Wills, N. M. | bioRxiv Science

bioRxiv2024

Evidence for the pairing of mRNA exiting ribosomes acting as a Driver of unidirectional forward bypassing: Its cessation permits Backwards scanning

Efficient translational avoidance of a 50 nucleotide non-coding insert in phage T4 gene 60 sequence involves dissociation of peptidyl-tRNA pairing from the mRNA take-off codon, GGA. This tRNA is retained in the ribosomal P-site during bypassing. Its anticodon does not scan the 5 part of the non-coding sequence, which contains a cognate GGG codon, but it does scan the main and 3 part of the coding gap for potential complementarity. Re-pairing to a matched GGA landing site codon occurs exclusively 48-50 nt after take-off. Coding resumption ensues. Here we analyze proteins synthesized from mutants that abrogate the potential for re-pairing at the canonical landing site. The results show that some ribosomes move backwards 39 nt allowing re-pairing to the initially hidden GGG or forwards at least 56 additional nt before resumed anticodon: codon pairing at a downstream matched codon. We discuss the nature of the driving force for the initial unidirectional movement and why it becomes dissipated when the site of landing in WT is reached. Also presented is an analysis of the relative importance of recoding signals located 6 nucleotides upstream (5) and close downstream (3) of the WT landing site, for re-pairing to mRNA and coding resumption.

molecular biology↗

bioRxiv2020

Tissue-specific dynamic codon redefinition in Drosophila

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.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