Genetic Information Research Institute, Mountain View, California
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Abstract |
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Key Words: SINE non-LTR retrotransposon CR1 clade LINE 5S rRNA pol III transposable element
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Introduction |
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SINE elements are 100-bp to 500-bp long and contain internal promoters for RNA polymerase III (Singer 1982; Okada and Ohshima 1995; Schmid 1996). The unusual feature of the internal pol III promoters is that their control regions are located downstream of the transcription start site (Paule and White 2000). Therefore, a retrotransposed cDNA copy of a RNA molecule transcribed by the polymerase III preserves the internal promoter. There are three general types of pol III promoters (Paule and White 2000). The first two types are formed from internal promoters, whereas the type 3 represents external promoters. Type 1 promoters are composed of the 15-bp A box, the
5-bp intermediate element IE, and the
18-bp C box, which form
50-bp internal control region (ICR) running between +50 and +90. The 5S rRNA gene, encoding a highly conserved 120-bp RNA component of the large ribosomal subunit, is the most prominent gene employing the type 1 pol III promoter (Paule and White 2000; Barciszewska et al. 2001). Type 2 promoters are composed of the
15-bp A box and
10-bp B box that form an ICR running from +10 to +65. Typical type II promoters are present in tRNA and animal 7SL RNA genes. All SINEs characterized to date can be divided into two classes. The major class includes SINEs derived from tRNA molecules (Okada and Ohshima 1995; Ogiwara et al. 1999). The minor one is composed the Alu and B1 families derived from 7SL RNA and present in the primate and rodent genomes, respectively (Weiner 1980; Ullu and Tschudi 1984). Therefore, all currently known SINEs utilize pol III promoters that belong to the type 2. Given the high abundance of 5S rRNA in eukaryotic cells, the apparent lack of SINEs derived from 5S rRNA remained a puzzle (Weiner 2002).
Here, we report a new class of SINE elements, called SINE3, derived from 5S rRNA and utilizing the type 1 pol III promoter in the zebrafish genome. We also show that a 70-bp 3' tail of SINE3 is similar to the 3' tail of CR1-like non-LTR retrotransposons. Furthermore, SINE3 elements are not flanked by the target site duplications (TSD). Therefore, we suggest that retrotransposition of SINE3 is catalyzed by the enzymatic machinery encoded by CR1-like retrotransposons.
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Materials and Methods |
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Using the SINE3 consensus sequence as a CENSOR query, we identified all SINE3-like elements present in GenBank sequences and in sequences from the Ensembl Zebrafish release 4.06.1, which comprises approximately 1% of the 1.7-Gb zebrafish genome (http://www.ensembl.org/Danio_rerio/). To identify insertions of SINE3 elements into copies of other known TEs, we expanded the GenBank-derived copies of SINE3 ±700-bp at both termini. Next, the internal portions of the expanded sequences similar to the SINE3 consensus sequence were masked out, and the remaining sequences were aligned to the sequences of transposable elements from the zebrafish section of Repbase Update (http://www.girinst.org/Repbase_Update [Jurka 2000]). We selected flanking regions similar to DNA sequences of known TEs in order to analyze target sites associated with SINE3 insertions.
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Results |
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We identified 14 insertions of SINE3 into 14 genomic copies of nine different DNA transposons and retrotransposons harbored by the zebrafish genome (table 1). Therefore, SINE3 is a transposable element. Analysis of DNA sequences harboring the identified insertions strongly indicates (data not shown) that SINE3 elements are not flanked by target site duplications (TSD), usually generated upon integration of most DNA transposons and retrotransposons into genomes. There are also no apparent open reading frames (ORFs), terminal inverted repeats, or direct repeats present in SINE3. Numerous SINE3 elements have been transposed recently (several MYA) because they are only 5% divergent from each other and are inserted into different nonhomologous regions. We identified two young subfamilies of SINE3 elements, called SINE3-1a and SINE3-2a (fig. 2). They are represented by 14 606-bp and 10
575-bp copies, respectively, identified in the current public set of zebrafish sequences. Therefore, the whole zebrafish genome is expected to harbor
1,400 and
1,000 copies of SINE3-1a and SINE3-2a elements, respectively. Their consensus sequences are 605-bp and 575-bp long, respectively. There is no significant similarity between the
30-bp 5' and 3' termini of SINE3-1a and SINE3-2a. Whereas the 3' tail of SINE3-1a is composed of the ACATT microsatellite, the 3' tail of SINE3-2a is composed of the ATT microsatellite. Excluding the termini, there is an 84% identity between the SINE3-1a (positions 33 to 573) and SINE3-2a (positions 32 to 550) consensus sequences. However, each consensus sequence is
98% identical to the elements from the same subfamily. Such a high intrasubfamily identity suggests that the retrotransposition of SINE3 elements is an ongoing process. For example, two SINE3-2a elements identified in the GenBank sequences AL831789 (positions 30523 to 31098) and AL844197 (positions 51654 to 51300) are only 1% different from each other.
