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- EMDB-26518: Rat 80S ribosome purified from brain RNA granules. Class 2 60S su... -

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Entry
Database: EMDB / ID: EMD-26518
TitleRat 80S ribosome purified from brain RNA granules. Class 2 60S subunit.
Map dataRat 80S ribosome purified from brain RNA granules. Class 2 60S subunit.
Sample
  • Complex: Rat 80S ribosome purified from brain RNA granules. Class 1 60S subunit.
Keywords80S / rat / RNA granule / RIBOSOME
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsOrtega J
Funding support Canada, 1 items
OrganizationGrant numberCountry
CIFAR Azrieli Global Scholars Canada
CitationJournal: J Neurosci / Year: 2023
Title: Ribosomes in RNA Granules Are Stalled on mRNA Sequences That Are Consensus Sites for FMRP Association.
Authors: Mina N Anadolu / Jingyu Sun / Senthilkumar Kailasam / Kleanthi Chalkiadaki / Konstanze Krimbacher / Jewel T-Y Li / Teodora Markova / Seyed M Jafarnejad / Francois Lefebvre / Joaquin Ortega / ...Authors: Mina N Anadolu / Jingyu Sun / Senthilkumar Kailasam / Kleanthi Chalkiadaki / Konstanze Krimbacher / Jewel T-Y Li / Teodora Markova / Seyed M Jafarnejad / Francois Lefebvre / Joaquin Ortega / Christos G Gkogkas / Wayne S Sossin /
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 ...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 , 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. 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.
History
DepositionMar 27, 2022-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26518.png

Downloads & links

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Map

FileDownload / File: emd_26518.map.gz / Format: CCP4 / Size: 240.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRat 80S ribosome purified from brain RNA granules. Class 2 60S subunit.
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.0192
Minimum - Maximum-0.040557567 - 0.11481529
Average (Standard dev.)-0.0016187839 (±0.0054912944)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions398398398
Spacing398398398
CellA=B=C: 433.82 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26518_additional_1.map
Projections & Slices
AxesZYX

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Half map: Rat 80S ribosome purified from brain RNA granules....

Fileemd_26518_half_map_1.map
AnnotationRat 80S ribosome purified from brain RNA granules. Class 2 60S subunit. Half map 1
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: Rat 80S ribosome purified from brain RNA granules....

Fileemd_26518_half_map_2.map
AnnotationRat 80S ribosome purified from brain RNA granules. Class 2 60S subunit. Half map 2
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Rat 80S ribosome purified from brain RNA granules. Class 1 60S su...

EntireName: Rat 80S ribosome purified from brain RNA granules. Class 1 60S subunit.
Components
  • Complex: Rat 80S ribosome purified from brain RNA granules. Class 1 60S subunit.

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Supramolecule #1: Rat 80S ribosome purified from brain RNA granules. Class 1 60S su...

SupramoleculeName: Rat 80S ribosome purified from brain RNA granules. Class 1 60S subunit.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Rattus norvegicus (Norway rat) / Organ: Brain

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.225 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

2938

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 269103
FSC plot (resolution estimation)

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