Evolution of serum albumin intron-1 is shaped by a 5′ truncated non-long terminal repeat retrotransposon in western Palearctic water frogs (Neobatrachia)

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Abstract

A 5′ truncated non-LTR CR1-like retrotransposon, named RanaCR1, was identified in the serum albumin intron-1 (SAI-1) of at least seven species of western Palearctic water frogs (WPWF). Based on sequence similarity of the carboxy-terminal region (CTR) of ORF2 and/or the highly conserved 3′ untranslated region (3′ UTR), RanaCR1-like elements occur also in the genome of Xenopus tropicalis and Rana temporaria. Unlike other CR1 elements, RanaCR1 contains a CA microsatellite in its 3′ UTR. The low nucleotide diversity of the 3′ UTR compared to the CTR and to SAI-1 suggests that this region still plays a role in WPWF, either as a structure-stabilizing element, or within a species-specific transcriptional network. Length variation of water frog SAI-1 sequences is caused by deletions that extend in some cases beyond the 5′ or 3′ ends of RanaCR1, probably a result of selection for structural and functional stability of the primary transcript. The impact of RanaCR1 on SAI-1 evolution is also indicated by the significant negative correlation between the length of both SAI-1 and RanaCR1 and the percentage GC content of RanaCR1. Both SAI-1 and RanaCR1 sequences support the sister group relationship of R. perezi and R. saharica, which are placed in the phylogenetic tree at a basal position, the sister clade to other water frog taxa. It also supports the monophyly of the R. lessonae group; of Anatolian water frogs (R. cf. bedriagae), which are not conspecific with R. bedriagae, and of the European ridibunda group. Within the ridibunda clade, Greek frogs are clearly separated, supporting the hypothesis that Balkan water frogs represent a distinct species. Frogs from Atyrau (Kazakhstan), the type locality of R. ridibunda, were heterozygous for a ridibunda and a cf. bedriagae specific allele.

Introduction

Spliceosomal introns are parts of all eukaryote genomes so far investigated. They are generally composed of quasi-random sequences and lack open reading frames (reviewed by Roy and Gilbert, 2006). Introns are excised (‘spliced’) out of RNA transcripts of coding genes prior to protein synthesis and thus are usually considered non-functional and selectively neutral, but conserved sequence motifs, especially around the intron–exon boundaries, indicate that there are functional constraints linked with the spliceosomal machinery. The formation of secondary structures in pre-mRNA may also be functional (reviewed by Buratti and Baralle, 2004).

The mechanisms of intron evolution are largely unknown. Comparative studies using intron sequences of closely and distantly related taxa may help to identify functional and non-functional structural motifs important for understanding these mechanisms. The western Palearctic water frog (WPWF) group includes at least seven closely related species (Plötner, 2005), most of which originated in the upper Miocene and middle Pliocene/early Pleistocene (Beerli et al., 1996, Plötner et al., in press). It thus provides an opportunity to study intron evolution on a ‘fine scale’, within the last 10 million years (My).

Here we present first results of such a comparative study of intron-1 of the serum albumin gene (SAI-1) of WPWFs. We also describe a non-long terminal repeat (non-LTR) retrotransposon that is embedded in the SAI-1 of the water frog species investigated. This non-LTR retrotransposon was classified as a chicken repeat (CR) 1-like long interspersed nuclear element (LINE). CR1 retrotransposons (Stumph et al., 1981) and related non-LTR retroelements are widely distributed in the genomes of vertebrates and invertebrates (e.g. Eickbush, 1994, Haas et al., 2001, Deininger and Batzer, 2002, Deininger et al., 2003, Shedlock, 2006, Kordiš et al., 2006, Novikova et al., 2007, Shedlock et al., 2007; and literature cited therein). They are increasingly used as characters for phylogenetic reconstructions (e.g., John et al., 2005, Kordiš et al., 2006, Shedlock, 2006, Watanabe et al., 2006, Kaiser et al., 2007, Shedlock et al., 2007, Treplin and Tiedemann, 2007). The primary advantage of retroelement insertions for such studies is the high likelihood that, if two genomes share a mobile element at the same locus, the mobile element and insertion are identical by descent (e.g., Deininger and Batzer, 2002).

We compared the structure and base composition of the SAI-1 and CR1-like sequences and discuss the impact that RanaCR1 has had on the evolution of SAI-1 in WPWF. We also used sequences of both SAI-1 and the inserted CR1-like retroelement to test hypotheses on water frog systematics proposed on the basis of protein electrophoretic data (Beerli et al., 1996) and mitochondrial sequences (Plötner, 1998, Plötner, 2005, Plötner and Ohst, 2001, Plötner et al., 2001, Plötner et al., 2007).

Section snippets

Samples

We analysed SAI-1 and RanaCR1 sequences of 24 WPWFs from 21 localities (Table 1) and one eastern Palearctic water frog (an R. nigromaculata from North Korea, EMBL FN432386).

DNA isolation and primer development

Total genomic DNA was extracted from pieces of muscle tissue taken from ethanol preserved specimens by use of a DNA extraction kit (Qiagen, Hilden, Germany) following the standard protocol for preserved tissues. Primers for PCR amplification and sequencing were designed using the serum albumin cDNA of Rana shqiperica. For

Structure of SAI-1 and RanaCR1

Intron-1 of the serum albumin gene in western Palearctic water frogs and the single eastern Palearctic water frog species (R. nigromaculata) examined has typical exon/intron splice site junction sequences (exon-1/GT for the donor site, AG/exon-2 for the acceptor site). An adenine (an essential element for splicing) is located in a pyrimidine rich region 13–14 nt upstream of the acceptor site. The BDGB program (http://www.fruitfly.org/seq_tools/splice.html) predicted several additional splice

Structure of RanaCR1 and its potential impact on SAI-1 evolution

RanaCR1 is the first non-LTR retroelement detected in the genome of neobatrachian anurans. Like most transposable elements (TEs), RanaCR1 is 5′ truncated probably resulting from abortive RNA reverse transcription, when the reverse transcriptase dissociates from its RNA template before having completed cDNA synthesis (e.g., Silva and Burch, 1989, Eickbush, 1994, Burch et al., 1993, Kajikawa et al., 1997, Poulter et al., 1999). As our data show, length variation of TEs may also be caused by

Acknowledgments

For providing water frog samples, we thank Rainer Günther (Berlin), Spartak N. Litvinchuk (St. Petersburg), and Dirk Schmeller (Saint Girons). Tissue samples from Greece were made available by the Greek Ministry of Rural Development and Food, kindly mediated by Doris Tippmann (Embassy of the FRG, Athens). We are grateful to Nils Hof (Berlin) for technical assistance. Two anonymous reviewers provided constructive criticism. This work was supported by the Deutsche Forschungsgemeinschaft (grants

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