Tissue-specific dynamic codon redefinition in Drosophila

Abstract

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3' of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue-and cell-specific dynamic stop codon redefinition in Drosophila. Stop codon readthrough | recoding | Drosophila | Kelch | central nervous system (CNS) †Correspondence: j.atkins@ucc.ie or lynn.cooley@yale.edu Hudson et al. | bioRχiv | June 22, 2020 | 1-11

Ir56b is an atypical ionotropic receptor that underlies appetitive salt response in Drosophila

“…Readthrough may occur when a near-cognate tRNA inserts an amino acid, and its efficiency varies across cell type and conditions. 42,43 We speculate that the conserved Ir56b PTC could provide a mechanism that allows the level of Ir56b to be modulated by environmental conditions such as salinity. Such a mechanism could have been lost in the cactophilic species that evolved in arid regions and that may have experienced a narrower range of salt concentrations or may have special needs to maintain salt balance.…”

Tissue-Specific Regulation of Translational Readthrough Tunes Functions of the Traffic Jam Transcription Factor

“…Subsequently, ribosome profiling revealed differential ribosomal footprints in several other genes that show significant TR in samples derived from early embryos and S2 cells (Dunn et al, 2013). More recently, several studies in Drosophila have reported highest levels of TR in tissues of the CNS, mainly neurons (Chen et al, 2020; Hudson et al, 2020). These studies are concurrent with findings in mice, where the incidence of cell-type-specific TR was detected by analysis of TR in neurons and astrocytes in the CNS as well as peripheral tissues (Palazzo et al, 2020; Sapkota et al, 2019).…”

Widespread readthrough events in plants reveal unprecedented plasticity of stop codons

“…In the yeast Saccharomyces cerevisiae, the [PSI+] strain forms prion-like polymers of the abnormal eukaryotic release factor 3 (eRF3), which is implicated in translation termination and peptide release, resulting in a high level of readthrough across many genes 20 , including Phosphodiesterase 2 (PDE2), Interacting with mpp10p 3 (IMP3), and Bypass of stop codon 4 (BSC4) 10,21,22 . In Drosophila melanogaster, readthrough is estimated to affect over 300 genes 23,24 , such as kelch (kel) 12,25 , Synapsin (Syn) 26 , headcase (hdc) 27 , and traffic jam (tj) 13 . In (which was not certified by peer review) is the author/funder.…”