Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
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Abstract |
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Introduction |
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The human Alu family accounts for more than 10% of the human genome (International Human Genome Sequencing Consortium 2001
) and is one of the most extensively characterized SINE families. The typical Alu sequence is a dimer consisting of two 7SL RNAderived monomer units, the left monomer and the right monomer, connected by an A-rich linker. It has been proposed that free left and right Alu monomers (FLAM and FRAM, respectively) are precursors of dimeric Alu elements (Jurka and Zuckerkandl 1991
; Quentin 1992a
). Moreover, an even more ancient fossil Alu element (FAM) is found within the human genome (Quentin 1992b
). The rodent B1 family was apparently generated by partial deletion and tandem duplication of a rodent PB1 that is almost identical to primate FLAM (Quentin 1994
). The galago Alu type II is a unique family in which the 5' tRNA-derived region and the 3' Alu right-monomer unit are fused. Members of the 7SL RNAderived SINE families not only have conserved sequences but also have conserved secondary structures, and the RNA secondary structures of the Alu and B1 families may have played an essential role in their amplification through retroposition (Labuda and Zietkiewicz 1994
).
Two groups recently proposed various phylogenetic relationships among higher-order mammals on the basis of extensive analyses of DNA sequences (Madsen et al. 2001
; Murphy et al. 2001a,
2001b
). They proposed a monophyletic clade that includes primates, the tree shrew, the flying lemur, rabbit, and rodents. These results provided the possibility that species including 7SL RNArelated SINEs such as the primate Alu family and the rodent B1 family are monophyletic. If this is indeed the case, then 7SL RNArelated SINEs must be present in the genomes of the tree shrew, flying lemur, and rabbit. Here, we report evidence for the fourth and fifth families of 7SL RNAderived SINEs in the genome of the tree shrew. We also report that the secondary structures of the Alu domains within these newly characterized SINEs have been conserved throughout mammalian evolution. This work helps clarify the evolutionary history of the 7SL RNAderived SINE superfamily and therefore constitutes an important step toward establishing a phylogenetic link between rodents and primates.
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Materials and Methods |
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For isolation of 7SL RNAderived SINEs from the tree shrew, we used a set of polymerase chain reaction (PCR) primers designated AluF1 (5'-TCACGCCTGTAATCCCAGCACT-3') and AluR1 (5'-ATCTCGGCTCACTGCAGCCT-3') to amplify a fragment corresponding to nucleotides 17214 of the Alu family using human DNA as a template (Labuda and Zietkiewicz 1994
). The tree shrew genomic library was then screened using the PCR-amplified human Alu fragments as probes. Probes were labeled by primer extension using the AluF1 and AluR1 primers in the presence of [
-32P]dCTP. Hybridization proceeded in a solution of 50% (v/v) formamide, 6 x standard saline citrate (SSC), 1% (w/v) sodium dodecyl sulfate (SDS), 2 x Denhardt's solution, and 100 µg/ml herring sperm DNA at 33°C overnight (1 x SSC consisted of 0.15 M NaCl and 0.015 M trisodium citrate, pH 7.0; 1 x Denhardt's solution consisted of 0.02% (w/v) Ficoll 400, 0.02% (w/v) polyvinylpyrrolidone, and 0.02% (w/v) bovine serum albumin). Washing was performed in a solution of 2 x SSC plus 1% SDS at 37°C for 20 min. Positive phage clones were isolated, and their inserts were subcloned into pUC18 or pUC19. Inserts were then sequenced with universal M4 and RV primers (TaKaRa, Japan).
Alignment of 7SL RNA and tRNA-derived SINE Sequences and Derivation of a Consensus Sequence
We manually aligned the 7SL RNA- and tRNA-derived SINE sequences and deduced consensus sequences for Tu type I, II, and III families from the alignment of six, eight, and six sequences, respectively. The consensus nucleotide at each position was chosen to be the one occurring most frequently in the aligned sequences. Nomenclature of nucleotides is according to the standard IUPAC code. The sequences were deposited in DDBJ/GenBank under accession numbers AB090247AB090266.
