Characterization of an active LINE-1 in the naked mole-rat genome

Naked mole-rats (NMRs, Heterocephalus glaber) are the longest-living rodent species. A reason for their long lifespan is pronounced cancer resistance. Therefore, researchers believe that NMRs have unknown secrets of cancer resistance and seek to find them. Here, to reveal the secrets, we noticed a retrotransposon, long interspersed nuclear element 1 (L1). L1s can amplify themselves and are considered endogenous oncogenic mutagens. Since the NMR genome contains fewer L1-derived sequences than other mammalian genomes, we reasoned that the retrotransposition activity of L1s in the NMR genome is lower than those in other mammalian genomes. In this study, we successfully cloned an intact L1 from the NMR genome and named it NMR-L1. An L1 retrotransposition assay using the NMR-L1 reporter revealed that NMR-L1 was active retrotransposon, but its activity was lower than that of human and mouse L1s. Despite lower retrotrasposition activity, NMR-L1 was still capable of inducing cell senescence, a tumor-protective system. NMR-L1 required the 3′ untranslated region (UTR) for retrotransposition, suggesting that NMR-L1 is a stringent-type of L1. We also confirmed the 5′ UTR promoter activity of NMR-L1. Finally, we identified the G-quadruplex structure of the 3′ UTR, which modulated the retrotransposition activity of NMR-L1. Taken together, the data indicate that NMR-L1 retrotranspose less efficiently, which may contribute to the cancer resistance of NMRs.

www.nature.com/scientificreports/ ORF2p is a protein required for endonuclease (EN) and reverse transcriptase (RT) activity for a "copy-and-paste" retrotransposition of L1s to new genomic loci 14,15 . L1 retrotransposition occurs by target-site primed reverse transcription (TPRT), during which ORF2p makes a nick in genomic DNA and synthesizes L1 cDNA using the 3′ hydroxyl group at the nick 16 . The active retrotransposition of L1 is considered a major source of endogenous mutagenesis that can promote cancer development [9][10][11][12][13] . Consistently, we have found that Kaposi's sarcoma-associated herpesvirus, an oncogenic virus, stimulates L1 retrotransposition, thereby enhancing transformation 17 . Together with the extraordinary resistance of NMR to cancer and the fewer transposon-derived sequences in the NMR genome, as described above, we reasoned that the retrotransposition of L1s in the NMR genome is less efficient than those in other genomes, which might contribute to cancer resistance. In this study, we cloned an active L1 from the NMR genome and characterized it to evaluate our hypothesis.

Results
Identification of an L1 sequence from the NMR genome. To identify an intact L1 sequence, which contained both potential ORF1p and ORF2p sequences, from the NMR genome, we searched the NMR genome database for nucleotide sequences with similarity to ORF2p of human L1 RP (accession no.: AAD39215) and mouse L1 spa (accession no.: AAC53542) using tblastn. The search using human L1 RP led to the identification of many potential ORF2p sequences (~ 3 kB) with a potential ORF1p (~ 1 kB) in the upstream region, while the search using mouse L1 spa found only short ORFs in the top 30 candidate sequences. Among these candidates identified by the search using human L1 RP , we chose an intact L1 sequence and named it NMR-L1 (Fig. 1A). The accession number of the complete sequence of NMR-L1 identified in this study is BR001516. The nucleotide sequence identity of NMR-L1 with human L1 RP (accession no.: AF148856) was 63%. A comparison of the amino acid sequences of each gene showed 33% sequence identity and 79% similarity in the ORF1 gene and 63% identity and 92% similarity in the ORF2 gene. Phylogenetic analysis of the nucleotides coding ORF1p and ORF2p showed that NMR-L1 and human L1 Hs constituted a clade that was different from that of mouse L1s (Fig. S1). We compared the amino acid residues of NMR-L1 to those important for retrotransposition of human L1 RP . All the residues we compared were conserved in the NMR-L1 ORF1p and ORF2p (Fig. 1, B and C). These results suggest that NMR-L1 likely exhibits intact retrotransposition activity.  of NMR-L1, we constructed NMR-L1 retrotransposition reporter plasmids ( Fig. 2A). According to a previously established dual luciferase-based L1 retrotransposition assay 18 , we inserted the sequences of NMR-L1 (from ORF1 to the 3′ UTR, pMY07 and pMY07′; from ORF1 to ORF2, pMY04 and pMY04′) downstream of the 5′ UTR of L1 RP ( Fig. 2A). We also inserted a Firefly luciferase (FLuc) reporter cassette in the antisense orientation, in which the FLuc gene was interrupted by an antisense intron, downstream of the 3′ UTR ( Fig. 2A, pMY07 and pMY07′) or ORF2 ( Fig. 2A, pMY04 and pMY04′). Thus, the FLuc activity was expected to be detected only after the donor L1 had undergone one round of retrotransposition. The Renilla luciferase (RLuc) gene in the plasmid was used to normalize the transfection efficiency. We also constructed FLuc-tagged NMR-L1 reporters with an D714A mutation in ORF2p ( Fig. 2A, pMY07′ and pMY04′), which was expected to be defective in reverse transcription since an Asp in position 714 of the NMR-L1 ORF2p, which corresponds to an Asp in position 702 of the human L1 ORF2p, a critical residue for RT activity 19 , was substituted with an Ala residue (Fig. 1C). We used pYX14 as the reference human L1 reporter and pYX15 as the retrotransposition-defective L1 reporter ( Fig. 2A) 18 . We then evaluated L1 retrotransposition using these constructed plasmids (Fig. 2B).
