www.nature.com/scientificreports OPEN Characterization of the Long Terminal Repeat of the Endogenous Retrovirus-derived microRNAs in Received: 31 May 2018 Accepted: 12 September 2019 the Olive Flounder Published: xx xx xxxx Hee-Eun Lee1,2, Ara Jo1,2, Jennifer Im1,2, Hee-Jae Cha 3, Woo-Jin Kim4, Hyun Hee Kim5,6, Dong-Soo Kim7, Won Kim8, Tae-Jin Yang 9 & Heui-Soo Kim2,10 Endogenous retroviruses (ERVs) have been identifed at diferent copy numbers in various organisms. The long terminal repeat (LTR) element of an ERV has the capacity to exert regulatory infuence as both a promoter and enhancer of cellular genes. Here, we describe olive founder (OF)-ERV9, derived from chromosome 9 of the olive founder. OF-ERV9-LTR provide binding sites for various transcription factors and showed enhancer activity. The OF-ERV9-LTR demonstrates high sequence similarity with the 3′ untranslated region (UTR) of various genes that also contain seed sequences (TGTTTTG) that bind the LTR-derived microRNA(miRNA), OF-miRNA-307. Additionally, OF-miRNA-307 collaborates with transcription factors located in OF-ERV9-LTR to regulate gene expression. Taken together, our data facilitates a greater understanding of the molecular function of OF-ERV families and suggests that OF- miRNA-307 may act as a super-enhancer miRNA regulating gene activity. Paralichthys olivaceus, known as olive founder (OF) is an economically important marine fatfsh which is exten- sively cultured in Korea, China and Japan. Due to their high economic value, there are several selective breed- ing programs in place, such as those involving sex manipulation, owing to diferences in growth speed and size between male and female olive founders1–3. However, due to farming conditions, olive founders are prone to fatal infections or diseases caused by various bacteria, viruses and parasites4–7. Te olive founder RNA sequencing program has provided important information regarding known alternative splicing patterns and gene duplica- tion events within the olive founder8. However, there are few studies which characterize olive founder from a molecular biology approach. Endogenous retroviruses (ERVs) are one of the transposable elements (TE) which are inherited as stable genomic components throughout the evolution of a species. Depending on the species, copy number and chro- mosomal distribution may vary9. ERVs mediate structural variation and genomic instability based on their copy number and reverse transcriptase activity10. ERV elements are highly defective, containing large deletions, stop codons, and frameshifs in their open reading frames (ORFs). Moreover, structural genes from some ERV families are preferentially expressed in various tissues and cancer cell lines11,12. Multiple-copy of ERV families scattered throughout the genome have been reported to regulate the expression of neighbouring genes13–15. Long terminal repeat elements (LTR) recruit transcription factors and thus can enhance the transcription of host cell genes11,15. 1Department of Integrated Biological Science, Pusan National University, Busan, 46241, Republic of Korea. 2Institute of Systems Biology, Pusan National University, Busan, 46241, Republic of Korea. 3Department of Parasitology and Genetics, Kosin University College of Medicine, Busan, 49267, Republic of Korea. 4Biotechnology Research Divisions, National Fisheries Research and Development Institute, Busan, 46083, Republic of Korea. 5Departments of Life Science, Sahmyook University, Seoul, 01795, Republic of Korea. 6Chromosome Research Institute, Sahmyook University, Seoul, 01795, Republic of Korea. 7Department of Marine Bio-Materials & Aquaculture, Pukyong National University, Busan, 48513, Republic of Korea. 8School of Biological Sciences, Seoul National University, Seoul, 08826, Republic of Korea. 9Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea. 10Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, 46241, Republic of Korea. Hee-Eun Lee and Ara Jo contributed equally. Correspondence and requests for materials should be addressed to H.-S.K. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:14007 | https://doi.org/10.1038/s41598-019-50492-7 1 www.nature.com/scientificreports/ www.nature.com/scientificreports (A) ğ.64 )CI 2QN 'PX 226 ğ.64 (B) Figure 1. (A) Te schematic structure of OF-ERV9, created using RetroTector. In addition to a 5ʹ and 3- LTR, this sequence includes genes encoding the gag, pol, env and ppt proteins. (B) Karyogram of a female olive founder showing 5S (blue) and 45S (orange) rDNA, OF-ERV5-LTR (green), and OF-ERV9-LTR (red). Colocalization of two LTR elements is indicated in yellow. LTR elements may contain intrinsic enhancer activity; however, base substitutions, transcription factor bind- ing sites (TFBS), bi-directional transcription start sites (TSS), and open chromatin may increase the enhancer activity of LTR elements16. Tissue-specifc promoter and enhancer activity of human endogenous retrovirus (HERV) -K LTR has been shown in several human and CHO cell lines17. Furthermore, studies have shown that species-specifc ERV enhancer activity is generally restricted to hypomethylated tissues, suggesting that ERV fam- ilies are an important mediator in evolutionary regulation18. In the case of olive founder, chromosome 5-derived OF-ERV5-LTR has shown promoter activity in HepG2 and HINAE cells which prompted us to investigate the role of OF-ERV9-LTR in this study19. MicroRNAs (miRNAs) are small noncoding RNA molecules made up of 19 to 22 nucleotides (nt), that play important roles in gene regulation20–22. Primary miRNAs (pri-miRNAs) are a long double-stranded RNA (dsRNA) transcript with a hairpin structure, which is identifed by the nuclear protein DiGeorge Syndrome Critical Region 8 (DGCR8). Te RNA-binding protein DGCR8 together with the RNase III enzyme Drosha recognize and cleave the hairpin RNA, thereby turning pri-miRNA into pre-miRNA. Te formed pre-miRNA also adopts a hairpin structure which is processed into mature miRNAs by Dicer. Individual miRNAs bind to 3′ untranslated region (UTR) of target gene through a critical region called the “seed region”, in order to regu- late gene expression by either translational repression or mRNA degradation20. Previously, TE-derived human miRNA genes were discovered, and it was shown that several miRNA genes share their sequences with TEs23,24. In this study, the genomic structure, chromosomal location and enhancer activity of OF-ERV9-LTR have been analysed. The enhancer activity of OF-ERV9-LTR was controlled by TFBSs. In addition, a novel OF-ERV9-LTR-derived miRNA was discovered. The relative expression and functional studies of the OF-ERV9-LTR-derived microRNA-307 (OF-miR-307) suggest that OF-ERV-LTR elements may contribute to several biological functions in olive founder. Results Structure and chromosomal location of OF-ERVs. Te schematic structure of OF-ERV9 shows that each terminal contains an LTR, with the gag, pol, env, and ppt genes placed between them (Fig. 1A). Te reference 5S and 45S rDNA repeats were distinctly observed in the metaphase chromosome spread of the olive founder, with one pair of each 5S and 45S rDNA located side by side on the short arms, near the centromeric region of the sub-telocentric chromosome 2. Tree pairs of OF-ERV5-LTR signals were observed on the short arm of chromo- some 4 and 5 and on the long arm of chromosome 13 (Fig. 1B). Meanwhile, four pairs of OF-ERV9-LTR signals were observed, which were localized on chromosomes 4, 6, 9 and 13. Te signals on chromosome 6 and 9 were detected in the pericentromeric region. In the case of the two homologous chromosome pairs on chromosomes 4 and 13, the yellow signal suggested that OF-ERV5-LTR and OF-ERV9-LTR were located very close to each other, due to the overlap of the green and red signals. Sequence analysis of OF-ERV9-LTR and enhancer activity analysis in SW620 cells. Te TFBSs of OF-ERV9-LTR constructs were analysed by using MATCH in TRANSFAC v8.0. (Suppl Fig. 1). Te TRANSFAC program was used to predict TFBSs and those that had a threshold value higher than 0.95 for both core match and matrix match were selected and labelled. Constructs containing the OF-ERV9-LTR region and various permu- tations were cloned into the pGL-4.23 vector (Promega) and checked for enhancer activity (Fig. 2). Te plasmid containing the OF-ERV9-LTR region showed high enhancer activity. Tis enhancer activity was downregulated when the construct excluded the binding site for the transcription factor (TF) Sox-5. In contrast, the activity SCIENTIFIC REPORTS | (2019) 9:14007 | https://doi.org/10.1038/s41598-019-50492-7 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. Te enhancer activity analysis of OF-ERV9-LTR. Each OF-ERV9-LTR region was cloned into an enhancer vector, as indicated by the schematic structures, and assayed in a cell culture. Te structure on the lef side describes the vector as well as which transcription factors were cloned. Each arrow represents diferently designed primers, described in detail in the Materials and Methods section of the main text. Te graph shows the enhancer activity of each cloned plasmid. Te data presented represent the mean ± standard error (Student’s t-test vs. control; *p < 0.01). increased slightly when Sox-5, GATA-1 and HNF-6 binding sites were excluded. Lastly, point mutations were induced in the binding sites of FOXO1 and HFH-3, enabling the exclusion of all the identifed and selected TFBSs. In this instance, enhancer activity was further downregulated. Identification and phylogenetic relationship of OF-ERV9-LTR sequences. OF-ERV9-LTR sequences were analysed using the NCBI database to identify genes with similar sequences. As a result, the olive founder genes mkln1, slc37a3, LOC109642524, LOC109644478, LOC109626170, and LOC109636349 were matched with the OF-ERV9-LTR sequence. Te identifed conserved region is highlighted by a red box in Fig.