pairing can eliminated by the action of cellular antioxidant systems, some occur at both the DNA and RNA levels, thus causing base substi- ROS still remain within the cell and damage various biologically tutions in DNA and translation errors, respectively. However, little attention has been paid to the biological significance of oxidized important molecules, including proteins, lipids, and nucleic acids mRNA or to the clearance mechanism for such RNA lesions in (1–3). Among the various types of oxidized purine and pyrimi- mammalian cells. Here, we developed a sensitive reporter system dine bases thus produced, 8-oxo-7,8-dihydroguanine (8-oxoG) to capture sequence-altered proteins that are translated from 8- appears to be the most important with respect to the mainte- oxoG–containing mRNAs. Also, we applied a deep-sequencing nance and transfer of genetic information (4). Unlike other types method to measure the content of these sequence-changed of oxidized bases, 8-oxoG does not block nucleic acid synthesis mRNAs to correlate the relationship between 8-oxoG–containing but rather induces alternate base mispairing. When such mispair- mRNAs and sequence-altered proteins. Additionally, we found ing occurs during DNA replication, base substitution mutations that the accumulation of 8-oxoG in RNA can accelerate the pro- may result (5, 6). Likewise, the 8-oxoG mispairing that occurs duction of pathogenic amyloid β peptides in mammalian cells. during RNA synthesis may induce errors in gene expression (7). Organisms are equipped with elaborate mechanisms for Author contributions: M.S. and J.-P.C. designed research; D.-P.D., W.G., J.-L.Z., T.X., Z.-L.J., counteracting the deleterious effects of 8-oxoG (6, 8). One of B.N., Y.Z., Jin Li, and N.Y. performed research; G.-X.H. contributed new reagents/analytic these well-known mechanisms involves MTH1 enzymatic activity tools; D.-P.D., W.G., H.H., J.-L.Z., X.-Q.Z., T.X., Z.-L.J., L.-Q.Z., X.-D.H., B.N., Y.Z., Jin Li, X.-Y.Z., Jian Li, T.-M.Z., Q.H., D.-G.L., H.-B.C., N.Y., P.-P.Z., Z.-X.Z., H.-M.Y., Y.W., S.H.W., where deoxyribonucleoside triphosphate, 8-oxo-dGTP, is hy- Y.-X.Z., J.-Y.W., and J.-P.C. analyzed data; and D.-P.D., W.G., H.H., J.-L.Z., S.H.W., M.S., and drolyzed to the monophosphate 8-oxo-dGMP, preventing 8- J.-P.C. wrote the paper. oxoG incorporation into DNA (9). The importance of MTH1 in The authors declare no conflict of interest. the maintenance of DNA integrity was shown through a study of This article is a PNAS Direct Submission. MTH1-deficient cells that revealed an increased frequency of Published under the PNAS license. spontaneous mutations compared with wild-type cells and that 1D.-P.D., W.G., H.H., J.-L.Z. and X.-Q.Z. contributed equally to this work. MTH1−/− mice developed more tumors than wild-type mice (3, 2To whom correspondence may be addressed. Email: [email protected] or mseki@ 10). In addition to protecting DNA, MTH1 also plays a role in college.fdcnet.ac.jp. maintaining RNA integrity through its ability to degrade the 8- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. oxoG–containing ribonucleoside triphosphate, 8-oxoGTP. Thus, 1073/pnas.1718363115/-/DCSupplemental. MTH1 can prevent an alteration in gene expression due to Published online April 2, 2018. 4218–4222 | PNAS | April 17, 2018 | vol. 115 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1718363115 Downloaded by guest on September 27, 2021 infected with an shRNA retroviral vector containing a MTH1- targeting sequence and then selected for stable knockdown cells (Fig. 1A). To examine the biological effects of 8-oxoG in RNA, we introduced 8-oxoGTP into living cells by a hypotonic shift method (11). The addition of 8-oxoGTP to these MTH1-knockdown cells led to an increase in the 8-oxoG content in RNA (Fig. 1B). Two to four hours after the administration of 8-oxoGTP, the 8- oxoGsn/106 Gsn (guanosine) value in the MTH1-knockdown cells was twofold higher than in control cells. We noted that the RNA level of 8-oxoG detected in the 8-oxoGTP–treated cells decreased gradually to the control level within 12 h after the in- troduction of 8-oxoGTP. This decrease may be related to the preferential degradation of 8-oxoG–containing messenger RNA in HeLa cells (12). Gene Expression Alteration via Insertion of 8-oxoG into Messenger RNA. Since 8-oxoG is able to pair with both adenine and cytosine (6, 8), the incorporation of 8-oxoG into messenger RNA can alter its coding properties. When an amber mutation was in- troduced into mRNA, we assumed that the °GAG codon (where – °G represents an 8-oxoG containing nucleotide) would pair with Fig. 2. Expression of GaLuc and CLuc in response to 8-oxoG. (A) The relative GaLuc signals in the MTH1-knockdown (iMTH1, closed bars) and control (iGFP, open bars) cells upon the application of 8-oxoGTP. Twenty-four hours after transfection with the reporter, the cells were permeabilized in the presence of various concentrations of 8-oxoGTP and then were cultured in medium for 12 h. The relative GaLuc signal was calculated by dividing the value for the 8-oxoGTP–treated cells by that for the nontreated control cells. (B) The relative CLuc signals in the MTH1-knockdown and control cells. The CLuc signal was calculated in a similar manner to the GaLuc signal. (C) The relative GaLuc signals in the MTH1-knockdown cells transfected with an empty vector (open bar) and those transfected with MTH1 cDNA to over- produce MTH1 proteins (closed bar). The experimental conditions were as described in A.(D) The relative CLuc signals in the MTH1-knockdown cells transfected with an empty vector (open bar) and those transfected with MTH1 cDNA to overproduce MTH1 protein (closed bar).