BMC Genomics 2008, 9:122 http://www.biomedcentral.com/1471-2164/9/122 sequence unaffected (see Methods for an explicit exam- ple), denoted here Nstop. Furthermore, we estimated a probability of seeing Nstop given the parent amino acid sequence, and considering codon bias (see Methods), but not taking into account the effects of bi-codon bias men- tioned above (see 'Open reading frame translation' sec- tion). To gain clues as to the possible function of matreshkas, all matreshka protein sequences were analysed with func- tional motif and signal peptide prediction programs. Matreshka nucleotide sequences were also screened for tandem repeats, to exclude artifactual sequences which pass the length filter (e.g., if the repeat contains no stop codon in the matreshka frame). Matreshkas with tandem repeats in any one of the three organisms were discarded from in-depth analysis, even though Alex itself was found to contain such repeats. In addition, matreshkaM nucle- otide sequences were scanned for consensus Kozak trans- lation initiation motifs [14,15], to help determine whether leaky ribosomal scanning [16] may be occuring. To estimate how frequently Kozak motifs appear by chance, we scanned the 9163 human coding sequences used for building matreshkas, and counted how fre- quently ATG occurred versus full Kozak sequences (all frames included). On average, ~30 ATGs, of which ~5 (16%) were Kozak motifs, were observed per transcript. Of the 1793 matreshkaM, only 217 (12%) have a Kozak motif exactly at the start codon (data in Additional file 3). However, of the 15 longest matreshkaM, 8 have a Kozak motif exactly at the start codon, which is a significant enrichment of true positives (exact binomial test, N = 15, P = 0.0001). The matreshka Nstop versus length distribution can be seen in Fig. 4, with outliers of interest highlighted (data availa- ble in Additional file 3). Details on the highlighted matreshkas can be found in Table 3. Some matreshkas can be extended into 5' or 3' untranslated regions (UTRs) or (in the case of KCNK12 or POLG) into flanking genomic sequence (in total, 365 matreshkas extend 5' past the boundary of the parent RefSeq). Of particular interest is the THPO matreshka, which is of the rare methionine- start, frame -1 variety. The matreshkas in Table 3 which don't begin with methio- nine are unlikely to represent complete proteins, since the human matreshka sequence either contains no methionines (KCNK12, GPR135, POLG, FOXA2) or methionines only very close to the 3' end of the matreshka (GPR27, TAF6). As with Alex and virtually all matreshka predictions, none of the candidates in Table 3 contain known protein functional motifs, as ascertained by pat- tern matching to Prosite motifs. The following sections describe matreshkas of particular interest. Case study 1: Matreshka framing super conserved receptor expressed in brain 1 (GPR27) The first example presented here is of a particularly large matreshka associated with the GPR27 transcript, also called super conserved receptor expressed in brain 1. GPR27 is an aminergic G-protein coupled receptor, cloned from human brain cDNA and part of a family of three members which are highly conserved between verte- brates [17]. This single-exon coding sequence is entirely contained within a frame -1 matreshkaX, which begins and ends in the flanking UTRs (Fig. 5A). The matreshkaX varies in length between 407 and 418 amino acids depending on the species, and in the RefSeq coding region alone has between 53 (mouse) and 60 (human) positions where a stop codon could have truncated the matreshkaX, suggesting selection pressure to maintain it. the probabil- ity in human of a stop codon truncating the matreshka, Pstop, was estimated at 0.999997. It seems possible from our analysis that the high level of conservation of this gene may be necessary to maintain the protein sequences of both GPR27 and the matreshka. Figure 5B shows the matreshkaX amino acid sequence, which like Alex is basic (predicted pI 12.9) and enriched in proline residues (~3- fold more than average). The GPR27 matreshka is unlikely to be translated from a leaky scanning event [16], since it contains only one methionine residue 30 amino acids from its C-terminus. It may be part of the exon of a longer, unknown transcript, in which case the AG strad- dling its 5' end could be a canonical splice signal. Case study 2: Matreshka contained in thrombopietin (THPO) Thrombopoietin is the major regulator of platelet produc- tion by megakaryocytes, with associated disorders includ- ing thrombocytosis and thrombocytopenia. Splice variant 1 of THPO contains a matreshka of the scarce frame -1 methionine-start type, which in human is 254 amino acids long, and covers 72% of the length of THPO (Fig. 6A). It contains 55 positions where a stop codon could have occurred without changing the parent RefSeq protein sequence (Pstop = 0.999999). For comparison, in frame +1 there are 30 positions where a stop codon could occur, and 12 stop codons actually did. Like Alex, the THPO matreshka is proline-rich (Fig. 6B). The genomic region upstream of the matreshkaM start was computationally scanned for transcription start sites (TSS), in case it is in fact derived from a separate transcript overlapping with the THPO sequence: none was found. The matreshkaM start is 212 nucleotides downstream from the parent start, and the initiator ATG lies in a strong Kozak context (Fig. 6A) in all three organisms. This sug- gests that the matreshka could be produced from a leaky ribosomal scanning event [16]. 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