BMC Genomics Research article Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene Ping Liang*1, Jayakumar R Nair2, Lei Song1, John J McGuire2 and Bruce J Dolnick*2 BioMed Central Open Access Address: 1Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, USA and 2Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, USA Email: Ping Liang* - Ping.Liang@roswellpark.org; Jayakumar R Nair - R.Jayakumar@RoswellPark.org; Lei Song - Lei.Song@RoswellPark.org; John J McGuire - John.McGuire@RoswellPark.edu; Bruce J Dolnick* - Bruce.Dolnick@RoswellPark.org * Corresponding authors Published: 14 September 2005 BMC Genomics 2005, 6:125 doi:10.1186/1471-2164-6-125 This article is available from: http://www.biomedcentral.com/1471-2164/6/125 Received: 20 April 2005 Accepted: 14 September 2005 © 2005 Liang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. Results: Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N- terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. Conclusion: The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis. Background The rTS (ENOSF1) gene, a member of the enolase family, was initially identified in Homo sapiens by the discovery of an RNA with extensive complementarity to the mRNA for the DNA biosynthetic enzyme thymidylate synthase[1,2]. The rTS gene was later shown to code for two proteins Page 1 of 10 (page number not for citation purposes)