Although the retrotransposition activity of NMR-L1 was markedly lower than that of a human L1, we detected significant retrotransposition activity by comparing the intact NMR-L1 (pMY07) with the NMR-L1-D714A mutant (pMY07′) (Fig. 2B). To evaluate the relevance in other cell lines, we investigated the retrotransposition activity of NMR-L1 in NMR SV40ER (an NMR fibroblast cell line; Fig. S2), 3T3 (a mouse fibroblast cell line), and OL (a human oligodendroglioma cell line) cells. In NMR SV40ER and 3T3 cells, we could not detect substantial retrotransposition activity of both human and NMR L1s (data not shown). On the other hand, in OL cells, we detected the retrotransposition activity of a human L1 but not NMR-L1 (Fig. S3), confirming that the retrotransposition activity of NMR-L1 is lower than that of a human L1. For comparison, we also constructed an FLuc-tagged reporter of mouse L1 (ORFeus-Mm) 20 , pMY10, and its defective mutant (ORFeus-Mm-D212G/ D709Y) 20 , pMY11 (Fig. S4A). Mouse L1 showed more robust retrotransposition activity than a human L1 (Fig.  S4B). These results suggest that retrotransposition activity of NMR-L1 is still low even if compared to a mouse L1. Then, to evaluate the contribution of EN activity to NMR-L1 retrotransposition, we constructed another reporter with an D217A mutation in ORF2p, which was expected to be defective in EN activity since an Asp in position 217 of the NMR-L1 ORF2p, which corresponds to an Asp in position 205 of the human L1 ORF2p 21 , a critical residue for EN activity, was substituted with an Ala residue (Fig. S5A). Using this reporter, we found that www.nature.com/scientificreports/ NMR-L1 defective for the EN activity showed lower retrotransposition activity than the wild-type (Fig. S5B), suggesting a critical role of EN activity in NMR-L1 retrotransposition. Furthermore, to test whether the cognate 3′ UTR was required for retrotransposition activity, we conducted an L1 retrotransposition assay using a FLuctagged NMR-L1 reporter lacking the cognate 3′ UTR ( Fig. 2A, pMY04 and pMY04′). The FLuc activity levels of pMY04 and pMY04′ were comparable, suggesting that the NMR-L1 lacking the cognate 3′ UTR is retrotransposition incompetent (Fig. 2B). Taken together, our data show that we successfully cloned an active L1 sequence in the NMR genome, which had lower retrotransposition activity than human and mouse L1s and required the cognate 3′ UTR for retrotransposition.
Characterization of the 5′ UTR of NMR-L1. The 5′ UTRs of L1s are promoters of L1 mRNA transcription 9 . Using JASPAR 2018 22 , we searched putative transcription factor-binding sites in the 5′ UTR of NMR-L1 and identified several transcriptional factors that may bind to the 5′ UTR, including Atoh1, Erg, Pitx1, NFkB1, and RUNX1 (Fig. 3A). We then subcloned the cognate 5′ UTR of the NMR-L1 into a pGLuc-Basic plasmid and measured the NMR-L1 5′ UTR promoter activity. Although the activity seemed lower than that of a human L1, we detected substantial promoter activity of the NMR-L1 5′ UTR (Fig. 3B). These results suggest that the NMR-L1 5′ UTR contains sequences with promoter activity.
Characterization of the 3′ UTR of NMR-L1. Primate L1s contain guanine-rich sequences with the ability to fold into G-quadruplex (G4) structures in the 3′ UTR 23 . Because NMR-L1 is phylogenetically located near a human L1 (Fig. S1), we reasoned that NMR-L1 may contain guanine-rich sequences that can fold into G4 structures in the 3′ UTR, similar to human L1s. Consistent with our assumption, we found several guaninerich sequences capable of forming G4 structures in the 3′ UTR of NMR-L1 (Fig. 4A). Stabilization of the G4 structures of human L1s reportedly stimulates L1 retrotransposition 23 . We therefore evaluated the effect of pyridostatin trifluoroacetate (PDS), a stabilizer of G4 structures, on NMR-L1 retrotransposition. NMR-L1 retrotransposition was enhanced by PDS treatment when NMR-L1 with the cognate 3′ UTR (pMY07) was used for the assay (Fig. 4B). On the other hand, the retrotransposition was unaffected by PDS when NMR-L1 without the 3′ UTR (pMY04) was used (Fig. 4C). These results suggest that the 3′ UTR of NMR-L1 contains G4 structures that modulate its retrotransposition activity.

