Characterization of Novel Alu- and tRNA-Related SINEs from the Tree Shrew and Evolutionary Implications of Their Origins

Hidenori Nishihara, Yohey Terai and Norihiro Okada

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 
We characterized two novel 7SL RNA–derived short interspersed nuclear element (SINE) families (Tu types I and II) and a novel tRNA-derived SINE family (Tu type III) from the tree shrew (Tupaia belangeri). Tu type I contains a monomer unit of a 7SL RNA–derived Alu-like sequence and a tRNA-derived region that includes internal RNA polymerase III promoters. Tu type II has a similar hybrid structure, although the monomer unit of the 7SL RNA–derived sequence is replaced by a dimer. Along with the primate Alu, the galago Alu type II, and the rodent B1, these two families represent the fourth and fifth 7SL RNA–derived SINE families to be identified. Furthermore, comparison of the Alu domains of Tu types I and II with those of other 7SL RNA–derived SINEs reveals that the nucleotides responsible for stabilization of the Alu domain have been conserved during evolution, providing the possibility that these conserved nucleotides play an indispensable role in retropositional activity. Evolutionary relationships among these 7SL RNA–derived SINE families, as well as phylogenetic relationships of their host species, are discussed.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 
Retroposons are genetic elements that are propagated through the process of retroposition in which RNA transcripts are reverse-transcribed and reinserted as DNA at various sites within the genome (Jagadeeswaran, Forget, and Weissman 1981Citation ; van Arsdell et al. 1981Citation ). Short interspersed nuclear elements (SINEs; Okada 1991a,Citation 1991b;Citation Okada and Ohshima 1995Citation ; Shedlock and Okada 2000Citation ) and long interspersed nuclear elements (Fanning and Singer 1987Citation ; Eickbush 1994Citation ) constitute two major classes of retroposons (Rogers 1985Citation ; Weiner, Deininger, and Efstratiadis 1986Citation ; Singer and Berg 1991Citation ). SINEs have been characterized in many multicellular organisms including mammals (Schmid and Maraia 1992Citation ; Batzer, Schmid, and Deininger 1993Citation ; Smit and Riggs 1995Citation ; Smit et al. 1995Citation ), a reptile (Endoh, Nagahashi, and Okada 1990Citation ), fishes (Kido et al. 1991Citation ; Takahashi et al. 1998Citation ), and invertebrates (Ohshima et al. 1993Citation ). All SINEs are derived from tRNAs (Sakamoto and Okada 1985Citation ; Endoh and Okada 1986Citation ; Okada 1991a,Citation 1991b;Citation Okada and Ohshima 1995Citation ) with the exception of the primate Alu and rodent B1 families that are derived from 7SL RNA (Weiner 1980Citation ; Ullu and Tschudi 1984Citation ).

The human Alu family accounts for more than 10% of the human genome (International Human Genome Sequencing Consortium 2001Citation ) and is one of the most extensively characterized SINE families. The typical Alu sequence is a dimer consisting of two 7SL RNA–derived 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 1991Citation ; Quentin 1992aCitation ). Moreover, an even more ancient fossil Alu element (FAM) is found within the human genome (Quentin 1992bCitation ). 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 1994Citation ). 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 RNA–derived 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 1994Citation ).

Two groups recently proposed various phylogenetic relationships among higher-order mammals on the basis of extensive analyses of DNA sequences (Madsen et al. 2001Citation ; Murphy et al. 2001a,Citation 2001bCitation ). 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 RNA–related SINEs such as the primate Alu family and the rodent B1 family are monophyletic. If this is indeed the case, then 7SL RNA–related 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 RNA–derived 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 RNA–derived SINE superfamily and therefore constitutes an important step toward establishing a phylogenetic link between rodents and primates.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 
Construction and Screening of Genomic Libraries, Subcloning, and Sequencing
The tree shrew (Tupaia belangeri) was purchased from Kasyo (Japan). Genomic DNA was extracted from liver tissue and digested with EcoRI. Digested DNA was size-fractionated by passage through a SizeSep 400 spin column (Amersham Biosciences, U.K.). DNA fragments (<2 kb) were ligated with {lambda}gt10 arms (Stratagene, La Jolla, Calif.) and packaged in vitro. Novel 7SL RNA–derived SINEs were isolated from this genomic library and were characterized as described below.

