A Dual Origin of the Xist Gene Figure 5. Comparison of the human XIC region with its orthologous sequences in other species. Comparison of the human genomic region containing protein-coding genes CDX4, CHIC1, SLC16A2, RNF12, KIAA2022 (indicated as KIAA22) and ABCB7 that surround the pseudo (y)TSX, XIST, ENOX and FTX genes with its orthologous region in opossum (M. domestica), chicken (G.gallus), frog (X. tropicalis) and four fish species (Danio rerio, Oryzias latipes, Takifugu rubripes and Takifugu nigroviridis) (Table S5). Homologous genes are shown in the same colour. The orthologs of chicken Rasl11c and Uspl genes are detected on chromosome 18 of medaka fish (O. latipes) and on chromosome 7 of zebrafish (D. rerio). In fish, Cdx4, Chic1 and Lnx are not linked with Slc16a2–Rnf12 and Slc25a4 flanks Lnx in T.rubripes and O. latipe. Cdx4, Chic1 and Lnx genes are found at two different locations on D. rerio chromosome 14 and at one location these genes are involved in inversion duplication. Interestingly, two genes, Tex11 (testis expressed gene 11) and Dlg3 (discs, large homolog 3) that have orthologs on the human X chromosome in the region Xq13.1, are located downstream from Kdr (kinase insert domain receptor) in Danio rerio (not shown). doi:10.1371/journal.pone.0002521.g005 inactivation center. Cnbp2 is a retrotransposed protein-coding gene specific for eutherians [4]. Three exons of the protein-coding Tsx gene, which is expressed during spermatogenesis, originated from Fip1l2. The homology of Fip1l2 exon 17 is found in the region located 2 kb from the end of Xist exon 8. We have also found additional exons of this gene in the human sequence (Fig. 2). Two genes, Uspl and Wave4(Wasf3), provided the basis for development of Enox(Jpx) and Ftx, respectively. Both Enox(Jpx) and Ftx, contain exons homologous to those in cognate protein-coding genes Uspl and Wave4(Wasf3), respectively. Additionally Uspl contributed one exon to the promoter region of Enox(Jpx). Presumably, Rasl11c was omitted from the evolutionary process and its remains are located between Xist and Enox (Jpx). Analysis of Enox(Jpx) and Ftx ESTs has revealed overlapping variants of their RNAs (BC345566 (ENOX), BM546361 and CN412172 (FTX)) similar to Xist and Tsix RNAs. It cannot be excluded that this is another pair of regulatory genes, however, their function and possible role in X inactivation is as yet untested. As for the origin of the Xist gene itself, the following scenario of ancestral gene formation seems the most probable. The basis was the Lnx3 gene, containing Pdz and RING finger domains. Lnx3 exons 4, 5, and 11 gave rise to the exons 5, 6 and 7 of the Xist consensus gene (Fig. 2). The Xist promoter region originated from the 59-UTR of Lnx3 exons 1 and 2. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transpos- able elements and thus the Xist gene has originated from pseudogene Lnx3 and a set of various transposons. These mobile elements were integrated into the locus and subsequently their fragments were incorporated into the RNA. Simple tandem repeats A, B, C, D, E, and F contributed essentially to the formation of exons in the ancestral gene; their monomer units presumably originated from endogenous retrovi- ruses as weak similarity was observed in blocks of tandem repeats (Fig. 1). There are some cases of coding sequences entirely or partially derived from ancient transposable elements [13], and it has been reported that over 4.4% of Unigene transcripts contain significant matches to transposable elements within their coding regions [14]. Xist can be classified as an intermediate pseudogene, and represents a new type of ncRNA gene. Our data adds to what is already known regarding Xist evolution by showing that insertion of transposable elements within the ancestral protein- coding gene contributed to the creation of a novel ncRNA gene. In fact, other regions of the XIC have evolved similarly by transposable element insertion, mutating active protein-coding genes into non-coding pseudogenes. Our results constitute important findings into the origin of the unique Xist gene and suggest that acquisition of transposable elements by a protein- coding gene may be a more wide-spread phenomenon contribut- ing to ncRNA gene origin and evolution than previously thought. Species-specific mode of Xist gene evolution Comparative analysis across different species shows that eutherian Xist evolved in a species-specific manner. As reported previously the unique sequence of the Xist gene is not conserved and evolves very rapidly [2,15]. The exon-intron structure of Xist is also not strongly conserved. The interspecific differences in the unique sequence, length and structure of exons suggest that the length of the RNA (and, consequently, the sequences, like mobile elements and tandem repeats, contributing to the RNA size in different species) is either non-essential for function (neutral sequences) or these sequences became selectively adapted in a specific manner to the conditions of functioning in the genome and the X chromosome of particular species. Certain core sequences common for all species are essential for the regulation of gene activity and its function. The functional role of introns in the structure of the XIST/Xist gene is not well understood. The introns in protein-coding genes can separate structural or functional protein domains; however, it is unknown what their roles is in regulatory genes producing nuclear RNAs and how they differ from exons, as neither are protein-coding. Moreover, there are some cases in the evolution of the Xist gene when exons convert into introns and vice versa. We named the exons that are inactivated in some species but preserved in introns as pseudoexons (pEx). Three such pseudoex- ons, which are active in some species and inactivated in others, have been identified (Table 1, fig. 1). PLoS ONE | www.plosone.org 8 June 2008 | Volume 3 | Issue 6 | e2521