Puromycin reveals a distinct conformation of neuronal ribosomes.

Puromycin 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., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. 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 cryoelectron 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 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.

Ribosomes in RNA Granules Are Stalled on mRNA Sequences That Are Consensus Sites for FMRP Association.

Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.SIGNIFICANCE STATEMENT Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here, we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome-protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.