Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama
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Abstract |
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Introduction |
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Another structural feature of tRNA-related SINEs is their relationship to long interspersed elements (LINEs), which are also retroposons but which encode the reverse transcriptase that is necessary for their own retroposition. Many examples of sequence homology between the 3'-ends of a SINE family and a LINE family have been reported (Ohshima et al. 1996
; Okada and Hamada 1997
; Terai, Takahashi, and Okada 1998
; Ogiwara et al. 1999
). Thus, it has been proposed that the reverse transcriptase encoded by an active LINE is responsible for the retroposition of a partner SINE that coexists in the same genome and, moreover, that the 3'-end of the SINE is recognized by the LINE-encoded reverse transcriptase during this process (Okada et al. 1997
).
The evolution of sequences within a single family of SINEs can be considered in terms of the formation of subfamilies, each of which uniquely shares correlated changes in nucleotides, in other words, each of which includes certain diagnostic nucleotides. Subfamilies of SINEs appear to have been formed by the accumulation of mutations in a limited number of SINE sequences that were capable of retroposition, and these SINEs are called source genes (Schmid and Maraia 1992
). Subfamily structures have been described for the Alu family in primates (for review, see Jurka and Milosavljevic [1991]
; Shen, Batzer, and Deininger [1991]
; and Deininger and Batzer [1995]
), the B1 family in rodents (Zietkiewicz and Labuda 1996
), the S1 family in cruciferous plants (Lenoir et al. 1997
), and the HpaI family in salmon (Kido et al. 1994
, 1995
; Takasaki et al. 1994
, 1996
), among others, from alignments of sequences in each family. However, many other reports on SINE families have not focused on such structures, and details of the mechanisms of evolution of such subfamilies remain to be clarified.
The African cichlid (AFC) family (Takahashi et al. 1998
) of SINEs was first characterized in cichlid fish (Family Cichlidae) in the East African Great lakes. These fish are famous for the large number of species, the endemicity of species in each lake, and the diversity that has been acquired by explosive adaptive radiation (Fryer and Iles 1972
; Greenwood 1984
; Coulter 1991
). Recent reports (Takahashi et al. 1998
, 2001a,
2001b;
Y. Terai et al., unpublished data) have discussed the timing of retroposition of individual members of the AFC SINE family, which includes several dozen orthologous loci, in attempts to elucidate the phylogenetic relationships among cichlids (see Shedlock, Milinkovitch, and Okada [2000]
and Shedlock and Okada [2000]
for the methodology, which involves polymerase chain reactions [PCRs]). However, the cited analyses focused only on the presence or absence of SINE sequences themselves at genomic loci. The diagnostic nucleotides were not analyzed, although the existence of subfamilies was suggested by a preliminary analysis of a limited number of sequences (Terai, Takahashi, and Okada 1998
). In the present study, we reexamined many sequences in the AFC SINE family and found three divergent subfamilies that had not previously been reported, in addition to the three previously reported subfamilies. We then attempted to clarify the evolution and retropositional dynamics of these SINEs by comparing their sequences and analyzing the timing of their insertion at genomic loci by retroposition.
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Materials and Methods |
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Results |
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Mosaic Structures of Subfamilies
Considering the distribution of diagnostic nucleotides in the various subfamilies at a total of 93 sites (fig. 1
, panel A), we found that the 5'-regions, which corresponded to positions 1130 (referred to hereafter as the head region) of subfamilies Af1, Af2, and Af4 had almost identical patterns of diagnostic nucleotides (type A), whereas this region in each of the subfamilies Af3, Af5, and Af6 had a unique and different pattern of such nucleotides (types B, C, and D, respectively). To our surprise, the other regions (positions 131350; referred to hereafter as the tail region) of the various sequences exhibited a different pattern of relationships among the subfamilies. The subfamilies Af1 and Af6 had very similar diagnostic nucleotides in the entire tail region (type X), whereas subfamilies Af2, Af3, and Af5 shared another pattern of diagnostic nucleotides in this region (type Y). The diagnostic nucleotides in the tail region of the Af4 subfamily were identical to those of type Y at positions 131247, but those at subsequent positions (248350) were more similar to those of type X. Therefore, we designated this type of tail region (YX). Our analysis suggested that the subfamilies of AFC SINEs had a mosaic structure, with various combinations of four kinds of head region (types A through D) and three kinds of tail region [types X, Y, and (YX)], each of which was associated with the unique distribution of diagnostic nucleotides (fig. 1
, panel B).