For isolation of 7SL RNA–derived 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 17–214 of the Alu family using human DNA as a template (Labuda and Zietkiewicz 1994Citation ). 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 [{alpha}-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.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 
Isolation of Three Novel SINE Families from the Tree Shrew
To isolate Alu-related sequences from the tree shrew, we first performed PCR with AluF1 and AluR1 primers using human genomic DNA as template. The resulting PCR products were then used as probes to screen a tree shrew genomic DNA library for Alu-related sequences. Among the sequences isolated were two families of SINEs that contained a sequence similar to primate Alu. These sequences were designated Tu type I and Tu type II.

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 RNA–derived 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 RNA–derived 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 RNA–derived 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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Fig. 1.—The 3' halves of Tu types I and II SINEs are derived from 7SL RNA. Alignment of (a) six members of the Tu type I family of SINEs and (b) eight members of the Tu type II family of SINEs. The consensus sequence (cons) of each family is shown above the alignment. Dots indicate nucleotides identical to those in the consensus sequence, whereas dashes indicate gaps inserted to improve the alignment. Sequence positions are indicated to the left and right. Schematic representations of the sequences are shown above each consensus sequence. Gray boxes indicate the 7SL RNA–derived regions of each type of SINE, whereas white boxes indicate tRNA-derived regions. Black boxes that connect two white boxes indicate internal RNA polymerase III promoters (Box B), and solid lines indicate A-rich linkers

 
To identify other SINEs in the tree shrew genome, we looked for repetitive sequences within the large number of tree shrew genomic sequences that we determined. Six members of a repetitive family were isolated, aligned, and designated as the Tu type III family (fig. 2a ). A 252-bp consensus sequence was then deduced and compared with consensus sequences for RNA polymerase III promoters (Box A and Box B). The presence of these promoters suggests that the Tu type III family of repetitive sequences represents a novel SINE family. The copy number of the Tu type III SINEs within the tree shrew genome was estimated at ~2 x 105 by a ratio of the sequence of the Tu type III among all the sequences determined.



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Fig. 2.—The 5' halves of Tu types I and II were derived from another SINE family, designated Tu type III. (a) Six members of the Tu type III family are aligned with the consensus sequence (cons) shown above the alignment. Dots indicate nucleotides identical to those in the consensus sequence, whereas dashes indicate gaps inserted to improve the alignment. Nucleotides that are homologous to the consensus sequence are shown in uppercase, and others are shown in lowercase. Direct repeats in the flanking regions are underlined. Sequence positions are indicated to the left and right of the consensus sequence. Box A and Box B indicate the consensus sequences of RNA polymerase III promoters of various SINEs (Borodulina and Kramerov 1999Citation ). (b) Possible secondary structures of tRNA-derived regions of the Tu types I–III, galago Monomer SINE, and galago Alu type II. Dashes indicate gaps in the sequences. Classical base pairs and G-U pairs are indicated by black lines and dots, respectively. A white line indicates a possible base pair in the consensus sequence in cases where the consensus contains two or more nucleotides. Nucleotides in Tu types I and II that are identical to those in Tu type III are boxed. The bases in bold italics are common to various tRNAs, and the numbering system parallels that for tRNA (Gauss, Grüter, and Sprinzl 1979Citation )

 
Structures of Tu type I, II, and III SINEs
We attempted to determine the possible ancestral tRNA from which the Tu type III SINEs originated by direct comparison with tRNA species. These efforts, however, proved difficult because of the extensive divergence of these Tu sequences from the original tRNA. Therefore, secondary structures of Tu types I–III sequences were modeled to determine whether they are structurally related to tRNA. Figure 2b (upper right) shows a secondary structure of the consensus sequence of the Tu type III family. In tRNAs, there are many conserved or semiconserved nucleotides (Gauss, Grüter, and Sprinzl 1979Citation ), and in generating this Tu type III structure, several conserved nucleotides were positioned so as to be consistent with those conserved in tRNA. These Tu type III nucleotides are U8, C11, A14, A15, G18, G19, G24, G26, C32, U33, A37, C48, G53, U54, U55, C56, G57, A58, U60, and C61 as indicated in bold italics (fig. 2 ; numbering parallels that for tRNAs). Even though no particular species of tRNA could be identified by comparing Tu type III and tRNA sequences, the fact that a tRNA-like structure can be generated for the Tu type III consensus sequence suggests that the Tu type III family was derived from a tRNA.