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The 5' End of SINE3 Is a Former 5S rRNA Gene
The very 5' end of SINE3 is 75% identical to the 5S rRNA gene (fig. 3). The 120-bp 5S rRNA is highly conserved in different species. It is believed that tRNA and 5S rRNA belong to the most abundant classes of RNA molecules in eukaryotic cells. Usually, the 5S rRNA gene is present in multiple copies tandemly repeated in the genome. The zebrafish 5S rRNA gene was not studied and characterized in depth, but it was mapped recently to the long arm of chromosome 3 (Gornung et al. 2000; Phillips and Reed 2000). We identified 145 copies of 5S rRNA in the AL645691 HTGS GenBank sequence. The sequence is composed of two contigs, the order of which has not yet been determined (AL645691, positions 1 to 145784 and 145885 to 158047). Sequence data show that the zebrafish 5S RNA genes form at least two separate clusters. The first cluster is sequenced completely and is composed of 76 copies of 5S rRNA (AL645691, positions 97294 to 78116). The second cluster is sequenced only partially and it is represented by the whole second contig composed of 69 tandemly repeated copies of 5S rRNA. Flanking regions of the last cluster are not known. In both clusters, the repeat unit is 177-bp long and is composed of the 120-bp 5S rRNA gene and a 57-bp NTS. There is
99% identity between these 5S rRNA genes. Similar identity characterizes the spacer copies. The GCTT-3' terminus of the 5S rRNA gene and the 5'-TTCG terminus of the spacer (fig. 3) form the GCTTTTCG signal that works presumably as a terminator of the pol III transcription (Sajdak, Reed, and Phillips 1998; Paule and White 2000). Interestingly, this signal is missing in SINE3 (figs. 2 and 3). All three functional sites, Box A, IE, and Box C, which constitute the pol III internal promoter of the 5S rRNA gene (Sajdak, Reed, and Phillips 1998), are surprisingly well preserved in SINE3 (fig. 3). Given this conservation, we assume that the type 1 internal promoter of pol III is involved in the SINE3 transcription, which is necessary for its transposition. Due to the loss of the 5S rRNA pol III terminator (fig. 3), the pol III transcription does not stop at the 3' end of the 5S rRNA-related region and goes further to the 3' terminus of SINE3.
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Origin of SINE3
Figure 5 illustrates a putative model of the origin of SINE3. According to this model, SINE3 has emerged accidentally as a composite retroelement, whose 5' and 3' ends were derived from a 5S rRNA gene/pseudogene and a CR1-like non-LTR retrotransposon's 3' terminal portion, respectively, separated by a short fragment of the host genome. If so, the SINE3 internal region is a copy of the last genomic fragment (fig. 5). The model does not assume too many unlikely events necessary for the origin of SINE3. Typically, retrotransposed copies of CR1-like elements are 5' truncated and preserve their 3' termini, which are necessary for retrotransposition, given their transcription and availability of reverse transcriptase expressed by corresponding full-length CR1-like non-LTR retrotransposons. It is believed that the CR1-like reverse transcriptase recognizes specifically DNA sequences similar to the 3' end of the CR1-like retrotransposon encoding the reverse transcriptase. Due to the deleted promoter, further transcription and retrotransposition of the 5' truncated copy would be suppressed (fig. 5b), unless this copy is close to a pol III promoter carried by 5S rRNA (fig. 5c). Presumably, the pol III transcription of zebrafish 5S rRNA genes produces a mature RNA because the pol III terminator is formed at the border of the 120-bp 5S rRNA coding sequence and NTS. However, as seen for SINE3, the transcription terminator can be inactivated by a few mutations. Alternatively, it can be inactivated by an abnormal 3' processing of the 5S rRNA or by integration of 5S rRNA into some random sites. As a result, the read-through transcription of the 5S rRNA pseudogene can be extended to the 3' termini of the CR1-like element only, forming a proto-SINE3 mRNA (fig. 5d). Given presence of the cognate CR1-like reverse transcriptase, the new SINE3 mRNA can be retrotransposed and give rise to a new SINE family.
