Publications (15)103.16 Total impact
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Article: Low dependency of retrotransposition on the ORF1 protein of the zebrafish LINE, ZfL2-1.
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ABSTRACT: The zebrafish long interspersed element (LINE), ZfL2-1, which belongs to the L2 clade, contains two open reading frames, ORF1 and ORF2. ORF1 encodes a protein containing a coiled-coil motif and an esterase domain, whereas ORF2 encodes a protein containing an endonuclease and a reverse transcriptase domain. To elucidate the functional significance of ORF1 in retrotransposition, we constructed many variants of ZfL2-1 and examined their retrotransposition ability. We concluded: 1) the ORF1 protein is not essential for ZfL2-1 retrotransposition in cultured cells; 2) the translation of ORF1 is required for the translation of ORF2; and 3) ORF2 translation probably occurs via suppression of the ORF1 stop codon, the efficiency of which is influenced by the context of the sequence juxtaposed to the 3' side of the stop codon. These results offer a new perspective on the evolution of the L2 clade LINEs.Gene 03/2012; 499(1):41-7. · 2.34 Impact Factor -
Article: A new mechanism to ensure integration during LINE retrotransposition: a suggestion from analyses of the 5' extra nucleotides.
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ABSTRACT: Long interspersed elements (LINEs) are transposable elements that exist in the chromosomal DNA of most eukaryotes; as such, they have a large impact on the genome evolution of their hosts. LINEs mobilize by a mechanism called retrotransposition in which the LINE RNA is reverse-transcribed into DNA and then integrated into the host chromosome. The integration of the 3' end of the LINE element simultaneously occurs with the initiation of reverse transcription; this process is called target-primed reverse transcription and is one of the important characteristics of LINEs. However, the molecular mechanism of the integration of the 5' end is not well understood. Here, we show that, in cultured cells, the integrants of the zebrafish ZfL2-2 LINE produce extra nucleotides at their 5' ends, and the extra nucleotides originate from their flanking sequences. We also found that, in cultured cells, some integrants of the human L1 LINE and, in their native hosts, some endogenous elements of two other LINEs also contain 5' extra nucleotides of similar origin, suggesting that the mechanism for generation of the 5' extra nucleotides is universal among various LINEs. From these data, we propose a general mechanism for 5' integration in LINE retrotransposition.Gene 03/2012; 505(2):345-51. · 2.34 Impact Factor -
Article: Self-interaction, nucleic acid binding, and nucleic acid chaperone activities are unexpectedly retained in the unique ORF1p of zebrafish LINE.
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ABSTRACT: Long interspersed elements (LINEs) are mobile elements that comprise a large proportion of many eukaryotic genomes. Although some LINE-encoded open reading frame 1 proteins (ORF1ps) were suggested to be required for LINE mobilization through binding to their RNA, their general role is not known. The ZfL2-1 ORF1p, which belongs to the esterase-type ORF1p, is especially interesting because it has no known RNA-binding domain. Here we demonstrate that ZfL2-1 ORF1p has all the canonical activities associated with known ORF1ps, including self-interaction, nucleic acid binding, and nucleic acid chaperone activities. In particular, we showed that its chaperone activity is reversible, suggesting that the chaperone activities of many other ORF1ps are also reversible. From this discovery, we propose that LINE ORF1ps play a general role in LINE integration by forming a complex with LINE RNA and rearranging its conformation.Molecular and cellular biology 11/2011; 32(2):458-69. · 6.06 Impact Factor -
Article: Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.
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ABSTRACT: Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.PLoS Genetics 05/2009; 5(4):e1000461. · 8.69 Impact Factor -
Article: A new system for analyzing LINE retrotransposition in the chicken DT40 cell line widely used for reverse genetics.
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ABSTRACT: Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.Gene 07/2007; 395(1-2):116-24. · 2.34 Impact Factor -
Article: Functional splice sites in a zebrafish LINE and their influence on zebrafish gene expression.
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ABSTRACT: Long interspersed elements (LINEs) are transposable elements that exist in many kinds of eukaryotic genomes, where they have a large effect on genome evolution. There are several thousands to hundreds of thousands of LINE copies in each eukaryotic genome. LINE elements are amplified by a mechanism called retrotransposition, in which a LINE-encoded protein reverse transcribes (copies) its own RNA. We previously isolated two retrotransposition-competent LINEs, ZfL2-1 and ZfL2-2, from zebrafish. Although it has generally been thought that LINEs do not have 'introns' (because the LINE RNA is used as the template during retrotransposition), we now show that these two LINEs contain multiple putative functional splice sites. We further show that at least one pair of these splice sites is actually functional in zebrafish cells. Moreover, some of these splice sites are coupled with the splicing signal of a host endogenous gene, thereby generating a new chimeric spliced mRNA variant for this gene. Our results suggest the possible role of these LINE splice sites in modulating retrotransposition and host gene expression.Gene 05/2007; 390(1-2):221-31. · 2.34 Impact Factor -
Article: Novel sex pheromone desaturases in the genomes of corn borers generated through gene duplication and retroposon fusion.
