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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.

Viral infection triggers several dsRNA sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, RNase L, that cleaves single stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here we show that this fragmentation induces the Ribotoxic Stress Response via ZAKα, potentially through ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes, including antiviral IFIT mRNAs and GADD34 that encodes an antagonist of the Integrated Stress Response. Intriguingly, we found the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.