Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development

Abstract

Background A number of cellular processes have evolved in metazoans that increase the proteome repertoire in relation to the genome, such as alternative splicing and translation recoding. Another such process, translational stop codon readthrough (SCR), generates C-terminally extended protein isoforms in many eukaryotes, including yeast, plants, insects, and humans. While comparative genome analyses have predicted the existence of programmed SCR in many species including humans, experimental proof of its functional consequences are scarce. Results We show that SCR of the Drosophila POU/Oct transcription factor Ventral veins lacking/Drifter (Vvl/Dfr) mRNA is prevalent in certain tissues in vivo, reaching a rate of 50% in the larval prothoracic gland. Phylogenetically, the C-terminal extension is conserved and harbors intrinsically disordered regions and amino acid stretches implied in transcriptional activation. Elimination of Vvl/Dfr translational readthrough by CRISPR/Cas9 mutagenesis changed the expression of a large number of downstream genes involved in processes such as chromatin regulation, neurogenesis, development, and immune response. As a proof-of-principle, we demonstrate that the C-terminal extension of Vvl/Dfr is necessary for correct timing of pupariation, by increasing the capacity to regulate its target genes. The extended Vvl/Dfr isoform acts in synergy with the transcription factor Molting defective (Mld) to increase the expression and biosynthesis of the steroid hormone ecdysone, thereby advancing pupariation. Consequently, late-stage larval development was prolonged and metamorphosis delayed in vvl/dfr readthrough mutants. Conclusions We demonstrate that translational recoding of a POU/Oct transcription factor takes place in a highly tissue-specific and temporally controlled manner. This dynamic and regulated recoding is necessary for normal expression of a large number of genes involved in many cellular and developmental processes. Loss of Vvl/Dfr translational readthrough negatively affects steroid hormone biosynthesis and delays larval development and progression into metamorphosis. Thus, this study demonstrates how SCR of a transcription factor can act as a developmental switch in a spatiotemporal manner, feeding into the timing of developmental transitions between different life-cycle stages. Graphical abstract

Transcriptional Regulators of Ecdysteroid Biosynthetic Enzymes and Their Roles in Insect Development

“…A recent study demonstrated that the vvl gene produces long and short isoforms through a stop codon readthrough process in the PG during larval development ( Zhao et al, 2021 ). Stop codon readthrough is a phenomenon in which near-cognate tRNAs are able to decode stop codons as sense codons, resulting in a read-frame extension even in a conventional 3′ untranslated region.…”

“…Stop codon readthrough is a phenomenon in which near-cognate tRNAs are able to decode stop codons as sense codons, resulting in a read-frame extension even in a conventional 3′ untranslated region. While the error rate of translational termination should be less than 0.1%, surprisingly, the stop codon readthrough of vvl mRNA reaches a rate of 50% in the PG ( Zhao et al, 2021 ). Consequently, the C-terminal extension of Vvl produces a long isoform, which possesses an extra 286 amino acid residues compared with the short isoform (427 amino acid length) encoded by the conventional vvl coding sequence.…”

“…(Vvl-L&S) nvd, spok, sro, phm, dib, sad (Vvl-L) nvd, spok, dib, sad spo, phm phm, dib, sad spo, dib + D. melanogaster D. melanogaster T. castaneum B. mori Oncopeltus fasciatus Cheng et al, 2014;Danielsen et al, 2014;Meng et al, 2015;Sarwar et al, 2020;Zhao et al, 2021 …”

The opportunities and challenges of using Drosophila to model human cardiac diseases

“…For example, the highly conserved NKX2.5 ( tinman in fly) which plays a crucial role in heart development is also a hotspot for genetic variants that have been linked to CHD ( McElhinney et al, 2004 ; Pashmforoush et al, 2004 ). In addition, the fly has an unprecedented arsenal of genetic tools that enable precise genetic manipulation to target specific developmental time points, or specific tissues and even cell-types within those tissues ( Hales et al, 2015 ; Zhao et al, 2021 ). Over the years many techniques to assay heart structure and function in the fly have been developed; these have consistently shown the physiological similarities of Drosophila and human heart development and function, as well as dysfunction when studying disease-associated genes and variants ( Wolf and Rockman, 2008 ; Manivannan et al, 2020 ).…”

Tissue-specific regulation of translational readthrough tunes functions of the traffic jam transcription factor

“…Recently, tissue-specific TR was identified in a transcription factor Ventral veins lacking/Drifter ( Vvl/Dfr ). TR in Vvl/Dfr is necessary for regulation of several downstream genes and is crucial for larval development ( 31 ). For the majority of the candidates, the efficiency and the biological relevance of TR are not known.…”