Specific binding of PCBP1 to heavily oxidized RNA to induce cell death

Takashi Ishiia, Hiroshi Hayakawaa, Tatsuhiro Igawab, Takeshi Sekiguchic, and Mutsuo Sekiguchib,1

aDepartment of Biochemistry, Fukuoka Dental College, 814-0193 Fukuoka, Japan; bFrontier Research Center, Fukuoka Dental College, 814-0193 Fukuoka, Japan; and cDepartment of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, 812-8582 Fukuoka, Japan

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved May 23, 2018 (received for review April 22, 2018) In aerobically growing cells, the guanine base of RNA is oxidized to in addition to a greater amount of pathogenic Aβ peptides that have 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in thus far been characterized. These results suggest that organisms as their expression. We previously demonstrated that the well as cell constituents that use oxygen as the source of energy human AUF1 binds to 8-oxoG in RNA to induce the must have some mechanisms to eliminate the hazardous effects of selective degradation of oxidized messenger RNA. We herein oxidized RNA. In a previous study (12), we identified AUF1 as a report that the poly(C)-binding protein PCBP1 binds to more protein that specifically binds to poly(A)-tailed RNA that has been severely oxidized RNA to activate -related reactions. treated with H2O2. Subsequently, it was revealed that AUF1 is in- While AUF1 binds to oligoribonucleotides carrying a single 8- volved in the selective degradation of oxidatively damaged mes- oxoG, PCBP1 does not bind to such oligoribonucleotides but senger (13). We now ask how human cells respond when instead binds firmly to oligoribonucleotides in which two 8-oxoG their RNAs are more severely damaged. This paper describes the residues are located nearby. PCBP1-deficient cells, constructed results of our investigation to answer this question. from the human HeLa S3 line using the CRISPR-Cas9 system, exhibited higher survival rates than HeLa S3 cells when small Results doses of hydrogen peroxide were applied. The levels of caspase-3 The Specific Binding of PCBP1 to Heavily Oxidized RNA. To iden- activation and PARP-1 cleavage in the PCBP1-deficient cells were tify the involved in processes to deal with 8-oxoG- significantly lower than those in wild-type cells. The structure– containing RNA, a 30-mer oligoribonucleotide derived from function relationship of PCBP1 was established with the use of rabbit β-globin messenger RNA 3′-UTR, which is known to PCBP1 mutant proteins in which the conserved KH domains were contain no AUF1-binding sites (14), was used as a probe. We defective. Human cells appear to possess two distinct mechanisms, converted a single guanine residue in the sequence to 8-oxoG one controlled by AUF1 and the other by PCBP1, with the former (for 8-oxoG x1), and two guanine residues located at nearby sites functioning when messenger RNA is moderately oxidized and the were changed to 8-oxoG (for 8-oxoG x2) (Fig. 1A). To screen for latter operating when the RNA is more severely damaged. proteins that bind to heavily oxidized RNA, two types of oli- goribonucleotides (normal control and 8-oxoG x2) were conju- oxidative stress | RNA-binding protein | apoptosis gated at their 3′-ends to magnetic beads with the aid of biotin and avidin. An extract of human HeLa S3 cells was applied to eactive oxygen species (ROS) are generated in mammalian these probes, and proteins that attached to the probes were Rcells as byproducts of ATP production and oxygen utiliza- separated by SDS-gel electrophoresis followed by silver staining tion. Although most of these radicals are eliminated by the ac- (Fig. 1B). At positions where the proteins of ∼37 kDa would tion of cellular antioxidant systems, some ROS remain within the migrate (shown by an arrowhead), strong signals were detected cell and damage various biologically important molecules, in- in the 8-oxoG x2 sample but not in the normal sample. The pro- cluding proteins, lipids, and nucleic acids (1, 2). Among the teins in this region were subjected to a mass spectroscopic analysis,