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Results and Discussion |
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Figure 1a and b
show alignments of sequences of members of the Tu type I and Tu type II families, respectively. The 3' halves of both consensus sequences are quite similar to that of human 7SL RNA (>85% similarity excluding gaps), suggesting that the 3' halves of these two novel SINE families are derived from 7SL RNA. Therefore, the Tu types I and II represent the fourth and fifth families of 7SL RNAderived SINEs identified to date. Schematic representations of Tu type I and II sequences are shown above each consensus sequence (fig. 1
). Tu type I SINE is a hybrid consisting of a 5' region of 96 bp and a 3' region derived from 7SL RNA. Tu type II SINE has a similar hybrid structure consisting of a 5' region of 86 bp that is very similar to that of Tu type I SINE and a 3' region that is a dimer of a 7SL RNAderived monomer that is similar to the primate Alu sequence. The 5' regions of Tu types I and II may have originated from a common ancestral sequence that contained RNA polymerase III promoters (black boxes in fig. 1a and b
). In the screening for these 7SL RNAderived SINEs, the isolation frequency of positive plaques was very low, and the copy number of both families was estimated to be 102. Furthermore, the SINE sequences from both families accumulated many mutations, and the average sequence divergences among members of the Tu types I and II were calculated to be 77.6% and 78.1% excluding gaps, respectively. These data suggest that contemporary members of both these families may have lost retropositional activity.
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Figure 2b (upper left) shows that the consensus 5' half of Tu type I can also be fitted to a tRNA-like structure. Surprisingly, comparison of the consensus secondary structures of the 5' halves of Tu type I and type III SINEs reveals that several nucleotides are well matched (boxed nucleotides in fig. 2b ), suggesting that these sequences might be evolutionarily related. Like Tu type I, the Tu type II consensus sequence can also be fitted to a tRNA-like structure (fig. 2b, upper center), and the nucleotides that match the Tu type III family are boxed. Although no significant sequence similarities were evident on comparing tRNAs with both Tu type III and the 5' halves of Tu type I and type II (using Genetyx software), the similarity between the putative tRNA-like secondary structures of the Tu SINEs strongly suggests a tRNA-like origin for each of these SINEs.
The hybrid structure of tRNA- and 7SL RNAderived regions in Tu type I SINEs is similar to that of the galago Alu type II which consists of a tRNA-like region and a right monomer of the dimeric Alu family (Daniels and Deininger 1983
). The tRNA-like region originated from a tRNA-derived SINE family (galago Monomer SINE) that is apparently unique to the galago genome (Daniels and Deininger 1991
). To look for possible evolutionary relationships between tRNA-related regions of the galago and Tu SINEs, we constructed secondary structures for the tRNA-derived regions of both the galago Alu type II SINE and the galago Monomer SINE and compared them with that of Tu type III (fig. 2b,
lower; as above, the nucleotides that match the Tu type III family are boxed). Both tRNA-like structures of galago SINEs are quite similar to that of Tu type III.
Figure 3a
shows an alignment of sequences from several 7SL RNAderived SINE families. Figure 3b
shows the structural relationships among several 7SL RNAderived SINE sequences and the putative 7SL RNA progenitor. It is likely that FAM, FRAM, FLAM, the primate Alu family, galago Alu type II, the rodent B1 family, and the rodent PB1D are all derived from the Alu domain of 7SL RNA (Ullu and Tschudi 1984
; Weichenrieder et al. 2000
). Evolutionary relationships among these families are discussed below.
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During the evolution of the primate Alu family, FAM was probably the first SINE generated from 7SL RNA, with the subsequent generation of dimeric Alu from FRAM and FLAM (Quentin 1992a,
1992b;
see fig. 3b
), each of which was generated from FAM. The rodent B1 family originated from PB1, which is almost identical to primate FLAM (Quentin 1994
). PB1D was then generated by an internal deletion of PB1, and the rodent B1 family was established by the internal duplication of PB1D (Quentin 1994
). Therefore, it is reasonable to speculate that at least the two precursors of the primate Alu, namely FAM and FLAM, might have been created in a common ancestor of primates and rodents. Subsequently, the primate Alu and rodent B1 appear to have evolved differently in each lineage. It follows that the tree shrew genome might also contain FAM and FLAM elements, both of which we attempted to identify. But these elements could not be detected presumably because of their high sequence divergence.