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Discussion |
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As reported in this manuscript, SINE3 and zebrafish CR1-like non-LTR retrotransposons share common structural features. Neither CR1-like elements nor SINE3 are flanked by target site duplications, theirs 3' termini are composed of 3-bp to 5-bp microsatellites, and they share common 3' ends. All these features suggest that transpositions of SINE3 depend on the enzymatic machinery encoded by CR1-like elements.
Different classes of transposable elements, including endogenous retroviruses and LTR retrotransposons (Jin and Bennetzen 1989; Kapitonov and Jurka 1999; Witte et al. 2001), "cut and paste" DNA transposons (Fedoroff, Wessler, and Shure 1983; Smit and Riggs 1996; Kapitonov and Jurka 1999; Jurka and Kapitonov 2001), and "rolling-circle" DNA transposons (Kapitonov and Jurka 2001) are composed of autonomous and nonautonomous elements. An autonomous element encodes a complete set of enzymes catalyzing its transpositions, whereas some or all these enzymes are not encoded by a nonautonomous element, which can be mobilized only when the necessary enzymes are provided in trans by the autonomous element. Presumably, the "autonomous-nonautonomous" dyad (the A-N dyad) is a characteristic common to all classes of transposable elements. Consequently, all SINEs can be considered as nonautonomous non-LTR retrotransposons.
Since, as reported in this manuscript, the zebrafish genome harbors nearly 10,000 SINE3 elements, the "mystery" of the lack of SINEs derived from 5S rRNA (Weiner 2002) does not exist anymore. However, given the similar size and expression level of tRNA and 5S rRNA, the observed difference in a diversity of species colonized by SINEs derived from these molecules continues to be puzzling. SINEs derived from tRNA are present in mammals (Daniels and Deininger 1985; Smit and Riggs 1995; Shimamura et al. 1999), vertebrates (Kido et al. 1991; Ohshima et al. 1996; Ogiwara et al. 2002), invertebrates (Ohshima et al. 1993), and plants (Yoshioka et al. 1993; Deragon et al. 1994). However, SINEs derived from 5S rRNA are present in the zebrafish genome only. It is known that transcription of pol III internal promoters can be significantly modulated by DNA signals juxtaposed upstream of the transcribed region (Paule and White 2000). Presumably, the type 1 promoters in 5S rRNAs depend much more on these upstream signals than do type 2 promoters in tRNAs. As a result, the pol III promoter in a retroposed 5S rRNA copy remains silent or is expressed at a low level. If so, it is quite unlikely that 5S rRNA-derived SINE elements will be very common in eukaryotic species.
Although the last explanation is fairly simple and can be easily verified in experiments, there are other interesting biological considerations that may well apply. Efficient retrotransposition of tRNA-derived and 5S rRNA-derived SINEs would be unlikely unless the corresponding RNAs escaped their basic role in translation and avoided being bound by proteins and other RNA molecules interacting with and involved in regulation of functional tRNA/5S rRNA. Therefore, the observed common occurrence of tRNA-derived SINEs and scarcity of 5S rRNA-derived SINES can be explained by a presumption that tRNA molecules are much more capable "escapists and evaders" than 5S rRNA. In other words, tRNA molecules are not bound by as many different translation-related factors as 5S rRNAs, nor are they as tightly regulated in cells as 5S rRNAs. Interestingly, tRNA and 5S rRNA utilize two different pathways for export out of the nucleus into the cytoplasm (Izaurralde and Mattaj 1995; Pasquinelli et al. 1997; Arts et al. 1998; Ullman et al. 1999). It is possible that the tRNA-related pathway can be accidentally evaded more easily than the alternative one. Noteworthy, nucleocytoplasmic transport of 5S rRNA is mediated by L5 ribosomal protein that binds 5S rRNA at positions 27 to 44 (Scripture and Huber 1995). This region is highly modified in SINE3 (fig. 3). Presumably, it was necessary for evading the 5S rRNA nucleocytoplasmic transport.
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Acknowledgements |
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Footnotes |
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