various types of oxidized purine and pyrimidine bases thus pro- and the major protein was identified as poly(C)-binding protein CELL BIOLOGY duced, 8-oxo-7, 8-dihydroguanine (8-oxoG) appears to be the 1 (PCBP1). PCBP1 was first characterized as a poly(C)-binding most important with respect to the maintenance and transfer of protein belonging to a subfamily of KH domain RNA-binding genetic information (3). Unlike other types of oxidized bases, 8- oxoG does not block nucleic acid synthesis, rather it induces base Significance mispairing. 8-OxoG can take syn conformation that can pair with anti adenine whereas the configuration of 8-oxoG pairs with The binding of human PCBP1 protein to heavily oxidized RNA cytosine as does a normal guanine base. When the 8-oxoG is triggers the induction of apoptosis-related reactions, including produced, a pair is replicated through DNA synthesis, and GC → the activation of caspase-3 and the cleavage of PARP-1. The TA transversion may be induced (4–6). Likewise, the 8-oxoG introduction of amino acid substitutions in the PCBP1 abolishes mispairing that occurs during RNA synthesis may induce errors this, resulting in the failure of PARP-1 cleavage. Human cells in the (7). appear to possess a mechanism to induce cell death when their In this context, the accumulation of 8-oxoG in messenger messenger RNAs are severely damaged. This mechanism might RNA may cause the synthesis of abnormal proteins (8). Al- be related to the accumulation of abnormal proteins in long- though most such proteins are inactive and unstable, some lived cells, such as those in the nervous system. proteins with amino acid alterations may persist in the cell. The existence of erroneous proteins might be hazardous to cells, Author contributions: T. Ishii, H.H., and M.S. designed research; T. Ishii, H.H., T. Igawa, and particularly to those with long life, such as cells in the neuronal T.S. performed research; and T. Ishii and M.S. wrote the paper. system (9). Indeed, Nunomura et al. (10) demonstrated that The authors declare no conflict of interest. RNA oxidation is a prominent feature of neuronal vulnerability This article is a PNAS Direct Submission. in patients with Alzheimer’s disease. More recently, we showed Published under the PNAS license. that there is the casual relationship between RNA oxidation and 1To whom correspondence should be addressed. Email: [email protected]. Aβ peptide formation using CHO cells to which 8-oxoGTP is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. externally supplied (11). Cells carrying 8-oxoG in messenger 1073/pnas.1806912115/-/DCSupplemental. RNA secreted 28 types of previously unknown Aβ-derived peptides, Published online June 11, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1806912115 PNAS | June 26, 2018 | vol. 115 | no. 26 | 6715–6720 Downloaded by guest on October 2, 2021 To reveal the binding characteristics of PCBP1, we prepared two types of oligoribonucleotides: one containing a single 8- oxoG residue at position 14 (for 8-oxoG x1), the other carrying two 8-oxoG residues at positions 9 and 15 (for 8-oxoG x2) (Fig. 1A). When an extract of HeLa S3 cells was applied to the two types of probes, a distinct difference was observed (Fig. 1C). PCBP1 produced a strong binding signal for the 8-oxoG x2 probe, but only a very weak binding signal for the 8-oxoG x1 probe. This is quite distinct from the binding characteristics of AUF1, which showed positive binding signals for both types of probes. To show the relationship between the binding characteristics of PCBP1 and AUF1 more clearly, an extract of AUF1-deficient cells that had been constructed by gene targeting (13) was pre- pared. Using the 8-oxoG x2 probe, it was revealed that PCBP1 can bind to the probe in the absence of AUF1 (Fig. 1D). The independence of the binding capacities between the two types of proteins was further shown in another experiment, in which the binding to an 8-oxoG x2 probe was examined at various con- centrations of NaCl. The PCBP1 binding to the 8-oxo-G x2 probe occurred in the presence of 300 mM NaCl, whereas no binding of AUF1 to the probe was observed under the same condition (Fig. 1E).

