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

Abstract

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.

Puromycin reveals a distinct conformation of neuronal ribosomes

“…However, we did not observe any clear difference in the conformation of the nascent peptide or in the PTC with the structure previously obtained for the hybrid state ribosomes (class 1) in the untreated sample (17) (Supplementary Figure 6). Codons encoding acidic amino acids (Asp and Glu) are close to two-fold enriched in peaks of ribosome-protected mRNA fragments recovered from the granule fraction (17). This finding is consistent with a stalling mechanism where a neuronal-specific factor facilitates stalling of peptides rich in acidic residues.…”

“…The last row of the table shows the net increase (black numbers) or decrease (red numbers) for each class. The two percentage numbers in the '+ puromycin' and 'net increase or decrease line' refer to the classification results when the sample that was analyzed by cryo-EM was processed as described in Figure 1A & 1C (first set of percentages) or according to our previous analysis (17), in which after treating the granule fraction with puromycin and nuclease, samples were directly applied to cryo-EM grids.…”

“…RNA granules were enriched from whole brain homogenate harvested from five-day-old (P5) Sprague Dawley rats of both sexes (Charles River Laboratories), as previously described (17). 3141, Thermo Scientific], filled to top with additional RNA granule buffer and centrifuged for 2 hours in a Thermo Scientific AH-629 swing-bucket rotor at 56660 x g at 4˚C to achieve the polysome pellet.…”

“…The initial 3D reference used for the exhaustive 3D classification was an 80S neuronal ribosome map from rats low-pass filtered at 60 Å created from EMD-28726 and EMD-28727 (17). The resulting maps from the exhaustive 3D classification were visually inspected in Chimera (48) and it was concluded that there were two main classes of particles.…”

“…The second dataset obtained from puromycin-treated monosomes purified as for Anadolu et al (17) was analyzed using an identical image processing pipeline. In this case, we collected 13,645 movies and use a subset of 207 movies to initially pick up 19,733 particles using the Laplacian picker and generate the templates for particle picking in the entire dataset.…”