Cell senescence induction by NMR-L1 retrotransposition.
Cell senescence is one of tumor-protective mechanisms and also a hallmark of aging 24 . Although L1 retrotransposition drives oncogenic insertions, it can also induce cell senescence at least through stimulation of IFN responses 25 . To evaluate whether NMR-L1 induces cell senescence, we measured senescence-associated β-galactosidase (SA-β-gal) activity. As reported   www.nature.com/scientificreports/ previously, we detected L1 retrotransposition-dependent cell senescence induction by a human L1 (Fig. 5A). Although NMR-L1 exhibited lower retrotransposition activity, it still induced cell senescence in a retrotransposition-dependent manner (Fig. 5B). These results suggest that NMR-L1 is less oncogenic because it is less competent to drive oncogenic insertions and does not affect the cell senescence-based tumor-protection.

Discussion
NMRs are believed to have secrets of cancer resistance because they rarely develop cancer 1 . Previous studies have proposed several possible reasons for this resistance, including a highly efficient surveillance system of cell cycles and resistance to multiple stressors 5,26 . In addition, we reasoned that such secrets likely include low retrotransposition activity since the NMR genome contains fewer retrotransposons than other mammalian genomes 7 . L1 is a retrotransposon that is considered to be an endogenous mutagen, which can cause oncogenic mutations and contribute to cancer development [9][10][11][12][13] . In this study, we, for the first time, cloned an intact L1 from the NMR genome and determined its retrotransposition activity. The amino acids important for L1 retrotransposition in a human L1 appeared to be conserved in NMR-L1 (Fig. 1), suggesting that NMR-L1 can undergo retrotransposition in a manner similar to human L1s, i.e., possibly by TPRT 16 . The retrotransposition activity of NMR-L1 was indeed detectable but only at the minimum level compared to that of a human L1 (Fig. 2). These results are consistent with our hypothesis that L1s in the NMR genome undergoes retrotransposition less efficiently, which may contribute to the cancer resistance of NMRs, although we cannot exclude the possibility that other L1s in the NMR genome may exhibit strong retrotransposition activity. Alternatively, L1s in the NMR genome may have evolved to function within the confines of the biology unique to NMRs. Although we sought to evaluate L1 rtrotranspositon activity in mouse and NMR cells, we could not detect substantial retrotransposition activity of both a human L1 and NMR-L1 in mouse 3T3 and NMR SV40ER cells (data not shown). This might be due to strong anti-L1 activity in NMR cells. In any case, our results at least indicate that NMR-L1 does not exhibit robust retrotransposition even in NMR cells. The 3′ UTR of human and primate L1s is dispensable for retrotransposition; however, we found that the 3′ UTR was required for NMR-L1 retrotransposition (Fig. 4), suggesting that NMR-L1 is a stringent-type L1 that strictly recognizes its own 3′ UTR for retrotransposition. The RNAfold web server (http://rna.tbi.univi e.ac.at/ cgi-bin/RNAWe bSuit e/RNAfo ld.cgi) predicted the secondary structure of the 3′ UTR of NMR-L1 (Fig. S6). Since the 3′ UTRs of primate L1s are devoid of canonical secondary structures 23 , the 3′ UTR secondary structures likely play a role in NMR-L1 retrotransposition, and thus, the 3′ UTR was required for it. Although the 3′ UTRs of primate L1s are thought to lack a secondary structure, they reportedly contain conserved guanine-rich sequences that can form G4 structures 23 . Similarly, we found that the 3′ UTR of NMR-L1 contained G4 structures (Fig. 4A). G4 structures are noncanonical secondary structures formed by guanine-rich nucleic acids and stabilized by stacks of guanine tetrads held together by Hoogsteen base pairing 27 . Stabilization of the G4 motif in a human L1 stimulates its retrotransposition 23 . Consistent with this, the stabilization of the G4 motif in the 3′ UTR of NMR-L1 also stimulated retrotransposition (Fig. 4, B and C). Together with the enrichment of G4 motifs in human L1s, nonautonomous Alu and SVA elements, and retroviral long terminal repeats, our finding supports the idea that the G4 motifs may be drivers of gene copy-number variation and horizontal gene transfer 23 .
