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Cited by 161RNA and protein synthesis mechanismsRNA Research and SplicingNeuroscience and Neuropharmacology Research

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Neuronal ribosomes exhibit dynamic and context-dependent exchange of ribosomal proteins

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Fusco, Claudia M., Desch, Kristina, Dörrbaum, Aline R., Wang, Mantian, Staab, Anja, Chan, Ivy C. W., Vail, Eleanor, Villeri, Veronica, Langer, Julian D., Schuman, Erin M.·2021·Nature Communications
DOI 10.1038/s41467-021-26365-xPMC PMC8531293

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Abstract

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.

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Fig. 1 RP mRNAs are present in hippocampal dendrites. a Overlap of RP mRNAs detected in neuronal processes from three different studies: dendritic arbors (ref. 26, in purple), purified synaptosomes (ref. 4, in green), developing axons (ref. 27, in yellow) of rodent neurons. See also Supplementary Data 1 for additional studies and data. b Schematic representation of a hippocampal slice. Three layers in area CA1 are shown: somata (stratum pyramidale, rich in cell bodies) and neuropil (strata radiatum et lacunosum-moleculare, rich in axons and dendrites77. c MA plot depicting the expression of mRNAs from the somata and neuropil of hippocampal slices14. Each mRNA is represented by a single data point. The y-axis depicts the relative expression in the neuropil or somata and the x-axis depicts the mean expression of each mRNA. Unlike mRNAs for other macromolecular complexes (e.g., the proteasome, nuclear pore complex and RNA polymerase I–III), mRNAs coding for ribosomal proteins are consistently enriched in the neuropil. d FISH detection of indicated RP mRNAs in dendrites (magenta MAP2, white FISH, blue DAPI) in hippocampal slices. Images are oriented with the somata layer at the top and the dendrites extending below. Scale bar = 50 µm. See Supplementary Fig. 1a, b for additional RPs and analysis. Representative images from n ≥ 7 field of views, acquired over ≥2 biologically and technically independent experiments, e FISH detection of indicated RP mRNAs as well as CamkIIα and His3 mRNA (magenta MAP2, white FISH) in cultured hippocampal neurons. Scale bar = 50 µm. See Supplementary Fig. 2 for additional RPs. Representative images from n ≥ 6 field of views. f Analysis of FISH data shown in Fig. 1e and Supplementary Fig. 2. Percentage of indicated mRNA signal in dendrites over the total detected in single neurons. Transcripts are ranked according to average value. The distribution of each mRNA was compared to the largely somatically localized transcript His3 (Ordinary one-way ANOVA (p < 0.0001) followed by a Dunnett’s multiple comparison test using His3 as control group, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Center of the box plots represents the median, hinges include first and third quartiles, and whiskers extend up to the smallest/largest value included in 1.5-fold the interquartile range (IQR). n of cells between 6 and 22 (as indicated by the dot plots).
