Fig. 10

Translational stop codon readthrough alters the output of Dfr as a transcriptional activator, and modulates hormone-regulated timing of developmental transitions. a Top: SCR of dfr mRNA varies in a spatiotemporal manner with high rates in larval brain and ring gland and low in adult trachea and ejaculatory duct. All these tissues confer high level expression of dfr mRNA (Fig. 3), indicating tissue-specific regulation of SCR. dfr SCR is prominent in brain and ring gland during early stages of larval development, then ceases with larval developmental time (Fig. 4), indicating that SCR is temporally controlled. Middle: Conventional translation of dfr mRNA ends at the first canonical stop codon (Stop), producing Dfr-S with 427 amino acids (Fig. 1). Translational SCR of the same dfr mRNA continues past the first stop codon by incorporation of a glutamine, and ends at the 2nd stop, producing Dfr-L with 713 amino acids (Fig. 1a). A predicted RNA hairpin structure just 3′ of the first stop codon [18] is likely to be involved in the readthrough mechanism. Bottom: The C-terminal extension of Dfr-L alters the transcriptional output and changes expression of genes involved in downstream biological processes (boxed), as shown by RNA-seq analyses of the transcriptome of dfr¹⁴ mutant larvae vs controls (Fig. 5). Loss of dfr SCR as, in dfr¹⁴ mutant larvae, negatively affects steroidogenesis. b, c Model of dfr SCR-dependent modulation of ecdysone-regulated developmental transitions. b Normal expression of the ecdysone biosynthetic pathway genes requires both Dfr-S, Dfr-L and Mld (Fig. 6). Dfr-L and Mld activate nvd and spok expression in a synergistic manner (Fig. 9), contributing to high ecdysone titers. This ensures correct timing of developmental progression from larval to adult stages. c Elimination of dfr SCR and production of Dfr-L (e.g., in the dfr¹⁴ mutant) compromises ecdysone biosynthesis, causing prolonged larval development, delayed pupariation, and increased adult size (Figs. 4 and 6). Thus, dfr SCR modulates hormone-regulated timing of developmental transitions