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ABSTRACT: The biosynthesis of female moth sex pheromone blends is controlled by a number of different enzymes, many of which are encoded by members of multigene families. One such multigene family, the acyl-CoA desaturases, is composed of certain genes that function as key players in moth sex pheromone biosynthesis. Although much is known regarding the function of some of these genes, very little is known regarding how novel genes have evolved within this family and how this might impact the establishment of new sex pheromone blends within a species. We have discovered that several cryptic Delta11 and Delta14 desaturase genes exist in the genomes of the European and Asian corn borers (Ostrinia nubilalis and Ostrinia furnacalis, respectively). Furthermore, an entirely novel class of desaturase gene has arisen in the Ostrinia lineage and is derived from duplication of the Delta11 desaturase gene and subsequent fusion with a retroposon. Interestingly, the genes have been maintained over relatively long evolutionary time periods in corn borer genomes, and they have not been recognizably pseudogenized, suggesting that they maintain functional integrity. The existence of cryptic desaturase genes in moth genomes indicates that the evolution of moth sex pheromone desaturases in general is much more complex than previously recognized.Proceedings of the National Academy of Sciences 04/2007; 104(11):4467-72. · 9.68 Impact Factor -
Article: Novel retrotransposon analysis reveals multiple mobility pathways dictated by hosts.
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ABSTRACT: Autonomous non-long-terminal-repeat retrotransposons (NLRs) proliferate by retrotransposition via coordinated reactions of target DNA cleavage and reverse transcription by a mechanism called target-primed reverse transcription (TPRT). Whereas this mechanism guarantees the covalent attachment of the NLR and its target site at the 3' junction, mechanisms for the joining at the 5' junction have been conjectural. To better understand the retrotransposition pathways, we analyzed target-NLR junctions of zebrafish NLRs with a new method of identifying genomic copies that reside within other transposons, termed "target analysis of nested transposons" (TANT). Application of the TANT method revealed various features of the zebrafish NLR integrants; for example, half of the integrants carry extra nucleotides at the 5' junction, which is in stark contrast to the major human NLR, LINE-1. Interestingly, in a cell culture assay, retrotransposition of the zebrafish NLR in heterologous human cells did not bear extra 5' nucleotides, indicating that the choice of the 5' joining pathway is affected by the host. Our results suggest that several pathways exist for NLR retrotransposition and argue in favor of host protein involvement. With genomic sequence information accumulating exponentially, our data demonstrate the general applicability of the TANT method for the analysis of a wide variety of retrotransposons.Genome Research 02/2007; 17(1):33-41. · 13.61 Impact Factor -
Article: Isolation and characterization of retrotransposition-competent LINEs from zebrafish.
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ABSTRACT: Long interspersed elements (LINEs) are a type of retroposon and are widely distributed in most eukaryotic genomes. LINEs are classified into two groups, the stringent type and relaxed type, based on the recognition of the 3' tail of their own RNA by reverse transcriptase (RT) during retrotransposition. Although most LINEs are thought to belong to the stringent type, retrotransposition studies of the stringent type LINEs are relatively limited compared with those of the relaxed type. We have now isolated two retrotransposition-competent LINEs (ZfL2-1 and ZfL2-2) from the zebrafish genome. Both ZfL2-1 and ZfL2-2 are members of the L2 clade; ZfL2-1 encodes two open reading frames (ORFs) and ZfL2-2 encodes one ORF, and each of the ORFs is required for retrotransposition. Using a retrotransposition assay in HeLa cells, we established that both ZfL2-1 and Zfl2-2 belong to the stringent type. We also demonstrated that an esterase (ES) domain encoded by ZfL2-1 ORF1 strongly enhances its own retrotransposition. The ES domain is encoded only in ORF1 of LINEs classified in the CR1 and L2 clades, although its function or significance in retrotransposition has not been elucidated. Thus, this is the first experimental evidence that the ES domain has an enhancing function during retrotransposition. These zebrafish LINEs will be useful for determining the function of ORF1 and the retrotransposition mechanism of stringent-type LINEs.Gene 02/2006; 365:74-82. · 2.34 Impact Factor -
Article: Probing the secondary structure of salmon SmaI SINE RNA.
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ABSTRACT: SmaI is a short interspersed element (SINE) of the salmon genome, and is derived from tRNA(Lys). We probed the secondary structure of SmaI SINE RNA by enzymatic cleavage and found that the RNA structure comprises three separate domains. The 5'-terminal region (the 5' domain) forms a tRNA-like cloverleaf structure, whereas the 3'-terminal region (the 3' domain) forms an extended stem-loop. The loop region is thought to be recognized by the reverse transcriptase (RT) encoded by the long interspersed element (LINE). The two structural domains are linked by a single-stranded region (the linker domain). Our melting profile analyses indicated the presence of two structural domains having different thermal stabilities, thus supporting the domain composition described above. Based on these results, we discuss the structural generality and evolutionary advantage of the domain composition of SINE RNA.Gene 02/2006; 365:67-73. · 2.34 Impact Factor -
Article: Solution structure and functional importance of a conserved RNA hairpin of eel LINE UnaL2.