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In order to characterize the sequences of the head region in greater detail, we subjected the tRNA-related region of each SINE (Takahashi et al. 1998), which corresponded to positions 585, to a homology search in the nucleotide sequence database. In figures 2
and 3
, the tRNA-related regions of the Af3 and Af6 subfamilies are, respectively, compared with those of other subfamilies, as well as with genes for tRNAs that were identified in the nucleotide sequence database from their high homology scores. In this comparison, the sequence of the Af3 subfamily (type C) was most similar (81.9%) to the type-A sequence, which was shared by subfamilies Af1, Af2, and Af4. By contrast, the type-C sequence was less similar to the sequences of genes for tRNAs. The similarity scores were 74.0% with a gene for tRNALeu from Spinacia oleracea (AJ400848; Schmitz-Linneweber et al. 2001
), 72.6% with a gene for tRNAThr from Neisseria meningitidis Z2491 (AL162752; Parkhill et al. 2000
), and 68.5% with a gene for tRNAHis from Mycobacterium leprae (U15186). The homology between the Af3 subfamily (type C) and the Af6 subfamily (type D) was also low (68.9%). When the tRNA-related region of the Af6 subfamily was compared with those of other subfamilies, it was clear that the homology was limited (65.3%69.9%; fig. 3
). However, much higher homologies were observed with genes for tRNAs: 79.5% with a gene for tRNAAla from Leptospira interrogans serovar (AB024693), 76.7% with a gene for tRNAThr from Caenorhabditis elegans (AF016671; The C. elegans Sequencing Consortium 1998
), and 75.3% with a gene for tRNALys from Zymomonas mobilis (AF088897). These results suggested that types A and C, together with type B, which was intermediate between types A and C, were more closely related to each other than to genes for tRNAs. By contrast, type D was only distantly related to other types of head region in the AFC family but was more similar to genes for several tRNAs.
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To examine the relative retropositional activities of the subfamilies at various stages in the evolution of cichlids in Africa, we plotted the timing of insertion of all 70 AFC SINEs on a previously established phylogenetic tree (fig. 4
, panel A; Takahashi et al. 1998
, 2001a,
2001b;
Y. Terai et al., unpublished data). Retroposition of members of the Af1 subfamily was detected at various sites on the tree. In particular, in the part of the tree that corresponded to an early period (stages IIV), this subfamily accounted for 14 of the 18 (77.8%) members of the AFC family. Retroposition of members of this subfamily was also apparent at all other later stages (VX) on the tree, as well as at the branch leading to the tribe Lamprologini in Lake Tanaganyika. Retroposition of the examined copies of the Af2 subfamily was evident at stages IVVII, as well as at the branches leading to the tribes Lamprologini and Ectodini in Lake Tanganyika. This subfamily seems to have been most active at stages V and VI, accounting for nine of the 24 (37.5%) members of the AFC family that were inserted in their specific loci during this period. Retroposition of the examined members of the Af3 subfamily was restricted to the recent part of the phylogenetic tree (stages VX, as well as the branches leading to the tribes Perissodini and Tropheini in Lake Tanganyika). Members of the Af6 subfamily were found to have been inserted at their specific loci at stages VIIX, which also correspond to a recent but more limited part of the tree. Retroposition of sequences in the minor subfamilies, that is, the two each copies of the Af4 and Af5 SINE, appeared at stages IIIII and VI, respectively.
