Fig. 1

Translational readthrough of dfr mRNA produces two alternative Dfr isoforms. a Schematic representation of the intronless dfr/vvl gene. The dfr open reading frame 1 (ORF1, 427 amino acids, red), ending at the first UAG stop codon, is followed directly by a second frame (ORF2, 286 amino acids, yellow), flanked by 5′ and 3′ untranslated regions (gray). Translational readthrough of the UAG produces Dfr-L containing a C-terminal extension. The following two stop codons have also been predicted to undergo stop codon readthrough extending into ORFs 3 and 4 [17]. Two independent Dfr antibodies were used, one recognizing the common part of Dfr-S and Dfr-L (ORF1; anti-Dfr-S/L) [29] and another (anti-Dfr-L), specific for the C-terminal extension (ORF2). b Immunoblots on embryo, adult, and larval extracts, using anti-Dfr-S/L. c Temporal RNAi-mediated knockdown of dfr in larvae for 3 days using Act-Gal4[ts] crossed to UAS-dfr-RNAi, compared to control (driver only). Bars represent means+SE (n = 4). d Schematic illustration of expression and isolation of Dfr-L followed by mass spectrometry. An in-frame Dfr-L-Myc fusion construct was transfected into S2 cells and the SCR product was isolated by pull-down using anti-Myc, followed by separation by electrophoresis. Digestion was performed with chymotrypsin prior to analysis by nano liquid chromatography-mass spectrometry (nLC-MS/MS). e Immunoblots using protein extracts from S2 cells, untreated or transfected with dfr plasmids, with or without a C-terminal Myc-tag. Left panel, anti-Dfr-S/L; right panel, anti-Myc. Note that S2 cells do not express Dfr endogenously. f nLC-MS/MS and MASCOT analysis returned 15 peptides matching Dfr, of which 4 in ORF2. The stop codon was interpreted as a glutamine codon (highlighted in blue; see Additional file 2 for more details). MASCOT analysis against the Dfr-L-Myc fusion protein additionally returned peptides matching the Myc peptide (green) and the Dfr-L-Myc fusion