The Binding Capacity of PCBP1 to Oligoribonucleotides and Oligodeoxyribonucleotides Carrying 8-oxoG. To define the binding characteristics of PCBP1 more precisely, we introduced a His6 tag to the C terminus of PCBP1 and the protein was purified to make it homogeneous. Using this refined system, we examined whether PCBP1 indeed required two 8-oxoG residues for its binding. As shown in Fig. 2A,PCBP1canbindtoanoligoribonucleotide carrying two 8-oxoG residues at the 9th and 15th positions but not to those with a single 8-oxoG residue at either of those sites. We then examined whether the distances between the two 8-oxoG residues were important for the specific binding of PCBP1. The result shown in Fig. 2B indicates that the two 8-oxoG residues must be located seven nucleotides apart from each other for PCBP1 binding to occur. No specific binding was observed when the two 8-oxoG residues were located more closely or Fig. 1. The specific binding of PCBP1 to heavily oxidized RNA. (A)Theoli- further apart. This implies that a single molecule of PCBP1 can goribonucleotide probe sequences. The control sequence was taken from a attach to the two 8-oxoG residues appropriately located on a part of the rabbit β-globin mRNA 3′UTR. In the 8-oxoG x2 sequence, the 9th ribopolymer. O and 15th guanine residues were replaced by 8-oxoG ( G), while in the 8-oxoG We were curious as to whether PCBP1 could bind to oligo- x1 sequence, only the 14th guanine was changed to 8-oxoG. (B) The detection deoxyribonucleotides carrying 8-oxoG. We therefore prepared of 8-oxoG–binding proteins. An extract of HeLa S3 was applied to magnetic beads to which control and 8-oxoG x2 probes were conjugated, and the oligodeoxyribonucleotides and oligoribonucleotides carrying two mixture was incubated at 4 °C for 90 min. After washing with HNTG buffer 8-oxoG residues at the 9th and 15th positions, and a binding [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1% (vol/vol) Triton X-100, 10% (wt/vol) assay was performed. As shown in Fig. 2C, almost the same high glycerol and protease inhibitors] three times, the materials were subjected to levels of PCBP1 binding were observed with both deoxyribonu- SDS gel electrophoresis, then silver staining was performed. The most intensive cleotide and ribonucleotide probes, whereas binding to control of the bands found specifically in the 8-oxoG x2 sample, marked by an ar- probes without 8-oxoG was very low. Since 8-oxoG can pair with rowhead, was selected for a further analysis. (C) The binding of PCBP1 and both cytosine and adenine (3, 4), we next examined whether AUF1 to two types of 8-oxoG–containing oligoribonucleotide probes. An ex- double-stranded oligodeoxyribonucleotides carrying two 8-oxoG tract prepared from HeLa S3 cells was applied to beads carrying control, 8- residues with such pairings exhibited PCBP1 binding. As shown in oxoG x1, and 8-oxoG x2 sequences, and the materials were processed as Fig. 2D, annealed duplexes with either an OG·CorOG·Apair, described above. Western blotting was performed using anti-PCBP1 and anti- O AUF1 antibodies. (D) The existence of PCBP1-binding activity in AUF1-deficient where G stands for 8-oxoG, exhibited no binding signal, whereas cells. Extracts prepared from HeLa S3 and its AUF1-deficient cells were mixtures of two types of single-stranded molecules showed strong applied to control and 8-oxoG x2 probes, and Western blotting was per- binding signals. In chromosomal DNA, single-stranded regions are formed with anti-PCBP1 and anti-AUF1 antibodies. (E) The binding po- seldom produced, so it is reasonable to assume that binding of tential of PCBP1 and AUF1 to 8-oxoG-containig oligoribonucleotide under PCBP1 may be limited to messenger RNA, which is mostly single- different ionic strengths. An extract prepared from HeLa S3 in HNTG buffer stranded. was applied to control and 8-oxoG x2 beads, and the samples were washed with HNTG buffer containing different concentrations of NaCl. The retained Apoptosis-Related Reactions in PCBP1-Deficient Cells. To observe the materials were analyzed by Western blotting using anti-PCBP1 and anti-AUF1 biological significance of PCBP1-associated events, we constructed antibodies. PCBP1-null cell lines via gene editing (24), as shown in Fig. 3A. Unlike other family , the PCBP1 gene is composed of a proteins (15–18). It was shown that these proteins play diverse single exon (15–18), and it was proposed that the sequence of roles in the gene expression at both the transcriptional and PCBP2 was processed by retrovirus transduction during mam- posttranscriptional levels (19–23). malian evolutionary radiation. Based on the Western blotting

