BMC Genomics 2008, 9:61 http://www.biomedcentral.com/1471-2164/9/61 Table 3: Gene Ontology term and InterPro domain overrepresentation in uORF dataset as determined by Genemerge GO term or Interpro reference number GO term or Interpro domain Genome frequency Frequency in uORF dataset Bonferroni corrected P- value GO:0008170 (MF) GO:0045039 (BP) IPR002296 IPR000241 IPR004217 N-methyltransferase activity protein import into mitochondrial inner membrane N6 adenine-specific DNA methyltransferase, N12 class Putative RNA methylase Zinc finger, Tim10/DDP-type 10/14601 6/14601 4/14040 3/14040 5/14040 MF, molecular function; BP, biological process 1 GO term or Interpro domain observed in CG9666 and CG9960 2 GO term or Interpro domain observed in CG9878 (Tim10) and CG17767 (Tim9b) 2/481 2/482 2/481 2/481 2/482 0.015 0.008 0.004 0.002 0.006 In support of a model in which mitochondrial proteins preferentially associate with other ORFs on a single tran- script, a further connection to the mitochondrial inner membrane is found when examining other genes in the uORF dataset. The CG8026 mORF encodes a putative mitochondrial folate transport protein [37,38] (Table 4). Interestingly, this trend may extend to the mammalian lineage, exemplified by the human Uncoupling protein 2 (UCP2) mORF, a putative inner mitochondrial mem- brane transporter. The UCP2 mORF is not only associated with what appears to be a CPuORF, but it is regulated by its uORF in a glutamine-dependent manner [39]. B-cell lymphoma 2 (BCL-2) is another mammalian oncogene that produces a protein from its mORF, BCL-2, which is local- ized to mitochondria [40] and is associated with a func- tional uORF [15]. Other Drosophila genes also have potential links to the mitochondrion, such as CG18624, a putative NADH dehydrogenase that is predicted to act in mitochondrial electron transport (Table 4). Also, uORF1 of CG9865 is a putative homolog of p8Mature T-Cell Proliferation 1 (p8MTCP1), an ORF that is transcribed on the same mRNA as p13MTCP1, is targeted to mitochondria [41], and may play a role in oncogenesis [42,43]. CG9865 uORF1 has a twin CX9C motif, as do p8MTCP1 and other proteins targeted to mitochondria, namely yeast proteins Mitochondrial Ribosomal Protein 10 (Mrp10p) [44], Cytochrome Oxidase 19 (Cox19p) [45], Cytochrome Oxi- dase 17 (Cox17p) [46], and Mitochondrial intermem- brane space Import and Assembly 40 (Mia40p) [47]. In humans, the twin CX9C motif found in Mia40p is required for import and stable accumulation of Mia40 in the intermembrane space [48]. Several genes in the uORF dataset contain ORFs with CX9C motifs, such as uORFs 1 and 3 of CG9865, the uORFs of CG30290 and CG9288, and the mORF of CG7950 (See Additional File 2). These open reading frames could be interacting with other ORFs on the same transcript to target them to the mitochondria or to form a stabilizing protein complex. It is possible that these ORF associations are vestiges of ancient prokaryotic operons that originated in the mito- chondrion and were transferred to the nuclear genome over time. This hypothesis runs counter to the prevailing thought that mitochondrial proteins involved in transport are generally of eukayotic origin [49]. Regardless of their origin, nuclear ORFs coding for mitochondrial proteins may maintain an association with other ORFs on a single transcript over long periods of evolutionary time for sev- eral reasons. Both ORFs may be co-regulated at the tran- scriptional level and be required at similar times in development, thus providing more efficient transcription of DNA. Alternatively, the uORF may be regulating expres- sion of the mORF with important biological conse- quences. These possibilities are not mutually exclusive and further experimentation will be required to determine whether this energy-producing organelle is influenced by the translational regulation of uORF-mORF pairs on sin- gle transcripts. Interestingly, the trend in animal mitochondrial ORFs was not observed in plants. Instead, plant uORFs tend to associate with mORFs encoding transcription factors [19]. Perhaps these unique characteristics reflect fundamental differences in the two eukaryotic lineages. Despite their differences, plants and animals both seem to contain uORF-mORF pairs involved in a wide range of biochemi- cal and regulatory pathways (Table 4). There is some evi- dence in the literature that transcripts with uORFs can occur in similar biochemical pathways, such as genes affecting the polyamine biochemical pathway [50], but this is the exception rather than the rule and no additional examples have been born out by our analyses. To facilitate future studies of these elements, all CPuORF annotations will be submitted to Flybase. Conclusion The identification and characterization of putative CPuORFs has established a knowledge base from which many hypotheses have been generated and can now be Page 8 of 14 (page number not for citation purposes)