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, cryo-EM and ribosome profiling. We find that this fraction, isolated from P5 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 homolog (UPF1). Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (i) an enrichment for footprint reads of mRNAs that interact with FMRP and that are associated with stalled polysomes, (ii) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and (iii) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared to 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.

Ribosome assembly is orchestrated by many assembly factors, including ribosomal RNA methyltransferases whose precise role is poorly understood. Here, we leverage the power of cryo-EM and machine learning to discover that the bacterial methyltransferase KsgA performs a novel "proofreading" function in assembly of the ribosomal small subunit by recognizing and partially disassembling particles that have matured but are not competent for translation. We propose that this activity allows inactive particles an opportunity to reassemble into an active state, thereby increasing overall assembly fidelity. Detailed structural quantifications in our datasets additionally enabled expansion of the Nomura assembly map to highlight rRNA helix and r-protein interdependencies, which newly details how binding and docking of these elements are tightly coupled. These results have wide-ranging implications in our understanding of the quality control mechanisms governing ribosome biogenesis, and showcase the power of heterogeneity analysis in cryo-EM to unveil functionally relevant information in biological systems.

A series of generation 3−5 dendrons based on a bis(2,2hydroxymethylpropionic acid) (bis-MPA) scaffold bearing three respective lengths of linear poly(ethylene glycol) at their periphery and a dibenzocyclooctyne unit at their core was prepared. These dendrons were appended to the surface of azide-decorated α-chymotrypsin (α-CT) via strain-promoted azide−alkyne cycloaddition to yield a library of dendron-protein conjugates. These conjugates were characterized by FT-IR and NMR spectroscopy and were imaged using cryo-electron microscopy. The activity of the PEGylated α-CT-dendron conjugates was investigated using a small molecule (benzoyl-L-tyrosine p-nitroanilide) as well as different proteins of different sizes and crystallinities (casein and bovine serum albumin) as substrates. It was found that the activity of the conjugates toward the small molecule was largely retained, while the activity toward the proteins was significantly diminished. Furthermore, the results indicate that for most of the conjugates the PEG length had a more pronounced impact on enzyme activity than the dendron generation. Overall, the highest sieving ratios were found for α-CT-dendron conjugates decorated with G3-PEG 2000 , G4-PEG 2000 , and G5-PEG 1000 , with the latter two structures offering the best combination of sieving ratio and small molecule activity.

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and this event is normally followed by the dissociation of the puromycylated peptide. It was postulated that blocking the translocation of the ribosome with emetine could trap the puromycylated peptide on the ribosome, but evidence against this has recently been published (Hobson et al., 2020 https://doi.org/10.7554/eLife.60048; Enam et al., 2020 https://doi.org/10.7554/eLife.60303). In neurons, puromycylated nascent chains appear to remain in the ribosome even in the absence of emetine, yet direct evidence for this had 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 ribosomes, anisomycin, which normally blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains in neurons 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.