Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously we reported tissue-specific regulation of readthrough of the first kelch stop codon. Here, we characterize major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3′ of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3′ of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, we show that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.
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
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