uPEPperoni: An online tool for upstream open reading frame location and analysis of transcript conservation

Background: Several small open reading frames located within the 5' untranslated regions of mRNAs have recently been shown to be translated. In humans, about 50% of mRNAs contain at least one upstream open reading frame representing a large resource of coding potential. We propose that some upstream open reading frames encode peptides that are functional and contribute to proteome complexity in humans and other organisms. We use the term uPEPs to describe peptides encoded by upstream open reading frames. Results: We have developed an online tool, termed uPEPperoni, to facilitate the identification of putative bioactive peptides. uPEPperoni detects conserved upstream open reading frames in eukaryotic transcripts by comparing query nucleotide sequences against mRNA sequences within the NCBI RefSeq database. The algorithm first locates the main coding sequence and then searches for open reading frames 5' to the main start codon which are subsequently analysed for conservation. uPEPperoni also determines the substitution frequency for both the upstream open reading frames and the main coding sequence. In addition, the uPEPperoni tool produces sequence identity heatmaps which allow rapid visual inspection of conserved regions in paired mRNAs. Conclusions: uPEPperoni features user-nominated settings including, nucleotide match/mismatch, gap penalties, Ka/Ks ratios and output mode. The heatmap output shows levels of identity between any two sequences and provides easy recognition of conserved regions. Furthermore, this web tool allows comparison of evolutionary pressures acting on the upstream open reading frame against other regions of the mRNA. Additionally, the heatmap web applet can also be used to visualise the degree of conservation in any pair of sequences. uPEPperoni is freely available on an interactive web server at http://upep-scmb. biosci. uq. edu. au.