6716 | www.pnas.org/cgi/doi/10.1073/pnas.1806912115 Ishii et al. Downloaded by guest on October 2, 2021 Fig. 2. The binding capacity of PCBP1 to oligoribonucleotides (shown in red) and oligodeoxyribonucleotides (shown in black) carrying 8-oxoG at different positions. (A) The binding capacity of PCBP1 to oligoribonucleotides carrying one or two 8-oxoG residues. The oligoribonucleotides shown in the top and middle possess an 8-oxoG residue at the 9th or 15th position from the 5′-terminus. The sequence shown in bottom harbors two 8-oxoG residues at the 9th and 15th sites. Control (without 8-oxoG) and three oligoribonucleotides carrying one or two 8-oxoG residues at different positions were conjugated to magnetic beads and used as probes. These RNA beads were mixed with a purified preparation of C-terminally His-tagged PCBP1 and incubated for 90 min at 4 °C for binding. The beads were then washed with HNTG buffer three times, and the retained materials were subjected to SDS gel electrophoresis, followed by Western blotting using an anti-His antibody. (B) The effect of the distances between the two 8-oxoG residues on the specific binding. A purified PCBP1 protein was applied to beads carrying oligoribonucleotides with two 8-oxoG residues at different positions. The binding and subsequent processing were the same as described above. (C) The binding capacities of PCBP1 to oligoribonucleotides (red) and oligodeoxyribonucleotides (black) with two 8-oxoG residues at the 9th and 15th positions. The binding and subsequent treatments were the same as described above. (D) The binding capacity of PCBP1 to double-stranded de- oxyribonucleotides carrying 8-oxoG/C or 8-oxoG/A pairs at the 9th and the 15th sites. Four types of oligodeoxyribonucleotides were synthesized, andtwo complementary strands were hybridized to produce double-stranded probes carrying 8-oxoG/C or 8-oxoG/A at predesigned sites. Each single-stranded probe CELL BIOLOGY and mixture of oligodeoxyribonucleotides with or without 8-oxoG residues were investigated for His-PCBP1 binding.

results, a candidate clone was isolated as a knockout cell line (Fig. execute the later steps of apoptosis while being defective in trig- 3B). A DNA sequence analysis revealed that the thin bands found gering RNA damage signals. This notion was strengthened by an in the clone were nonfunctional deletion type fragments; thus, this experiment in which the induction of caspase-3 was measured clone can be regarded as a PCBP1-deficient line (SI Appendix, Fig. (Fig. 3G). In this case too, PCBP1-deficient cells showed a lower S1). PCBP1-deficient cells are viable but exhibit slower growth level of caspase-3 activity after H2O2 treatment while exhibiting than parental HeLa S3 cells (Fig. 3C). Since PCBP1 is suggested the high level of caspere-3 activity observed in actinomycin D- to be involved in various cellular processes, the slow growth may treated cells. Thus, the action of PCBP1 to induce apoptosis seems be attributed to one or a combination of deficiencies in these specific for RNA damage. processes. When wild-type and PCBP1-deficient cells were treated – with low levels of H2O2, the survival curves shown in Fig. 3D were The Structure Function Relationship of PCBP1. PCBP1 contains obtained. PCBP1-deficient cells seem to be slightly more resistant three KH domains, each consisting of ∼70 amino acid residues A to H2O2 than wild-type cells. (Fig. 4 ), and it is implied that these domains are essential for We next measured the apoptosis-related activities in PCBP1- multiple PCBP1 functions (15–18). We therefore introduced proficient and PCBP1-deficient cells. The two types of cells were amino acid substitutions to each of the three domains to see grown in the presence of 0.2 mM H2O2, and the amounts of PARP- which domains were important for specific binding to oxidized 1 and its cleaved forms were determined by Western blotting (Fig. RNA. As shown in Fig. 4B, amino acid changes in the KH1 and 3E).ThecleavageofPARP-1occursat a significantly lower rate in KH3 domains abolished the binding capacity for 8-oxoG–contain- PCBP1-deficient cells than in PCBP1-proficient cells. Such dif- ing RNA, while changes in the KH2 domain showed no significant ferences were not observed when PCBP1-deficient cells were effect. Finally, we asked whether the binding of PCBP1 to oxidized treated with actinomycin D, which damages DNA (Fig. 3F). This RNA is required for the activation of apoptosis-related reactions. implies that PCBP1-deficient cells possess a normal capacity to For this, stable cell lines expressing WT or KH mutant-type PCBP1

Ishii et al. PNAS | June 26, 2018 | vol. 115 | no. 26 | 6717 Downloaded by guest on October 2, 2021 Fig. 3. Apoptosis-related reactions in PCBP1-deficient cells. (A) A schematic illustration of the process of PCBP1 gene editing using the CRISPR/Cas9 system. sgRNA designed for genome editing was introduced into HeLa S3 cells. The red arrowheads (small and large) indicate the site of cutting by Cas9 nuclease. Cleavage of genomic DNA causes insertion-deletion mutation by inaccurate DNA repair and results in frameshift in the coding region. The thin line and closed box represent the genomic DNA and exon, respectively. The green boxes represent the methionine codon, and the red box represents the stop codon. The blue characters indicate the target sequence; the violet characters indicate the PAM sequence in the CRISPR/Cas9 system. (B) The establishment of a PCBP1- deficient cell line. HeLa S3 and HeLa S3-derived PCBP1-deficient cells were lysed in SDS sample buffer and subjected to SDS/PAGE. The expression of PCBP1 and actin protein was determined by Western blotting using anti-PCBP1 and anti-actin antibodies. (C) Growth curves of HeLa S3 and HeLa S3-derived PCBP1- deficient cells. Ten thousand HeLa S3 (●) and PCBP1-deficient (○) cells were plated and incubated under normal conditions. At the indicated times, the cells were detached by trypsin and counted. The data represent the mean ± SEM of three independent experiments. (D) Cell viability under oxidative stress. Appropriate numbers of HeLa S3 and PCBP1-deficient cells were seeded onto plates and incubated overnight. The cells on the dishes were treated with