The 5′ UTR of NMR-L1 contained ~ 30-bp repeat sequences (Fig. 3A). Mouse L1 Tf contains a tandem repeat of monomer unit (~ 200 bp) in the 5′ UTR, and an eel LINE, UnaL2, has a series of 39-bp repeats 28,29 . These repeat sequences are important for promoter activity 29 , suggesting that the 5′ UTR of NMR-L1 may also have activity. Indeed, the 5′ UTR promoter activity of NMR-L1 was demonstrated by NMR-L1 promoter assay (Fig. 3). We found the binding sites of 12-O-tetra-decanoylphorbol-13-acetate (TPA)-related transcription factors, RUNX1 and Erg 30 , in the 5′ UTR of NMR-L1 (Fig. 3A), suggesting that NMR-L1 was actively transcribed possibly through the binding of these transcriptional factors. Since TPA is a potent activator of protein kinase C, which regulates various biological processes, such as cell proliferation and vascular development, NMR-L1 expression might be upregulated during developmental processes, such as angiogenesis 31 .
Although enhanced L1 retrotranspositon is considered to promote oncogenic transformation by insertional mutations, it is also reported that L1 retrotransposition induces cell senescence, a tumor-protective system and also a hallmark of aging. In this study, we demonstrated that NMR-L1 can induce cell senescence, similarly to a human L1 despite its low retrotransposition activity. This suggests that NMR-L1 is less oncogenic in total than a human L1 because it has lower retrotransposition activity but still can stimulate a tumor-protective system. Since we could not activate L1 retrotransposition in NMR cells, it is unclear whether enhanced L1 retrotransposition indeed promotes transformation in NMR cells. However, as we have already demonstrated that enhanced L1 retrotranspositon induces transformation in human cells 17 , it is reasonable to speculate that lower retrotranspositon of NMR-L1 contributes to low incidence of cancer in NMRs.
In conclusion, we successfully developed an L1 retrotransposition reporter system for NMR-L1. Using this system, we demonstrated that NMR-L1 exhibited lower retrotransposition activity than other L1s, which might be a reason of the cancer resistance of NMRs. If this is the case, then our findings suggest the importance of the regulation of L1 retrotransposition for preventing cancer development, and screening of chemicals that suppress L1 retrotransposition, such as capsaicin 32 , may facilitate the development of ways of cancer prevention.

Materials and methods
Cells. 293T cells (a human embryonic kidney cell line from ATCC), OL cells (a human oligodendroglioma cell line 33 ), and 3T3 cells (a mouse fibroblast cell line from ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5%, 5%, and 10% fetal bovine serum (FBS), respectively. NMR SV40ER cells (an NMR fibroblast cell line; also see below) were cultured in DMEM supplemented with 15% FBS, nonessential amino acids (Gibco) and L-glutamine (Nakalai Tasque, Kyoto, Japan). Plasmids. pYX014 and pYX015, reporter plasmids for a human L1 retrotransposition, were kindly provided by Dr. Wenfeng An (South Dakota State University, USA) 18 . pYX015 is a mutant L1 construct defective for L1 retrotransposition 18 . The reporter plasmids for a mouse L1 retrotransposition were prepared by substituting a human L1 sequence of pYX14 with ORFeus-Mm or ORFeus-Mm-D212G/D709Y, which was kindly provided by Dr. Jef D Boeke (NYU Langone Health, USA) 20 . The reporter plasmids for NMR-L1 retrotransposition were prepared by substituting human L1 sequences with partially codon-optimized NMR-L1 ORF1 and ORF2 sequences with or without the 3′ UTR of NMR-L1. The reporter plasmids with a putative retrotransposition-defective mutation were prepared by site-directed mutagenesis using PrimeSTAR Max DNA polymerase (TaKaRa, Shiga, Japan). Summary descriptions of the constructed plasmids are shown in Figs. 2A, S4A, and S5A. The reporter plasmid for NMR-L1 promoter activity, pGLuc-NMR-5′-UTR, was generated by subcloning the 5′ UTR of NMR-L1 into a pGLuc-Basic plasmid (New England Biolabs, Ipswich, MA, USA). The reporter plasmid for human L1 promoter activity, pGLuc-5′-UTR, was generated previously 32 . Cell senescence assay. 293T cells were transfected with pYX14, pYX15, pMY07, or pMY07′ using Lipofectamine 2000 reagent. At 3 days after transfection, SA-β-gal activity was measured using a 96-Well Cellular Senescence Assay Kit (Cell Biolabs, San Diego, CA) according to the manufacturer's instructions. SA-β-gal activity level was normalized to the corresponding Renilla luciferase activity level. Then, the activity was further normalized to that of pYX14 or pMY07.
Statistics. Statistical significance was assessed using a two-tailed Student's t-test with a threshold of P < 0.05.