Research project Regulation of Programmed Stop Codon Readthrough (SCR) in space and time
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. The aim of this project is to gain mechanistic understanding of how cis- and trans-acting factors regulate SCR in a spatio-temporal manner. We address this by using Drosophila as a powerful model for genetic, molecular and structural dissection in combination with analyzing biological function of SCR in vivo.
We recently characterized cis-regulatory elements controlling programmed SCR of the Drosophila POU3F ortholog drifter/ventral veins lacking (dfr/vvl) (Kansara et al, 2026).Phylogenetic prediction identified an mRNA stem-loop in the 3' UTR and mutational analysis showed that stem-loop stability, but not the underlying sequence identity, critically impact dfr SCR. This mRNA stem-loop also promoted SCR in vivo in Drosophila. We further refined computational prediction of SCR-associated mRNA stem-loops and show that these elements effectively promote SCR of heterologous mRNAs.
Ongoing work aims at identifying tissue-specific trans-acting factors involved in regulation of SCR in a spatiotemporal manner. We utilize classical and molecular genetics in Drosophila, in combination with RNA-omics, mass spectrometry, RNA-biochemistry, and structural analyses in cells and in vitro.
Keywords: Stop codon readthrough, Genetic re-coding, Protein translation, Ribosome, POU/Oct factors, mRNA secondary structure.
Programmed SCR is regulated by mRNA sequence and structures. (a) Increased thermodynamic stability, more SCR; decreased stability, less SCR. (b) SCR reporter in vivo; CD8/red =canonical termination; GFP/green = SCR. (c) Constructs with or without stem-loop, stabilized stem (SV2), or no stop (UAG>CAG). (d-g) Expression of the constructs in larval ring glands, (d) control, (e) without stem,(f) stabilized stem and (g) with no stop codon. (h) Quantified SCR levels (GFP/CD8). Kansara et al, 2026