J Mol Evol (2006) 62:630–644 DOI: 10.1007/s00239-005-0201-5 Sauria SINEs: Novel Short Interspersed Retroposable Elements That Are Widespread in Reptile Genomes Oliver Piskurek,1 Christopher C. Austin,2 Norihiro Okada1 1 Faculty of Bioscience and Biotechnology, Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan 2 Museum of Natural Science, Louisiana State University, Baton Rouge, LA 70803, USA Received: 20 August 2005 / Accepted: 16 December 2005 [Reviewing Editor: Dr. Martin Kreitman] Abstract. SINEs are short interspersed retrotrans- ubiquity of Bov-B LINEs previously demonstrated in posable elements that invade new genomic sites. squamate genomes, suggests that these LINEs have Their retrotransposition depends on reverse trans- been an active partner of Sauria SINEs since this criptase and endonuclease activities encoded by SINE family was generated more than 200 million partner LINEs (long interspersed elements). Recent years ago. genomic research has demonstrated that retroposons account for at least 40% of the human genome. Key words: Sauria SINE — Bov-B LINE — tRNA Hitherto, more than 30 families of SINEs have been — Lizards — Snakes — Varanus — Squamata gen- characterized in mammalian genomes, comprising ome evolution 4600 extant species; the distribution and extent of SINEs in reptilian genomes, however, are poorly documented. With more than 7400 species of lizards and snakes, Squamata constitutes the largest and most diverse group of living reptiles. We have dis- Introduction covered and characterized a novel SINE family, Sa- uria SINEs, whose members are widely distributed SINEs (short interspersed elements) are nonviral among genomes of lizards, snakes, and tuataras. Sa- retrotransposable repetitive sequences with a length uria SINEs comprise a 5¢ tRNA-related region, a of 70–500 bp that are widespread among eukaryotic tRNA-unrelated region, and a 3¢ tail region (con- genomes (Weiner et al. 1986; Okada 1991; Schmid taining short tandem repeats) derived from LINEs. and Maraia 1992; Deininger and Batzer 1993). While We distinguished eight Sauria SINE subfamilies in some SINEs are derived from 7SL RNA (Ullu and genomes of four major squamate lineages and Tschudi 1984) or 5S rRNA (Kapitonov and Jurka investigated their evolutionary relationships. Our 2003), most SINEs are derived from tRNA (for a list data illustrate the overall efficacy of Sauria SINEs as of references see Ohshima and Okada 2005). Hence, novel retrotransposable markers for elucidation of the tRNA-like secondary structure as well as the squamate evolutionary history. We show that all conserved RNA polymerase III–specific internal Sauria SINEs share an identical 3¢ sequence with promoter sequences (designated A and B boxes) al- Bov-B LINEs and propose that they utilize the lows new SINE elements to be distinguished from enzymatic machinery of Bov-B LINEs for their own other repetitive elements in the genome. SINEs can retrotransposition. This finding, along with the amplify nonautonomously by a copy-and-paste mechanism, in which the initial amplification of a Correspondence to: Norihiro Okada; email: [email protected]. SINE at the parent locus is followed by integration of ac.jp a SINE copy at the genomic target site. This retro- 631 transposition of SINEs is dependent on autonomous Myr were addressed by several other investigators as partner LINEs (long interspersed elements) that en- well (Hamada et al. 1998; Hillis 1999; Miyamoto code reverse transcriptase (RT) and endonuclease 1999; Okada et al. 2004; Shedlock et al. 2004). (EN) for their own amplification (Eickbush 1992; Mammalian orders have received special attention Luan et al. 1993). Since most LINE/SINE pairs share with respect to the detection and characterization of an identical 3¢ tail sequence in eukaryotic genomes novel SINE families. To date, tRNA-derived SINE (Ohshima et al. 1996; Okada et al. 1997), RTs en- families are one of the most abundant genomic coded by LINE partners recognize the matching 3¢ components in species of all four major placental tails of SINEs and thus initiate SINE replication via mammalian clades proposed by Murphy et al. (2001). retrotransposition in trans. This mechanism of SINE They are present in genomes of laurasiatherians, amplification was demonstrated experimentally by which include carnivores, cetartiodactyls, chiropter- our group for active LINE/SINE pairs in the eel ans, eulipothyphlans, perissodactyls, and pholydo- genome (Kajikawa and Okada 2002; Kajikawa et al. tans (e.g., see Shimamura et al. 1997, 1999; Nikaido 2005). Okada et al. (1997) proposed that LINEs can et al. 1999; Kawai et al. 2002), in genomes of the be divided into two groups, the stringent type and the Euarchontoglires, namely, primates, dermopterans, relaxed