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ABSTRACT: The eel long interspersed element (LINE) UnaL2 and its partner short interspersed element (SINE) share a conserved 3' tail that is critical for their retrotransposition. The predicted secondary structure of the conserved 3' tail of UnaL2 RNA contains a stem region with a putative internal loop. Deletion of the putative internal loop region abolishes UnaL2 mobilization, indicating that this putative internal loop is required for UnaL2 retrotransposition; the exact role of the putative internal loop in retrotransposition, however, has not been elucidated. To establish a structure-based foundation on which to address the issue of the putative internal loop function in retrotransposition, we used NMR to determine the solution structure of a 36 nt RNA derived from the 3' conserved tail of UnaL2. The region forms a compact structure containing a single bulged cytidine and a U-U mismatch. The bulge and mismatch region have conformational flexibility and molecular dynamics simulation indicate that the entire stem of the 3' conserved tail RNA can anisotropically fluctuate at the bulge and mismatch region. Our structural and mutational analyses suggest that stem flexibility contributes to UnaL2 function and that the bulged cytidine and the U-U mismatch are required for efficient retrotransposition.Nucleic Acids Research 02/2006; 34(18):5184-93. · 8.03 Impact Factor -
Article: Isolation and characterization of active LINE and SINEs from the eel.
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ABSTRACT: Long interspersed elements (LINEs) and short interspersed elements (SINEs) are retrotransposons. These elements can mobilize by the "copy-and-paste" mechanism, in which their own RNA is reverse-transcribed into complementary DNA (cDNA). LINEs and SINEs not only are components of eukaryotic genomes but also drivers of genomic evolution. Thus, studies of the amplification mechanism of LINEs and SINEs are important for understanding eukaryotic genome evolution. Here we report the characterization of one LINE family (UnaL2) and two SINE families (UnaSINE1 and UnaSINE2) from the eel (Anguilla japonica) genome. UnaL2 is approximately 3.6 kilobases (kb) and encodes only one open reading frame (ORF). UnaL2 belongs to the stringent type--thought to be a major group of LINEs--and can mobilize in HeLa cells. We also show that UnaL2 and the two UnaSINEs have similar 3' tails, and that both UnaSINE1 and UnaSINE2 can be mobilized by UnaL2 in HeLa cells. These elements are thus useful for delineating the amplification mechanism of stringent type LINEs as well as that of SINEs.Molecular Biology and Evolution 04/2005; 22(3):673-82. · 5.55 Impact Factor -
Article: [Diversification of genomes by SINEs and LINEs].
Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 11/2004; 49(13):2080-9, 2078. -
Article: Solution structure of an RNA stem-loop derived from the 3' conserved region of eel LINE UnaL2.
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ABSTRACT: The eel long interspersed element (LINE) UnaL2 and its partner short interspersed element (SINE) share a conserved 3' tail containing a stem-loop that is critical for their retrotransposition. Presumably, the first step of retrotransposition is the recognition of their 3' tails by UnaL2-encoded reverse transcriptase. The solution structure of a 17-nucleotide RNA derived from the 3' tail of UnaL2 was determined by NMR. The GGAUA loop forms a specific structure in which the uridine is exposed to solvent with the third and fifth adenosines stacked. A sharp turn in the phosphodiester backbone occurs between the second guanosine and third adenosine. When the uridine is mutated (but not deleted), all mutants form the loop structure, indicating that the loop structure requires an exposed fourth residue. The retrotransposition assay in HeLa cells revealed that retrotransposition requires the second guanosine, although any nucleoside functions at the fourth position, suggesting that UnaL2 reverse transcriptase specifically recognizes the 5' side of the GGANA loop.RNA 10/2004; 10(9):1380-7. · 5.09 Impact Factor -
Article: LINEs mobilize SINEs in the eel through a shared 3' sequence.
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ABSTRACT: We characterized members of the LINE (UnaL2) and SINE (UnaSINE1) families from the eel genome and found that these LINE/SINE partners share similar 3' tails. A retrotransposition assay in HeLa cells demonstrated that the 3' conserved tail of UnaL2 is necessary for its retrotransposition. This 3' tail is recognized in trans by the UnaL2 reverse transcriptase at a surprisingly high rate, and that of UnaSINE1 can also be recognized, thus providing experimental evidence that a SINE can be mobilized by the retrotransposition machinery of a partner LINE. We also demonstrated that short repeats at the 3' end of UnaL2 are required for retrotransposition suggesting that UnaL2 retrotransposes in a manner reminiscent of the reverse transcriptase activity of telomerases.Cell 12/2002; 111(3):433-44. · 32.40 Impact Factor
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Institutions
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2002–2012
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Tokyo Institute of Technology
- Graduate School of Bioscience and Biotechnology
Tokyo, Tokyo-to, Japan
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