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The results of our analysis of head regions are shown in panel B of figure 4 . In most of this tree, retroposition of sequences with a type-A head region, in terms of diagnostic nucleotides (subfamilies Af1, Af2, and Af4), seemed active. However, the frequency of retroposition of type-A sequences relative to that of other types seemed to have decreased somewhat at more recent stages (VIIX, for example) as a result of retroposition of subfamilies with head regions of types C and D. The observed retroposition of sequences with head regions of types C and D (corresponding to subfamilies Af3 and Af6, respectively) was restricted to recent stages on the tree (stages VX and VIIX, respectively), as well as to branches leading to the tribes Perissodini and Tropheini, in the case of the type C. Panel C in figure 4 shows a similar analysis based on the tail regions of the AFC SINEs. In this panel, retroposition of sequences with either an X or a Y type of tail was broadly distributed on various parts of the tree. This observation suggests that sequences with either type of tail region have been active throughout almost the entire investigated evolutionary time frame of the cichlids. Thus, our analyses of heads and tails yielded contrasting results for the relative retropositional frequencies of sequences with different types of head and tail region during cichlid evolution. The frequencies seemed variable when we focused on differences among types of head region but constant when we focused on differences among types of tail region.
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Discussion |
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Possible Mechanism for and Role of Sequence Exchanges among Subfamilies
The mosaic structures of SINE sequences have been discussed in the reports on S1 elements in cruciferous plants (Lenoir et al. 1997
) and B1 elements in rodents (Zietkiewicz and Labuda 1996
). The authors of the cited reports suggested the possibility that such structures might have been formed by the exchange of sequences among different subfamilies by gene conversion. Kass, Batzer, and Deininger (1995)
proposed models for such gene conversion between SINEs. Their models include the following possible scenarios: (1) the cDNA of a SINE is used as a template to repair a double-strand break within the genomic sequence of another SINE and (2) unequal crossing-over, by homologous recombination, occurs between neighboring copies of SINEs. It is possible that gene conversion might also have been responsible for the mosaic structures of the subfamilies examined in the present study. In this case, the border between the head region and the tail region might be a hot spot for such double-strand breakage or recombination. However, gene conversion might not be the only explanation. We postulated that the mechanism of formation of the mosaic structures of AFC SINEs might be related somehow to the sharing by SINEs and LINEs of the retropositional machinery. As mentioned in the Introduction, the reverse transcriptase encoded by a LINE is considered to be responsible for the retroposition of a partner SINE. Moreover, recruitment of the 3'-ends of LINEs by SINEs has been suggested to be a general and important evolutionary phenomenon that allows SINEs to retain their retropositional activity (Ohshima et al. 1996
; Okada et al. 1997
; Terai, Takahashi, and Okada 1998
). Gilbert and Labuda (1999
, 2000)
found recently that many SINE families in the genomes of diverse animals share a conserved sequence, designated the core, adjacent to the 3'-end of their respective tRNA-related regions. The vast and ancient group of SINEs, which Gilbert and Labuda designated CORE-SINEs, includes various SINE families whose 3'-ends are homologous to those of LINE families. They proposed that the core region has provided CORE-SINEs with the ability to recruit the 3'-ends of active LINEs in various lineages of animals during the course of evolution, giving the SINEs the ability to exploit the active retropositional machinery of the LINEs. As a mechanism for recruitment of the 3'-ends of these LINEs by the SINEs, Gilbert and Labuda (2000)
proposed that template switching (Bowman, Hu, and Pathak 1998
) might occur between RNAs transcribed from a LINE and a CORE-SINE at the conserved core domain during the process of reverse transcription.
