Bousios et al. BMC Genomics 2010, 11:89 http://www.biomedcentral.com/1471-2164/11/89 of the RM cluster precisely define the borders of the CpG islands, which by itself supports the hypothesis that they may have a role in the Sirevirus regulatory net- work. Excluding one very long island in the ENV region of Usier that continues far beyond the last RM, the aver- age 400 bp Sirevirus CpG island starts and finishes approximately 45 bp upstream and downstream of a RM unit, respectively. In contrast to the positional stabi- lity of the Sirevirus CpG islands that imply their func- tionality, most classic elements do not contain a similar pattern (not shown). Only seven have LTR-derived islands, whilst nine lack such a domain and 14 have irregular conformations where CpG islands are present in varied non-LTR locations. A conserved TATA box lies shortly after the CpG island in the diverse Sirevirus LTR Another feature that differentiates Sireviruses from clas- sic retrotransposon genomes is the presence of a con- served TATA box motif in the Sirevirus LTRs. The promoter element 5’-cTATAA/TAT/AAg-3’, where lower case letters represent less critical bases [31], resides in all but two elements (Tnd-1 and ATCOPIA43) (Figure 5a) and is approximately located in the middle of each LTR (average nucleotide identity 76%). Intriguingly, in the RM-containing Sireviruses the TATA box occurs on average 37 bp after the CpG island, which suggests that the motif corresponds to an authentic promoter and the proximal RM units may regulate its activation (Figure 4b and Figure S4 in Additional file 2). Conservation of the Sirevirus 5’LTR/internal domain junction The second most popular sequence motif of the Sire- virus genome according to TEIRESIAS is the 5’LTR/ internal domain junction that accommodates the PBS. The highly conserved upstream junction is divided in three subdomains that in the 5’ to 3’ direction consists Page 8 of 14 of the universal CA end of the 5’LTR, the intervening sequence and the PBS (Figure 5b). The Sirevirus PBS is only 9 bp (5’-TATCAGAGC-3’), complements the 3’ end of the met tRNA, which is therefore the first primer during reverse transcription. The sharp decrease in sequence conservation after the ninemer implies that Sireviruses paradoxically use only a short segment of the tRNA molecule to prime cDNA synthesis. The interven- ing sequence is unusually long (5’-ATTGG-3’) and in six elements a G replaces the T at the second position. Consequently, the Sirevirus upstream attachment site is the heptamer 5’-CA/ATTGG-3’, whilst over the whole length of the 16 bp region the genus maintains 92.7% identity. In contrast, classic elements contain a more variable 5’LTR/PBS junction, although and due to the common met tRNA-derived PBS in many LTR retrotran- sposons [32], TEIRESIAS discovered a semi-conserved sequence motif in some elements (data not shown). A polyC-rich integrase singal is located at the 3’ end of the Sirevirus LTRs The successful integration of a new retrotransposon copy in the host genome is highly dependent on the interactions between INT and the attachment sites of the element [26]. However, research on retroviruses has shown that binding specificity of INT to the viral DNA is achieved through additional interactions that extend at least 25 bp inwards at the 3’ end of the LTR in a domain that is called the ‘integrase signal’ [12,33]. TEIR- ESIAS identified a conserved motif in this location in 17 Sireviruses. The putative Sirevirus integrase signal is approximately 19 bp long (nucleotide identity 88.2%) and contains a distinctive C-rich region in the central domain that varies in size (Figure 5c). The average dis- tance between the signal and the LTR end is 21 bp. To our knowledge this is the first time such a motif, which also characterizes a whole genus, is reported in the gen- ome of LTR retrotransposons. Figure 5 Alignment of the Sirevirus conserved TATA box region (A), 5’LTR/internal domain junction (B), and polyC-rich integrase signal (C).