To reconstruct the evolution of these 7SL RNAderived SINEs, we first have to understand the phylogenetic relationships among primates, rodents, and the tree shrew. There are two possible alternatives in this regard. One is a phylogeny in which the tree shrew as well as the flying lemur form a clade with primates that excludes rodents, as recently proposed by Murphy et al. (2001a,
2001b;
see fig. 5a
). The other alternative proposes that the tree shrew is phylogenetically closer to rodents than to primates, as shown in figure 5b
(Madsen et al. 2001
). Although we consider both phylogenetic hypotheses to be plausible, they are mutually exclusive and therefore need to be clarified. In each of these two phylogenetic hypotheses, we can postulate two possible evolutionary processes for the generation of SINEs, namely, generation in a common ancestor or independent generation in each lineage. In the case of the Tu type II and rodent B1, it is significant that the left-monomer unit that comprises two 7SL RNAderived regions of Tu type II is very similar to PB1D (fig. 3a and b
). Therefore, there are two possible evolutionary processes in which PB1D was generated in a common ancestor of the tree shrew and rodents or was generated in each lineage independently. In the case of the Tu type I and galago Alu type II, there is similarity not only between their composite structures but also between the secondary structures of their tRNA-derived regions (see figs. 2b
and 3b
). Therefore, there are also two evolutionary possibilities: that they were generated in a common ancestor of the tree shrew and galago or were generated independently in each lineage. Accordingly, there are several possible processes by which SINEs may have been generated depending on each of the phylogenetic hypotheses described above.
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The phylogenetic hypothesis in which the tree shrew is closer to rodents than to primates (fig. 5b ) also lends itself to the consideration of whether SINEs were generated independently or in a common ancestor. If the Tu types I and II and rodent B1 originated from a common ancestral SINE, then PB1D may have been generated in the ancestral genome of lineage F (fig. 5b ). Subsequently, each tree shrew and rodent SINE may have evolved independently from PB1D in each lineage. As in the phylogeny discussed above, this phylogeny suggests that the composite structures of tRNA- and 7SL RNAderived regions of the tree shrew and galago SINEs may have been generated independently in each lineage (lineages G and H in fig. 5b, respectively).
After considering all the possible processes by which these SINEs may have evolved, we conclude that figure 5b,
in which the tree shrew is more closely related to rodents than to primates, represents the most likely phylogeny. This phylogeny most readily explains the process by which PB1D may have been generated in a common ancestor of the tree shrew and rodents (lineage F in fig. 5b
) as well as how the joining of tRNA- and 7SL RNAderived regions may have occurred independently in the tree shrew and galago genomes (lineages G and H, respectively). These interpretations are in opposition to the recent phylogenetic hypothesis proposed by Murphy et al. (2001b)
in which the tree shrew is more closely related to primates than to rodents. In this case, the composite structure of tRNA- and 7SL RNAderived regions may at least have been created independently in tree shrew and galago genomes. Accordingly, we speculate that the joining of the 7SL RNAderived region to SINEs might have imparted some selectable advantage for retroposition of these SINE families in each genome. This speculation, together with the fact that the core of each Alu domain is well conserved (fig. 4
) in spite of much structural diversification in each lineage (fig. 3b
), reinforces the hypothesis that the protein-binding structure of Alu domains may have been maintained by selection and that the binding of SRP9-SRP14 to Alu domains may play an important role in retroposition.
By comparing the internal RNA polymerase III promoter sequence (Box B) of SINEs with that of 7SL RNAderived regions (see fig. 3a ), it is evident that several fatal mutations were introduced into the Box B sequences of the Alu right-monomer, galago Alu type II, and the latter of two promoters of rodent B1 that were generated by duplication. It is also interesting that the second promoter (Box B) of the left monomer of Tu type II also appears to be nonfunctional as a result of fatal mutations (from T to C at position 153 and from C to T at position 161 of Tu type IIleft, relative to 7SL; see fig. 3a ). In addition, the Box B regions are missing from 7SL RNAderived regions of Tu type I and from the right monomer of Tu type II. Therefore, all the putative second promoters in the Alu regions of Tu types I and II are most likely nonfunctional. But the RNA polymerase III promoters of 7SL RNAderived regions of ancient Tu types I and II must have been functional before fusion with the tRNA-derived region. After generation of the contemporary Tu types I and II, the Box B sequence of the 7SL RNAderived region may have been released from selective pressure and either deleted or mutated. This rationale also supports the notion that the Alu domain of Tu types I and II may have been conserved as a consequence of its possible role in retroposition through SRP9-SRP14 binding rather than for its RNA polymerase III promoter activity.
It may prove interesting to examine whether 7SL RNAderived SINE families are present in the genomes of other species in clade III (proposed by Murphy et al. 2001a
), namely, the flying lemur and rabbit. Characterization of 7SL-derived SINEs as well as their ancestral sequences in these animals will provide important clues to the evolutionary history of clade III species.
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Footnotes |
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Keywords: retroposon
Alu
SRP
7SL RNA
tRNA
tree shrew
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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