BMC Genomics 2008, 9:61 http://www.biomedcentral.com/1471-2164/9/61 Table 4: Predicted function and biological processes of uORF-mORF pairs in Drosophila (Continued) CG11989 CG7869 CG7628 CG9666 CG2128 CG9288 CG9924 CG5116 CG14550 CG7950 CG31241 CG31178 CG7071/CG34131c CG10238 Ard1b SuURb Hdac3b Rdxb DTLb MOCS2b Putative N-Acetyltransferase catalytic subunit DNA binding Phosphate transporter Putative methyltransferase Histone deacetylase Pyruvate kinase Unknown Putative RNA methylase Unknown Unknown Molybdopterin synthase large subunit (mORF) and small subunit (uORF) Putative GTP-binding protein Putative phosphatidylinositol N- acetylglucosaminyltransferase subunit P (mORF); Pcc1-like transcription factor (uORF) Putative tRNA processing enzyme subunit (uORF) Unknown [89]; Interpro domain Endoreplication [90, 91] Phosphate transport Interpro domain Unknown Interpro domain Wing development; [92, 93] Chromatin remodeling Unknown Interpro domain Regulator of Hedgehog [94, 95] response (growth and development) Late larval development [24]; Interpro domain Unknown Unknown Production of molybdopterin; [23, 96] Implicated in mammalian neurological damage Unknown Interpro domain Unknown Interpro domains tRNA processing (uORF) Interpro domain a refers to mORF unless otherwise noted bfend, Forked end; Tim9b, Translocase of inner membrane 9b; Rad1, Radiation insensitive 1; lmg, Lemming;Fatp, Fatty acid transport protein; LIG, Leucine-Rich Repeat and Immunoglobulin-containing protein (MacLaren et al, 2004); BicC, Bicaudal C; DmSNAP50/DmPBP49, snRNA activator protein 50/Proximal Sequence Element-Binding Protein 49;Galpha49B, G-protein alpha49B; Pepck, Phosphoenolpyruvate carboxykinase; Gint3, GDI interacting protein 3; Tim10, Translocase of inner membrane; SuUR, Suppressor of underreplication; Ard1, Arrest defective 1; Hdac3, Histone deacetylase 3; Rdx, Roadkill (Kent et al, 2006); DTL, Drosophila Tat-like; MOCS2, molybdopterin synthase 2 tested. CPuORFs in dipterans show similarities to their plant counterparts in terms of their prevalence within the genome and diversity of sequence, but differ in their greater average length, their genome clustering, and their preferential association with methyltransferases. In addi- tion, the present analysis has shown a significant correla- tion between mitochondrially-targeted proteins and transcripts containing uORFs, an observation that could lead to important discoveries impacting our understand- ing of human disease. Given the wealth of genetic tools available in Drosophila, this model system is ideally suited to the basic understanding of uORF-containing transcripts and post-transcriptional regulation. Methods Identification of conserved peptide uORFs Drosophila melanogaster transcript sequences, release 4.3 (19,389 sequences) were downloaded from Flybase [51], Anopheles gambiae transcript sequences, build 3.4 (14,127 sequences) were downloaded from Ensembl [52], and dipteran expressed sequence tags (ESTs) (666,153) were downloaded from NCBI [53] December 15, 2006. Because the melanogaster group members (includes D. simulans, D. yakuba, D. erecta, and D. ananassae) diverged from D. melanogaster relatively recently [28,29], their tran- script sequences are of limited use in detecting strong neg- ative selection over short sequence lengths due to the accumulation of few synonymous and non-synonymous substitutions. Therefore these species were excluded from this first comparison, as were D. melanogaster ESTs. Comparative analysis of D. melanogaster and A. gambiae sequences was performed using uORF-Finder [19], a pro- gram that identifies the longest open reading frame of a transcript in the first species (defined as the mORF), finds the putative homolog in the second species, and aligns all open reading frames upstream of these homologs to iden- tify putatively conserved uORFs. uORF-Finder was designed to compare full-length cDNA sequences from two species, therefore to accommodate a D. melanogaster full-length transcript-to-dipteran EST comparison, the program was modified and putative homologs in the ESTs were identified using the first 100 amino acids of the D. melanogaster mORFs. uORF size was also limited to 200 amino acids (no additional uORFs were found when uORF size was limited to 300 a.a.). The presence of putative CPuORFs was established in at least three different species by either extracting the first 100 amino acids of the D. melanogaster mORF sequence and searching the NCBI EST database using tBLASTn for putative homologs with conserved uORF sequences, or by scanning the UCSC D. melanogaster genome browser and inspecting other Drosophila genomes for conservation of Page 10 of 14 (page number not for citation purposes)