various concentrations of H2O2 for 1 h at 37 °C in FBS-free DMEM. The medium was then exchanged for fresh medium (DMEM containing 10% FBS) and cultured for 1 wk to form colonies. The colonies were fixed and stained with crystal violet. The number of stained colonies was counted, and the surviving fractions were calculated for both HeLa S3 (●) and PCBP1-deficient (○) cell lines. The data represent the mean ± SD of three experiments. (E) Cleavage of

PARP-1 after treatment with H2O2. HeLa S3 and PCBP1-deficient cells were incubated with 0.2 mM H2O2. At the indicated times, the cells were recovered and boiled in SDS lysis buffer. The samples were subjected to SDS/PAGE followed by Western blotting using anti-PARP-1 antibody. (F) PARP-1 cleavage on ap- plication of actinomycin D. To induce PARP-1 cleavage by DNA damage, HeLa S3 and PCBP1-deficient cells were treated with 10 μg/mL actinomycin D for the indicated times. The cells were recovered and processed as described in E.(G) The caspase-3 activity in cells exposed to oxidative stress. HeLa S3 and PCBP1-

deficient cells were treated with 0.2 mM H2O2 or 10 μg/mL actinomycin D for 4.5 h at 37 °C in DMEM containing 10% FBS. After incubation, the cells were lysed, and the lysates were separated into cytosol and organelle fractions. The cytosol fraction was used to measure a caspase-3 activity using DEVD-p- nitroanilide as a substrate. The data in the graph represent the mean ± SEM of three independent experiments.

6718 | www.pnas.org/cgi/doi/10.1073/pnas.1806912115 Ishii et al. Downloaded by guest on October 2, 2021 Fig. 4. The structure–function relationships of PCBP1. (A) The structure of PCBP1 and its mutation sites. The numbers of amino acid residues are shown above the domain structure of PCBP1. Below each domain, the amino acid substitution sites are shown. (B) The binding capacity of mutant forms of PCBP1 to 8-oxoG— containing oligoribonucleotide. Each purified KH domain mutant protein was mixed with beads attached to a 30-mer oligoribonucleotide carrying 8-oxoG at the 9th and 15th positions. After incubation, the beads were washed with HNTG buffer, then boiled in SDS sample buffer. The samples were separated using PAGE

and analyzed by Western blotting using an anti-His6 antibody. (C) A complementation assay using PCBP1 KH domain mutants. PCBP1-deficient cell line and stable cell lines expressing PCBP1 KH1 D31D32 or KH2 D115D116 mutant protein were incubated with 0.2 mM H2O2. At the indicated times, cells were collected, and lysed in SDS lysis buffer. The samples were applied to SDS/PAGE, followed by Western blotting using anti-PARP-1 and PCBP1 antibodies.