The AFC family from cichlids is also a member of the vast superfamily of CORE-SINEs (Gilbert and Labuda 1999
). AFC SINEs have a core domain adjacent to the 3'-end of the tRNA-related region and share the sequence of the 3'-end with the CiLINE2 family of LINEs (Terai, Takahashi, and Okada 1998
). We found that the border between the head region and the tail region of the AFC sequence is located within the core domain (fig. 1
, panel B). Thus, if template switching were to have occurred not only between RNAs transcribed from the AFC SINE and CiLINE2 but also between RNAs transcribed from the different subfamilies of AFC SINEs at the core domain, it is easy to see how the mosaic structures of SINE sequences, as observed in the present study, might have been generated. This hypothetical scheme can be summarized as follows. First, the reverse transcriptase encoded by an active copy of the CiLINE2 recognizes the 3'-end of the RNA transcribed from an AFC SINE in a certain subfamily. Second, the reverse transcriptase begins the synthesis of cDNA using the RNA as template. When the reverse transcription has proceeded to the region of the template RNA that corresponds to the core domain, the reverse transcriptase switches its template to the corresponding region of another RNA that has been transcribed from a SINE in a different subfamily of the AFC family. Finally, completion of reverse transcription and integration of the cDNA at a certain locus in the genome create a copy of the AFC family that is a mosaic, representing parts of each of the two subfamilies. If this new mosaic SINE continues to be actively transcribed, it can become the source gene for a new subfamily with a mosaic structure.
The present analysis suggests that different subfamilies were associated with different relative frequencies of amplification at each stage in the evolution of cichlids (fig. 4
, panel A). It has been proposed that the retropositional activity of a SINE is affected by various factors, such as chromatin structure, methylation, cis-acting promoter elements, trans-acting factors, and RNA processing (for review, see Schmid and Maraia 1992
). However, the primary prerequisite for retroposition is the integrity of the SINE sequence itself, even when the environment is ideal with respect to all these factors. Recent evidence for the possible involvement of the reverse transcriptase from a LINE in the retroposition of a SINE implies that the sequence at the 3'-end of the SINE itself, which is recognized by the reverse transcriptase, is especially important for retropositional activity. The observed generality of retroposition of sequences with either an X or a Y type of tail region throughout the cichlid phylogenetic tree (fig. 4
, panel C) seems to support this hypothesis, if we consider that the reverse transcriptase has a relatively strict preference for the sequence in the tail region and, hence, its sequence cannot easily be replaced by another related sequence that has accumulated mutations. By contrast, the observed variability in the relative retropositional activities of sequences with different types of head region at the various stages of the cichlid evolution (fig. 4
, panel B) is at least consistent with the notion that this region has more flexibility in terms of sequence because of weaker constraints on retroposition.
What factors might have influenced the relative retropositional activities of the different subfamilies of AFC SINEs during the course of evolution? Because retroposition of the Af3 and Af6 subfamilies was restricted to the recent part of the tree (fig. 4
, panel A), it seems that their source genes might have become active at around stages V and VI, respectively. The activation of their retroposition might have been related, directly or indirectly, to the emergence of these subfamilies themselves by putative recombination events, such as gene conversion or template switching. In this process, the active tails (types Y and X for subfamilies Af3 and Af6, respectively) were introduced into preexisting ancestral sequences, replacing tails that had been inactivated by the accumulation of mutations. If this hypothesis is correct, the recombination events that were responsible for the observed mosaic structures might have played a role in recycling of dead copies of AFC SINEs, contributing to the diversification of the AFC SINEs in the genomes of cichlids. Finally, this putative mechanism for the recent activation of the Af3 and Af6 subfamilies does not exclude the possibility that certain changes in the local environment of the genome (Schmid and Maraia 1992
; Takasaki et al. 1996
) might have triggered these events. For a further examination of the background associated with the changes in the retropositional activities of the various subfamilies, we need more data on the retropositional dynamics of the AFC SINEs, on mechanisms of regulation of retroposition by the local environment of the genome, and on biochemical interactions between SINEs and the reverse transcriptases encoded by LINEs.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut
Abbreviations: SINE, short interspersed element; LINE, long interspersed element.
Keywords: retroposon
AFC SINEs
cichlid
template switching
diagnostic nucleotides
Address for correspondence and reprints: Norihiro Okada, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. nokada{at}bio.titech.ac.jp
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References |
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