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Stop codon readthrough of a POU transcription factor regulates steroidogenesis and developmental transitions

ABSTRACT: Translational stop codon readthrough generates C-terminally extended protein isoforms. While evidence mounts of readthrough as a global phenomenon, proofs of its functional consequences are scarce. We show that readthrough of the Drosophila POU/Oct transcription factor gene drifter (dfr) occurs at a high rate and in a spatiotemporal manner in vivo, reaching more than 50% in the larval prothoracic gland. Phylogenetic analyses suggested that readthrough of dfr is conserved among Dipterans, with C-terminal extensions harboring intrinsically disordered regions and amino acids streches implied in transcriptional activation. The extended Dfr isoform is required for maintaining normal levels of the growth hormone ecdysone through regulation of its biosynthetic genes, acting in synergy with the transcription factor Molting defective (Mld). A 14-bp deletion that abolished readthrough, caused prolonged larval development and delayed metamorphosis. This study provides a striking example of alternative genetic decoding that feeds into the progression from one life cycle stage to another.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE149972 | GEO | 2020/08/01

REPOSITORIES: GEO

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Project description:Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3’-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features important for translation termination and readthrough.

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Stop codon readthrough of a POU transcription factor regulates steroidogenesis and developmental transitions

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