protein were isolated from a PCBP1-deficient cell line (SI Appen- specifically recognized by a mismatch repair (MMR) complex, dix,Fig.S2). In the case of the KH3 mutant, the cell line that was composed of MutSα, MutLα, and PCNA (26). Following this obtained hardly expressed the KH3 mutant protein due to the in- step, the activation of HMGA and the phosphorylation of stability of the protein. These cell lines were treated with H2O2, and the cleavage of PARP-1 was measured. As a result, cell lines expressing PCBP1 WT and KH2 mutant proteins were found to lead to the cleavage of PARP-1 (Fig. 4C). However, cleavage of PARP-1 remained at low levels in the KH1 mutant cell line. These results indicate that PCBP1 WT and KH2 mutant proteins are able to induce apoptosis-related reactions, whereas KH1 mutant protein is not. Taken together, the results suggest that the oxidized RNA- CELL BIOLOGY binding ability of PCBP1 is essential for the induction of apoptosis under oxidative stress. Discussion We herein show that human PCBP1 protein possesses an ability to bind specifically to RNA carrying two 8-oxoG residues at sites located in close proximity to each other. Since guanine residues are unevenly distributed in most mRNA and, moreover, many species of mRNA possess G-quadruplex sequences, composed of multiple repeats of guanine residues (25), it is difficult to esti- mate the number of PCBP1-target sites in the mRNA pop- ulation. Apart from this, the binding of PCBP1 to heavily oxidized RNA induces caspase-3 activation and the cleavage of PARP-1, which may be related to the induction of apoptosis (26, 27). In contrast, human AUF1 binds to RNA carrying only one 8- oxoG residue (12). It was shown that the AUF1 is involved in specific degradation of oxidatively damaged RNA when the cells were exposed to relatively low degrees of oxidative stress (13). Thus, humans apparently possess two distinct mechanisms, one controlled by AUF1 and the other by PCBP1, with the former Fig. 5. A model of the induction of apoptosis by RNA and DNA damage. RNA functioning when messenger RNA is moderately oxidized and damage caused by H2O2 is recognized by PCBP1 protein. Damaged RNA–PCBP1 the latter operating when the RNA is more severely damaged. complex thus formed delivers a signal to induce cell death. The RNA-initiated Cells carrying base mismatched DNA undergo apoptosis, in signaling merges with DNA damage-initiated one at some steps before the which many protein factors are involved. The base mismatch is activation of caspase-3.

Ishii et al. PNAS | June 26, 2018 | vol. 115 | no. 26 | 6719 Downloaded by guest on October 2, 2021 checkpoint kinases, such as ATR, CHK1, ATM, and CHK2, take cells possess four additional PCBP1-related proteins, PCBP2, place (27). Subsequently, the depolarization of the mitochon- PCBP3, PCBP4, and hnRNP K (11–14), one or more of these drial membrane and the activation of caspase-3 occur, which is proteins may function as a substitute for PCBP1. The expression followed by the cleavage of PARP-1 to accomplish apoptosis. In levels of these proteins differ considerably according to the cell types the present study, the caspase-3 activation and PARP-1 cleavage and growth conditions. To solve this problem, cell lines that are occur normally in PCBP1-defective cells exposed to actinomycin defective in one or more of the PCBP1-related proteins are needed. D, whereas such events are severely suppressed in cells demon- Overall, our work here show that human cells possess a strating RNA damage. Based on these results, we created the mechanism to induce cell death when their messenger RNAs are model shown in Fig. 5. severely damaged. This mechanism might be related to the ac- How the RNA damage recognized by PCBP1 is delivered to the cumulation of abnormal proteins in long-lived cells, being im- downstream steps remains an intriguing question. Some protein portant implications in the occurrence of age-related diseases. factors may interact with damaged RNA–PCBP1 complex to form a larger complex. Such complex may further activate proteins Materials and Methods functioning at the downstream steps, in a manner such as phos- Detailed information on methods, including the antibodies and chemicals, cell phorylation and/or other modification of the proteins involved. culture, preparation of RNA beads, identification of 8-oxoG-oligoribonucleotide- It is also important to clarify at what step in the two pathways, binding proteins, binding assay, preparation of the recombinant His-PCBP1 induced by RNA damage and DNA damage, respectively, would protein, establishment of the PCBP1-deficient cell line, growth-rate assay, H2O2 they merge. As described above, many sequential events take sensitivity assay, cleavage of PARP-1, the caspase-3 activity assay, cloning and place in the pathway initiated by DNA damage. There is a pos- sequencing of the chromosomal region of PCBP1 gene in a PCBP1-deficient cell sibility that the merging point of the signal delivered from RNA line, and establishment of PCBP1 KH mutant-expressing stable cell lines are in SI damage and that initiated by DNA damage may be some step Appendix, SI Materials and Methods. between the phosphorylation of checkpoint kinases and the depolararization of the mitochondrial membrane. Thus, cautious ACKNOWLEDGMENTS. We thank Masumi Hidaka, Ryosuke Fujikane, and examination of each step in the two pathways is required. Brian Quinn for useful comments and Hisako Takeuchi for her excellent Of note, a substantial degree of apoptosis-related reactions technical assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), includ- proceed, even when the PCBP1 activity is completely eliminated ing the MEXT-supported Program for the Strategic Research Foundation at by gene targeting. This insufficiency might be caused by the exis- Private Universities Grant S1411042 and Grants in-Aid for Scientific Research tence of related activities in PCBP1-knockout cells. Since human 16K07260, 22370003, and 26340040.

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