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 RNA–derived 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 1983Citation ). 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 1991Citation ). 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 RNA–derived SINE families. Figure 3b shows the structural relationships among several 7SL RNA–derived 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 1984Citation ; Weichenrieder et al. 2000Citation ). Evolutionary relationships among these families are discussed below.



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Fig. 3.—Evolution of 7SL RNA–derived SINE families. (a) Alignment of consensus sequences of 7SL RNA–derived SINEs. The sequence of human 7SL RNA is shown above the alignment (7SL). Dots indicate nucleotides identical to those in the consensus sequence, and dashes indicate gaps inserted to improve the alignment. Sequence positions of each SINE are indicated to the left and right. Nucleotides between positions 109 and 218 of the S domain of 7SL RNA were removed before alignment. Box A and Box B indicate the consensus sequences for RNA polymerase III promoters (Willis 1993Citation ; Labuda and Zietkiewicz 1994Citation ). The asterisks indicate nucleotides included in the Alu domain core (see fig. 4 ). (b) Schematic representation of the structures of 7SL RNA–derived SINEs and their 7SL RNA progenitor. Solid lines that connect blocks indicate either large deletions or dimerization. Common deletion breakpoints are indicated by vertical dotted lines that indicate corresponding positions in 7SL RNA. The black bars above the 7SL RNA indicate the Box B region and S domain, whereas the two white bars indicate the regions compared in figure 4 . The tRNA-derived regions of galago Alu type II and Tu types I and II are boxed

 
Structural Conservation of the Alu Domain of Mammalian 7SL RNA–derived SINE Families
7SL RNA is a component of the mammalian signal recognition particle (SRP) and comprises two domains, the S domain and the Alu domain (Walter and Johnson 1994Citation ; Lutcke 1995Citation ). The Alu domain forms a ribonucleoprotein complex that includes a heterodimer of the proteins SRP9 and SRP14 (Gundelfinger et al. 1983Citation ), and the crystal structure of this ribonucleoprotein complex has been reported (Weichenrieder et al. 2000Citation ). As shown in figure 3b, all members of 7SL RNA–derived SINE families share similar structural organization, each containing an Alu domain generated after deletion of the 7SL RNA S domain. Referring to Weichenrieder et al. (2000)Citation , we constructed secondary structures for the Alu domains of the Tu type I and II consensus sequences as well as the primate Alu, galago Alu type II, and rodent B1 families (fig. 4bf ) and compared them with the crystal structure of the Alu domain of 7SL RNA (fig. 4a ). The nucleotides in 7SL RNA responsible for folding of the Alu domain and to which the SRP9-SRP14 heterodimer binds were designated as the Alu core. The core consists of the nucleotide pairs C3-G44, G4-U23, and the four-nucleotide sequence G24, U25, R26, and R27 in the bulge (boxed nucleotides in fig. 4a ). Comparison of these Alu cores reveals that these nucleotides are well conserved in all the Alu domains, although G24 and G4 of 7SL RNA are replaced by U and A in mouse B1 and Tu type I, respectively (fig. 4e and f ). Therefore, it is likely that Alu domains within these SINEs could bind SRP9-SRP14 (although binding may be deficient in mouse B1 because G24 is directly involved in SRP9-SRP14 binding). The data suggest that the binding of SRP9-SRP14 to these Alu domains may play an important role in retroposition.