Fig. 1 RP mRNAs are present in hippocampal dendrites. a Overlap of RP mRNAs detected in neuronal processes from three different studies: dendritic arbors (ref. 26, in purple), purified synaptosomes (ref. 4, in green), developing axons (ref. 27, in yellow) of rodent neurons. See also Supplementary Data 1 for additional studies and data. b Schematic representation of a hippocampal slice. Three layers in area CA1 are shown: somata (stratum pyramidale, rich in cell bodies) and neuropil (strata radiatum et lacunosum-moleculare, rich in axons and dendrites77. c MA plot depicting the expression of mRNAs from the somata and neuropil of hippocampal slices14. Each mRNA is represented by a single data point. The y-axis depicts the relative expression in the neuropil or somata and the x-axis depicts the mean expression of each mRNA. Unlike mRNAs for other macromolecular complexes (e.g., the proteasome, nuclear pore complex and RNA polymerase I–III), mRNAs coding for ribosomal proteins are consistently enriched in the neuropil. d FISH detection of indicated RP mRNAs in dendrites (magenta MAP2, white FISH, blue DAPI) in hippocampal slices. Images are oriented with the somata layer at the top and the dendrites extending below. Scale bar = 50 µm. See Supplementary Fig. 1a, b for additional RPs and analysis. Representative images from n ≥ 7 field of views, acquired over ≥2 biologically and technically independent experiments, e FISH detection of indicated RP mRNAs as well as CamkIIα and His3 mRNA (magenta MAP2, white FISH) in cultured hippocampal neurons. Scale bar = 50 µm. See Supplementary Fig. 2 for additional RPs. Representative images from n ≥ 6 field of views. f Analysis of FISH data shown in Fig. 1e and Supplementary Fig. 2. Percentage of indicated mRNA signal in dendrites over the total detected in single neurons. Transcripts are ranked according to average value. The distribution of each mRNA was compared to the largely somatically localized transcript His3 (Ordinary one-way ANOVA (p < 0.0001) followed by a Dunnett’s multiple comparison test using His3 as control group, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Center of the box plots represents the median, hinges include first and third quartiles, and whiskers extend up to the smallest/largest value included in 1.5-fold the interquartile range (IQR). n of cells between 6 and 22 (as indicated by the dot plots).CC BY · PubMed Central
Fig. 2 RP mRNAs are translated in hippocampal dendrites. a Ribosome footprint coverage of RP mRNAs from the somata and neuropil of hippocampal slices14. Shown are the number of reads throughout the open reading frame (gray box) from the somata-enriched fraction (green) or the neuropil-enriched fraction (purple). Ribosome footprint coverage for additional RPs is shown in Supplementary Fig. 3. b Detection of nascent RPs (green) in dendrites (magenta for MAP2 immunostaining) of cultured hippocampal neurons using Puro-PLA37. Nascent proteins were labeled with puromycin (5 min), in the absence or presence (as indicated) of the protein synthesis inhibitor anisomycin (see methods). Scale bar = 50 µm. Data for additional nascent RP detection are shown in Supplementary Figs. 4b and 5. Representative images from n ≥ 7 field of views, acquired over ≥2 biologically and technically independent experiments. c Analysis of nascent RP detection shown in Fig. 2b, Supplementary Figs. 4b and 5. Analyzed are the newly synthesized RP punctae per 10 µm2 of dendrite, in control (green) or in the presence of protein synthesis inhibitor (gray). Each dot is the quantification from the whole dendritic arbor of one single neuron. Wilcox test, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Center of the box plots represents the median, hinges include first and third quartiles, and whiskers extend up to the smallest/largest value included in 1.5-fold the interquartile range (IQR). n of cells between 4 and 42 (as indicated by the dot plots), over at least 2 independent experiments.
Fig. 2 RP mRNAs are translated in hippocampal dendrites. a Ribosome footprint coverage of RP mRNAs from the somata and neuropil of hippocampal slices14. Shown are the number of reads throughout the open reading frame (gray box) from the somata-enriched fraction (green) or the neuropil-enriched fraction (purple). Ribosome footprint coverage for additional RPs is shown in Supplementary Fig. 3. b Detection of nascent RPs (green) in dendrites (magenta for MAP2 immunostaining) of cultured hippocampal neurons using Puro-PLA37. Nascent proteins were labeled with puromycin (5 min), in the absence or presence (as indicated) of the protein synthesis inhibitor anisomycin (see methods). Scale bar = 50 µm. Data for additional nascent RP detection are shown in Supplementary Figs. 4b and 5. Representative images from n ≥ 7 field of views, acquired over ≥2 biologically and technically independent experiments. c Analysis of nascent RP detection shown in Fig. 2b, Supplementary Figs. 4b and 5. Analyzed are the newly synthesized RP punctae per 10 µm2 of dendrite, in control (green) or in the presence of protein synthesis inhibitor (gray). Each dot is the quantification from the whole dendritic arbor of one single neuron. Wilcox test, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Center of the box plots represents the median, hinges include first and third quartiles, and whiskers extend up to the smallest/largest value included in 1.5-fold the interquartile range (IQR). n of cells between 4 and 42 (as indicated by the dot plots), over at least 2 independent experiments.CC BY · PubMed Central
