CG13749 looks like a paralog of IMPPP; other species have a longer C-terminus. In addition, in most species and also IMPPP, there are 4 exons with a different N-terminus, like this example in D. elegans: https://www.ncbi.nlm.nih.gov/gene/108149888. Alignments against the 6 D. melanogaster species currently available at NCBI show an insertion in 4/6 of those other assemblies vs Release 6, right near the end of the current CDS. That’s consistent with this gene being a segregating polymorphic pseudogene within Dmel populations. I Spligned NM_136584.2 to CM_010529.1 to figure out the revised sequence. The only difference is the CM sequence has an extra TCGACGTCGG insertion. So the corrected transcript is something like this (also removing the extra intron I mentioned based on the cross-species evidence): >NM_136584.2 Drosophila melanogaster uncharacterized protein (CG13749), mRNA TGACAATGCATTTGCTTGGACTTCTCGTGCCGATCGCTTGCGCTGTTTTGTGCGTTG GTGAAAAATCCCT TTACCATGGTCTCGCCCAAACGGAGACTAATCGACGATTCCATTGCTCCCTCAG CTGCCGATCTCCAGCG GACATACGACGGACATGATGGGCCTCACGTTTTCGGAACACCCGGCAATCAAGTCTACATTCGGGGGCAG AACGATGGCGTATACAAGGTGCCAGGAGTAGGTGGTCAGTTCCATCACGCATCGTCGCCAGCAGATCACG TCTATACGGATGAGCAGGGGATCACGTATGTGCACAAAAAAGACGCTGGAGGACCAGGAACACATACGCT TAGAGGACCAGGTCTAGCTGCTGTACAGCCCGCCTACTCCACTGTGCAGCCTGCGGGACTTCGCAATCCC CAGTTTCATGTGGAGCGAAGTGGTCGCACTGTGGATGTTGGTTCTGGAGGATTTGTCGTCCAAAGGAGCA GACGTAGTCCTCAGTTCCATGTGGAGCGTCCTGGTCGCACTGTGGATGTGGGTTCCGGAGGATTTTTTGT CCAAAGGGGCAGGCGTAGTCCCCAGTTTCCTGTGGAGCGACCTTGGCAAAGGG tcgacgtcgg CTTTGAAGGACTGATTG TTGAAAGGGACAGCCGCAGCATCAAAGATGGCCGCGTCCAGGGAGAAAACTTCGTGGCTCGTGACGATCA AGCAGGAATTTGGGATGATAGTGTGTCGGTGTGGAAGCGTCCGGATGGACGAACTGTCTCGGTGGGCAAT GACGGAAGTGTTATTGTTTCTGGACATGGGCACACGCTGCAATACTAATAAATAATGGGAAGCAGCCTTT TAATTTACAGAGATTAGGTAGCTAAAATTCCAATGATATTAAATGAACAAGATTCAC I ran that through ORFfinder to get the translation: https://www.ncbi.nlm.nih.gov/orffinder/ >lcl|ORF5 MHLLGLLVPIACAVLCVAADLQRTYDGHDGPHVFGTPGNQVYIRGQNDGV YKVPGVGGQFHHASSPADHVYTDEQGITYVHKKDAGGPGTHTLRGPGLAA VQPAYSTVQPAGLRNPQFHVERSGRTVDVGSGGFVVQRSRRSPQFHVERP GRTVDVGSGGFFVQRGRRSPQFPVERPWQRVDVGFEGLIVERDSRSIKDG RVQGENFVARDDQAGIWDDSVSVWKRPDGRTVSVGNDGSVIVSGHGHTLQ Y (251-aa) The revised N-terminus includes a predicted signal peptide mostly encoded in the revised first exon. From FlyBase (LC): Nice detective work! Our policy has been to annotate genes described as polymorphic pseudogenes as mutations in the strain (thus providing the correct CDS) and flagging with a comment that it may be a polymorphic pseudogene. However, I see no evidence in the data presented in GBrowse and JBrowse on FlyBase for the additional intron at the 5' end. I agree, sequence conservation supports it, but usually there would be additional support -- at least some vague evidence in the RNA-Seq. I would be inclined to annotate 2 isoforms, one with the intron and one without. Response from T. Murphy: The splice probably is rare. Here’s a view with the aggregate exon coverage in linear scale: https://www.ncbi.nlm.nih.gov/genome/gdv/browser/genome/?cfg=NCID_1_48358646_130.14.22.10_9146_1587743476_1511586678 There’s a bit of a coverage dip in that region, but lots of signal. So two isoforms with and without the retained intron make sense.