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Fig. 4.—Conservation of the Alu domain core of 7SL RNA–derived SINEs suggests its involvement in retrotransposition. (a) The secondary structure of the Alu domain of human 7SL RNA. Nucleotides of the 7SL RNA core to which the heterodimer SRP9-SRP14 binds are boxed. The structures are based on the crystal structure of SRP9-SRP14 with the Alu domain of 7SL RNA (Weichenrieder et al. 2000Citation ). The SINE sequence of each Alu domain conforms to the structure of the Alu domain of 7SL RNA (bh). Numbers along the sequences refer to the nucleotide sequence of 7SL RNA. Standard base pairs and G-U wobble pairs are shown as black dashes and dots, respectively. White bars indicate possible base pairs in the consensus sequence in cases where the consensus contains two or more nucleotides

 
Evolution of the 7SL RNA–derived SINE Superfamily and Mammalian Phylogeny
A mammalian phylogeny based on comprehensive sequence analysis was recently proposed (Murphy et al. 2001a,Citation 2001bCitation ). In this phylogeny, placental mammals are classified into four major clades, and the species belonging to clade III (Murphy et al. 2001aCitation ) include the flying lemur, tree shrew, rodents, rabbits, and primates. Interestingly, all the species (primates, rodents, and tree shrew) in which 7SL RNA–derived SINEs are present are included in clade III. Although Murphy et al. (2001b)Citation proposed that the tree shrew is more closely related to primates than to rodents, the precise phylogenetic position of this animal remains unclear.

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,Citation 1992b;Citation 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 1994Citation ). PB1D was then generated by an internal deletion of PB1, and the rodent B1 family was established by the internal duplication of PB1D (Quentin 1994Citation ). 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 RNA–derived 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,Citation 2001b;Citation 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. 2001Citation ). 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 RNA–derived 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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Fig. 5.—Two possible phylogenetic trees of primates, the tree shrew, and rodents and the origins of 7SL RNA–derived SINEs inferred from the SINE structures and sequences. The tree shrew is shown as being more closely related to primates than to rodents (a) or more closely related to rodents than to primates (b). The lineages A to I represent points in which new SINE families could have been generated according to our various hypotheses (see Results and Discussion). In all hypotheses, FAM and FLAM (PB1) were considered to have been generated in a common ancestral genome. Subsequently, the dimeric Alu and B1 families were generated in the lineages of primates and rodents, respectively

 
In the case of the phylogeny shown in figure 5a, let us consider the case in which PB1D may have been generated in a common ancestor of the tree shrew and rodents (lineage A). If this is indeed the case, then a PB1D-like SINE should have been discovered in the primate genome. But despite exhaustive efforts, no PB1D-like SINE sequence has been characterized in the human genome. Also, whereas it is possible that PB1D-like sequences were generated independently in the tree shrew and rodent lineages (lineages D and E in fig. 5a ), it is improbable because precise independent deletion of several nucleotides to generate PB1D is unlikely. In the case of the composite structures of Tu type I and galago Alu type II, these SINEs may have been generated in a common ancestor of primates and the tree shrew (lineage B in fig. 5a ). Subsequently, the 7SL RNA–derived region of the galago Alu type II may have been subjected to deletion in the tree shrew lineage, resulting in the generation of Tu type I. But this hypothesis is also unlikely, given that no galago Alu type II–like SINE has been detected in the human genome. Therefore, if we adopt the phylogeny in figure 5a as proposed by Murphy et al. (2001a,Citation 2001b)Citation , the composite structure of Tu type I and galago Alu type II is more likely to have been generated independently in each lineage (lineages C and D in fig. 5a, respectively). Therefore, the most likely process is that all these SINEs were generated independently in each lineage. This hypothesis is supported by a previous proposal that the generation of composite SINEs occurs more easily than previously predicted (Rogers 1985Citation ).

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 RNA–derived 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 RNA–derived 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)Citation in which the tree shrew is more closely related to primates than to rodents. In this case, the composite structure of tRNA- and 7SL RNA–derived regions may at least have been created independently in tree shrew and galago genomes. Accordingly, we speculate that the joining of the 7SL RNA–derived 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 RNA–derived 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 II–left, relative to 7SL; see fig. 3a ). In addition, the Box B regions are missing from 7SL RNA–derived 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 RNA–derived 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 RNA–derived 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 RNA–derived SINE families are present in the genomes of other species in clade III (proposed by Murphy et al. 2001aCitation ), 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.


    Footnotes
 
Dan Graur, Reviewing Editor

Keywords: retroposon Alu SRP 7SL RNA tRNA tree shrew Back

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 Back


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 Abstract
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 Materials and Methods
 Results and Discussion
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Accepted for publication July 17, 2002.