Puromycin reveals a distinct conformation of neuronal ribosomes

Abstract

AbstractPuromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published (Hobson et al., 2020https://doi.org/10.7554/eLife.60048; Enam et al., 2020https://doi.org/10.7554/eLife.60303). In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryo-electron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide new insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.Significance StatementPuromycin can be covalently linked to the nascent polypeptide chain on ribosomes, followed by dissociation of the puromycylated polypeptide. Here, we conclusively show that in stalled ribosomes isolated from neuronal RNA granules, the puromycylated peptide remains in the polypeptide exit tunnel of the ribosome. This validates previous data using this technique to localize stalled ribosomes in neurons and suggests a unique ribosomal conformation in these cells. Further evidence for the unique state of these ribosomes is the resistance of puromycylation to the inhibitor anisomycin, which prevents puromycylation in all other cellular contexts. These results provide insight into the mechanism underlying neuronal ribosome stalling and resolve a controversy about using puromycin to localize ribosome stalling in neurons.