type, depending on the relative specificity of scandentians, rodents, and lagomorphs (Cheng et al. recognition of their own RT 3¢ end. Most LINE 1984; Britten et al. 1988; Schmid 1996; Nishihara family RTs strictly recognize a specific sequence at et al. 2002; Schmitz and Zischler 2003; Piskurek et al. their 3¢ tail, whereas in the mammalian LINE family, 2003; Ray et al. 2005), as well as in genomes of the L1, no such 3¢ end-specific region (except the poly[A] afrotherian clade (Nikaido et al. 2003; Nishihara tail) is needed for RT recognition (Moran et al. 1996). et al. 2005) and in genomes of xenarthrans (Churakov The L1-encoded RT-dependent retrotranspositional et al. 2005). mechanism in trans is also believed to be the driving In contrast, very little progress in SINE research force of Alu-SINE amplification in primates (Jurka has been made for reptile genomes, although the 1997; Esnault et al. 2000; Ohshima et al. 2003) and Sauropsida represent the sister group of mammals. CYNt-SINEamplification in flying lemurs (Piskurek Whereas there are approximately 4600 species of et. al. 2003; Piskurek and Okada 2005). Retrotrans- mammals, more than 16,000 extant species of birds, posed CYNt-SINEsin flying lemurs are composed crocodiles, lizards, snakes, and turtles are known. exclusively of tRNA-related regions, along with a Hitherto, the only example of reptile SINEs that were poly(A) tail that is proposed to serve as a recognition characterized and applied as molecular markers to site for L1 RTs. Recently, Churakov et al. (2005) infer reptilian phylogeny of one turtle family (Batag- characterized tRNA-related mobile elements with uridae) is the tortoise polIII/SINE in hidden-necked poly(A) tails in the genome of armadillos and pro- turtles, the discovery of which dates back about 20 posed that DAS elements recruited the enzymatic years (Endoh and Okada 1986; Endoh et al. 1990; machinery of L1 LINEs as well. Ohshima et al. 1996; Sasaki et al. 2004). While the The retroposition site of offspring copies is almost Bataguridae represent the major group of turtles random, although a slight sequence preference for the (about 60 extant species), squamate reptiles, with target site has been reported (Jurka 1997). Also, the more than 7400 extant species of lizards and snakes, copy-and-paste mechanism of retrotransposition is are the largest and most diverse group of living rep- intrinsically unidirectional. In addition, no mecha- tiles (Zug et al. 2001). The order Sphenodontia rep- nism is known for the precise removal of SINEs from resents the sister group of the Squamata and consists any genome (Shedlock and Okada 2000; Batzer and of only two surviving species of tuatara (genus Deininger 2002). Thus, in phylogenetic studies the Sphenodon) from New Zealand (Zardoya and Meyer insertion of a SINE at a specific genomic location 1998; Rieppel and Reiz 1999; Rest et al. 2003). represents the derived character state for all species Sphenodontia and Squamata together form the Le- that share a SINE at an orthologous genome site. In pidosauria. contrast, the ancestral state is the absence of a SINE In this study we describe a novel tRNA-derived at a particular genomic location. Because of these SINE family that is widely distributed among lepi- characteristics, SINEs are thought to be homoplasy- dosaurian genomes. We first discovered mobile ele- free molecular markers for evolutionary studies ments in the genome of the common wall lizards (Okada et al. 1997; Shedlock and Okada 2000). In the (Podarcis muralis, suborder Sauria) and subsequently last decade, a growing body of literature has dem- characterized tRNA-derived SINEs of the same onstrated that SINEs are extremely effective markers family in two additional major lineages of lizards and for elucidating evolutionary history (Murata et al. in the genome of snakes. We designate this new SINE 1993; Takahashi et al. 1998; Nikaido et al. 1999; family as Sauria SINE. We examine and discuss Sa- Schmitz et al. 2001; Terai et al. 2004; Ray et al. 2005). uria SINE evolution, such as the genealogy of certain Their limitations for divergence times up to 150–200 SINE subfamilies in genomes of lizards and snakes, a 632 possible secondary structure for the tRNA-like 5¢ sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied end, and we discuss the short 3¢ sequence that origi- Biosystems), and partial SINE regions of additional Squamata nated from the Bov-B LINE. Furthermore, we give species were aligned with SINEs already identified in the common wall lizard. Subsequently, universal SINE primers were designed an example of how to use the Sauria SINE family as a for Sauria SINEs in lizards and snakes (SQ1F, CCCWG marker for the evolution of monitor