The mRNA architecture of the termination site primes programmed stop codon readthrough events in Drosophila

Abstract

Programmed stop codon readthrough (SCR) is a form of genetic re-coding, in which a near-cognate tRNA base-pairs with a stop codon, leading to the translation of a C-terminally extended protein. Recent studies revealed that SCR represents an evolutionarily conserved, spatio-temporally controlled mechanism of posttranscriptional gene regulation that requires cis-regulatory elements as well as trans-acting factors. In this study, we characterized cis-regulatory elements controlling programmed SCR of the Drosophila POU3-family member drifter/ventral veins lacking (dfr/vvl). Using S2 cell-based luciferase assays, we show that stop codon identity and the +4 to +9 nucleotide sequence are required but not sufficient for dfr SCR regulation. Phylogenetic prediction identified an mRNA stem-loop in the 3 UTR, proximal to the readthrough UAG codon. Mutational analysis revealed that the distance from the stop codon as well as stem-loop stability, but not the underlying sequence identity, critically impact dfr SCR. Similarly, the mRNA stem-loop promoted SCR in an in vivo Drosophila model. We applied this information to refine computational prediction of SCR-associated mRNA stem-loops and show that these elements effectively promote SCR of heterologous mRNAs. These findings increase our understanding of SCR and the underlying regulatory mechanisms. Key PointsO_LIStop codon readthrough is regulated both by mRNA sequence identity and mRNA structure elements. C_LIO_LIA 3 mRNA stem-loop and its thermodynamic stability determine stop codon readthrough efficiency. C_LIO_LImRNA stem-loops are frequently found in Drosophila genes that exhibit stop codon readthrough. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/685291v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@dbccf6org.highwire.dtl.DTLVardef@6d53f3org.highwire.dtl.DTLVardef@1a27deborg.highwire.dtl.DTLVardef@f8e5ae_HPS_FORMAT_FIGEXP M_FIG C_FIG