Fig. 3 Some RPs rapidly incorporate into neuronal ribosomes. a Schematic of the experimental design for measuring new RPs in assembled ribosomes from cultured neurons. Normal medium was replaced by medium containing heavy amino acids (dynamic SILAC) as indicated (gray boxes). Mature ribosomes were purified by sucrose cushion. New ribosomal proteins were quantified by mass spectrometry, measuring the heavy and light peak of each peptide. b Heatmap showing for each RP the fraction of new proteins (H/(H + L)) incorporated into assembled ribosomes. Pseudocells (median of peptides obtained per individual protein) are ordered according to unsupervised clustering, both for columns (biological replicate of each condition) and rows (individual ribosomal protein). Experimental conditions of the labeling are indicated at the bottom. c, d Scatterplots showing the fraction of new RPs (H/(H + L)) in assembled ribosomes after the different labeling conditions, as indicated by x- and y-axes. Points represent average ± standard deviation of three biological replicates. Proteins are colored according to clusters identified in Fig. 3b. Some RPs of interest are indicated by name. Dashed line represents x = y. Source data are provided as a Source Data file. e, f Average ± standard deviation of the fraction of new proteins in assembled ribosomes of RPs of the same cluster, as identified in Fig. 3b. The different labeling conditions are indicated on the x-axis. Cluster A, n = 3 RPs. Cluster B, n = 4 RPs. Cluster C, n = 5 RPs. Cluster D, n = 11 RPs. Cluster E, n = 26 RPs. Cluster F, n = 21 RPs.
Fig. 3 Some RPs rapidly incorporate into neuronal ribosomes. a Schematic of the experimental design for measuring new RPs in assembled ribosomes from cultured neurons. Normal medium was replaced by medium containing heavy amino acids (dynamic SILAC) as indicated (gray boxes). Mature ribosomes were purified by sucrose cushion. New ribosomal proteins were quantified by mass spectrometry, measuring the heavy and light peak of each peptide. b Heatmap showing for each RP the fraction of new proteins (H/(H + L)) incorporated into assembled ribosomes. Pseudocells (median of peptides obtained per individual protein) are ordered according to unsupervised clustering, both for columns (biological replicate of each condition) and rows (individual ribosomal protein). Experimental conditions of the labeling are indicated at the bottom. c, d Scatterplots showing the fraction of new RPs (H/(H + L)) in assembled ribosomes after the different labeling conditions, as indicated by x- and y-axes. Points represent average ± standard deviation of three biological replicates. Proteins are colored according to clusters identified in Fig. 3b. Some RPs of interest are indicated by name. Dashed line represents x = y. Source data are provided as a Source Data file. e, f Average ± standard deviation of the fraction of new proteins in assembled ribosomes of RPs of the same cluster, as identified in Fig. 3b. The different labeling conditions are indicated on the x-axis. Cluster A, n = 3 RPs. Cluster B, n = 4 RPs. Cluster C, n = 5 RPs. Cluster D, n = 11 RPs. Cluster E, n = 26 RPs. Cluster F, n = 21 RPs.CC BY · PubMed Central
Fig. 4 Some nascent RPs can incorporate into neuronal ribosomes independent of the canonical ribosome biogenesis pathway. a Schematic of the experimental design. Neurons were cultured on porous membranes where neurites can grow through the pores and be separately harvested. Normal medium was replaced by medium containing heavy amino acids for 2 days (filled boxes) in the presence (treated) or absence (control) of Leptomycin B (LMB) to prevent the export of nascent ribosomes from the nucleus and thus inhibit ribosome biogenesis. Mature ribosomes were purified from either compartment (top: cell bodies + neurites; bottom: neurites only) and new RPs were quantified by mass spectrometry as before. b Quantification by RT-PCR of new 18S rRNA in assembled ribosomes, relative to a non-labeled control (y = 1, dashed line). Two days of Leptomycin B (LMB) treatment significantly reduced the nascent rRNA levels in purified ribosomes (five paired biological replicates, paired t-test, two-tailed *p = 0.037). c Scatterplots showing fraction of new proteins (log2 of the H/L ratio) purified with assembled ribosomes from the cell bodies+neurites (left panel) or neurite-enriched (right panel) compartments of control or LMB-treated neurons. Ribosomal proteins are colored in green, all other proteins are indicated in gray. Dashed lines represent x = y. d Fold-change of the fraction of new RPs (H/(H + L)) detected in assembled ribosomes between control and LMB-treated samples, normalized to the median value obtained for each subunit (40S or 60S) (see methods). Ribosomes were purified from the cell bodies+neurites (dark green) or neurite-enriched (light green) compartments. Each point represents the mean ± standard deviation of four biological replicates. Exchanging RPs were identified as significant outliers by ROUT Method (*FDR ≤ 0.2%). See methods for information on statistical testing. Insert: Venn diagram of exchanging RPs in the two compartments. Source data are provided as a Source Data file. e Structure of human ribosome showing two views each for the large (top) and small (bottom) subunit (PDB: 4v6x). rRNAs in black, tRNA in yellow, stable RPs in gray, exchanging RPs in purple. Names are indicated for the exchanging RPs, as identified in Fig. 3d. f Fraction of solvent-accessible surface area for stable (n = 64 chains) and exchanging RPs (n = 16 chains), calculated for individual proteins within the structure of individual subunits of the human ribosome (PDB: 4v6x). Median ± interquartile range. Mann–Whitney test, two-tailed ***p = 0.0007.
Fig. 4 Some nascent RPs can incorporate into neuronal ribosomes independent of the canonical ribosome biogenesis pathway. a Schematic of the experimental design. Neurons were cultured on porous membranes where neurites can grow through the pores and be separately harvested. Normal medium was replaced by medium containing heavy amino acids for 2 days (filled boxes) in the presence (treated) or absence (control) of Leptomycin B (LMB) to prevent the export of nascent ribosomes from the nucleus and thus inhibit ribosome biogenesis. Mature ribosomes were purified from either compartment (top: cell bodies + neurites; bottom: neurites only) and new RPs were quantified by mass spectrometry as before. b Quantification by RT-PCR of new 18S rRNA in assembled ribosomes, relative to a non-labeled control (y = 1, dashed line). Two days of Leptomycin B (LMB) treatment significantly reduced the nascent rRNA levels in purified ribosomes (five paired biological replicates, paired t-test, two-tailed *p = 0.037). c Scatterplots showing fraction of new proteins (log2 of the H/L ratio) purified with assembled ribosomes from the cell bodies+neurites (left panel) or neurite-enriched (right panel) compartments of control or LMB-treated neurons. Ribosomal proteins are colored in green, all other proteins are indicated in gray. Dashed lines represent x = y. d Fold-change of the fraction of new RPs (H/(H + L)) detected in assembled ribosomes between control and LMB-treated samples, normalized to the median value obtained for each subunit (40S or 60S) (see methods). Ribosomes were purified from the cell bodies+neurites (dark green) or neurite-enriched (light green) compartments. Each point represents the mean ± standard deviation of four biological replicates. Exchanging RPs were identified as significant outliers by ROUT Method (*FDR ≤ 0.2%). See methods for information on statistical testing. Insert: Venn diagram of exchanging RPs in the two compartments. Source data are provided as a Source Data file. e Structure of human ribosome showing two views each for the large (top) and small (bottom) subunit (PDB: 4v6x). rRNAs in black, tRNA in yellow, stable RPs in gray, exchanging RPs in purple. Names are indicated for the exchanging RPs, as identified in Fig. 3d. f Fraction of solvent-accessible surface area for stable (n = 64 chains) and exchanging RPs (n = 16 chains), calculated for individual proteins within the structure of individual subunits of the human ribosome (PDB: 4v6x). Median ± interquartile range. Mann–Whitney test, two-tailed ***p = 0.0007.CC BY · PubMed Central
Fig. 5 The association of a subset of exchanging RPs is relatively enhanced after oxidative stress. a Schematic of the experimental design for measuring new RPs in assembled ribosomes after oxidative stress. Normal medium was replaced by medium containing heavy amino acids (SILAC) for a total of 3 h. To induce stress, H2O2 was added for 10 min to a final concentration of 0.1 mM (light green) or 1 mM (dark green) to the heavy medium, 2 h after the beginning of labeling. Mature ribosomes were purified and new RPs were quantified by mass spectrometry as before. b Volcano plots of significantly regulated ribosomal proteins (green, FDR < 0.01) in assembled ribosomes, comparing control and H2O2-treated neurons (see methods for more detailed information on normalization and statistical testing). Source data are provided as a Source Data file. c Normalized fold-change in incorporation levels (see methods) for representative ribosomal proteins, which are significantly regulated (RPS30, RACK1, RPLP2, RPLP0, and RPL10) or not (RPL24, RPL22, and RPS14) with H2O2 treatment. Three biological replicates. Center of the box plots represents the median, hinges include first and third quartiles, and whiskers extend up to the smallest/largest value included in 1.5-fold the interquartile range (IQR). Source data are provided as a Source Data file.
