Visualizing non-canonical translation events using single molecule reporters in live yeast and human cells

Ribosome recycling, the process of removing ribosomal subunits from an mRNA following translation termination, is a fundamental cellular process that maintains a proper free pool of ribosomal subunits. Aberrant ribosome recycling alters protein homeostasis and has been strongly implicated in aging of neurons. When ribosome recycling fails, ribosomes can “read through” the stop codon and translate the 3’ untranslated region (UTR) of mRNAs, leading to undesirable C-terminal extensions of proteins that can lead to their degradation or aggregation. However, some proteins undergo programmed stop codon readthrough to create alternative protein isoforms that alter their localization or function. The mechanisms that differentiate programmed stop codon readthrough from “normal” stop codons remains unclear. To investigate, we developed single molecule reporters that report on nascent translation of both the main open reading frame (ORF) and the 3’UTR on single mRNAs to monitor translation readthrough in live mammalian cells. We demonstrate that different stop codon sequence contexts lead to varied stop codon readthrough, as previously observed in vitro. We found that stop codon readthrough appears to have no effect on main ORF translation or mRNA stability. Interestingly, quantification of our results suggests that stop codon readthrough occurs in “bursts”, suggesting a type of signal, such as 3’UTR RNA structure, modulates readthrough. In future experiments, we will knock down known translation termination and recycling factors to determine their effect on translation termination and readthrough.