A B C D genomic DNA Transcripts MASK-BP3arf fusion transcript BMC Genomics 2008, 9:122 http://www.biomedcentral.com/1471-2164/9/122 [3]. Expression profiling of IGF-1 and MGF indicates that the variants have distinct physiological roles. The best-characterised case of overlapping tORFs in mam- mals is that of XL-alpha-s. This is a splice variant of a G protein alpha subunit, derived from the GNAS complex locus, which is expressed in neuroendocrine tissues and other tissues. The first exon of XL-alpha-s contains a downstream ORF which is frameshifted +1 relative to the XL-alpha-s initiator codon. This ORF gives rise to an entirely different protein called Alex, which is 356 amino acids long in rat [4]. Remarkably, XL-alpha-s and Alex interact, and this interaction can be disrupted by an inser- tion polymorphism in humans. The polymorphism leads to enhanced receptor-mediated cAMP formation in plate- lets and fibroblasts, increased trauma-related bleeding tendency, and in two families neurological problems and brachydactyly were observed [5]. Furthermore, the XL- alpha-s and Alex ORFs may extend in the 5' direction for several hundred nucleotides more [6], raising the possibil- ity that longer variants of XL-alpha-s and Alex exist. Although the Alex termination codon lies well within 50 base pairs (bp) of the next 3' splice junction, the XL-alpha- s transcript does not appear to be degraded according to the usual rules for nonsense-mediated decay [7]. Figure 1 summarises these cases of overlapping mammalian tORFs. During our own in silico comparative studies of entire translated human, mouse and rat genomes, we frequently observed overlapping tORFs conserved at the amino acid level. In an effort to explore this relatively uncharacterised aspect of gene structure and evolution, we screened for additional Alex-like cases in human and rodents using a bioinformatics approach. Specifically, we searched human, mouse and rat transcripts for frameshifted con- ARF MASK INK4A 317 intermediate exon EIF4EBP3 IGF-1Ea IGF-1Ec (MGF) XLαs Alex, 1068 26 172 FKingouwrne e1xamples of overlapping translated ORFs Known examples of overlapping translated ORFs. A) The second exon of the INK4A and ARF genes is shared but trans- lated in different reading frames. B) A transcript fusion can occur between the MASK and EIF4EBP3 genes, via an intermediate exon. In this case, the next exon is translated in a different frame from that in EIF4EBP3 transcripts. C) A splice variation in IGF-1 can cause a frameshift in the translation of C-terminal residues. D) Exon 1 of the G-protein subunit XL-alpha-s contains a second, frameshifted tRF which yields a distinct protein called Alex. Grey shading indicates overlapping frameshifted ORFs, exons and tORFs are drawn approximately to scale. Page 2 of 16 (page number not for citation purposes)