Fig. 5 The association of a subset of exchanging RPs is relatively enhanced after oxidative stress. a Schematic of the experimental design for measuring new RPs in assembled ribosomes after oxidative stress. Normal medium was replaced by medium containing heavy amino acids (SILAC) for a total of 3 h. To induce stress, H2O2 was added for 10 min to a final concentration of 0.1 mM (light green) or 1 mM (dark green) to the heavy medium, 2 h after the beginning of labeling. Mature ribosomes were purified and new RPs were quantified by mass spectrometry as before. b Volcano plots of significantly regulated ribosomal proteins (green, FDR < 0.01) in assembled ribosomes, comparing control and H2O2-treated neurons (see methods for more detailed information on normalization and statistical testing). Source data are provided as a Source Data file. c Normalized fold-change in incorporation levels (see methods) for representative ribosomal proteins, which are significantly regulated (RPS30, RACK1, RPLP2, RPLP0, and RPL10) or not (RPL24, RPL22, and RPS14) with H2O2 treatment. Three biological replicates. Center of the box plots represents the median, hinges include first and third quartiles, and whiskers extend up to the smallest/largest value included in 1.5-fold the interquartile range (IQR). Source data are provided as a Source Data file.CC BY · PubMed Central

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Authors
Fusco, C. M., Desch, K., Dörrbaum, A. R., Wang, M., Staab, A., Chan, I. C. W., Vail, E., Villeri, V., Langer, J. D., & Schuman, E. M.
Affiliations
  • Max Planck Institute for Brain Research (DE)
  • German Center for Neurodegenerative Diseases (DE)
  • Université de Bordeaux (FR)
  • Max Planck Institute of Biophysics (DE)
Corresponding author
Schuman, Erin M.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-10-21
Volume
12
Issue
1
Pages
6127-6127
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-26365-x
PMC
PMC8531293
OpenAlex
W3207078712
Cited by
157
References
84
Citations by year
2026: 23 · 2025: 43 · 2024: 38 · 2023: 27 · 2022: 23 · 2021: 3
FWCI
7.38
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DZNE Pub (submittedVersion, other-oa)
  • Publication Server of Goethe University Frankfurt am Main (Goethe University Frankfurt) (submittedVersion, cc-by)
  • Publication Server of Goethe University Frankfurt am Main (Goethe University Frankfurt) (submittedVersion, cc-by-nc-nd)
Funders
  • Deutsche Forschungsgemeinschaft (CRC 1080, DFG-CRC902, CRC902)
  • Max-Planck-Gesellschaft (DFG CRC 1080)
  • H2020 European Research Council (743216)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
ribosomal proteins, local protein synthesis, dendrites, axons, neuropil, ribosome biogenesis, oxidative stress, translation machinery, ribosome profiling, metabolic labeling
MeSH terms
Animals, Axons, Cells, Cultured, Female, Male, Neurons, Ribosomal Proteins, Ribosomes, RNA, Messenger, Protein Biosynthesis, Neurites, Rats, Sprague-Dawley, Neuropil, Rats

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