Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Schematic of immunoprecipitation-mass spectrometry (IP-MS) workflow. ( B ) Number of protein groups quantified per replicate from the tissue homogenate, synaptic fraction cushion and IP eluate fractions. ( C ) Distinct biochemical strategies (PL-MS and IP-MS) capture AMPA receptors as interactors of synaptic ribosomes. IP proteins were filtered based on uniqueness to the IP or >3-fold enrichment compared to the corresponding isotype control. Protein groups were collapsed to unique gene identifiers prior to overlap analysis. Values correspond to the number of unique gene identifiers. Right: Proteins found in the union of all three interactomes that are associated with the GO terms “AMPA glutamate receptor complex” or “Glutamate receptor signaling pathway”. ( D ) Relative intensities of six selected proteins that are enriched in the ribosome IP and GluA1 IP eluate fractions compared to the corresponding isotype control and are associated with the GO term “AMPA glutamate receptor complex”. Error bars show mean ± SEM. ( E ) GO overrepresentation analysis for proteins in the ribosome IP and GluA1 IP eluate fractions. Shown are the top 10 enriched GOMF terms. ( F ) Average log 2 protein intensities of ribosomal proteins (RPs) in the ribosome IP and GluA1 IP eluate fractions compared to the corresponding isotype control. Horizontal bars represent the median; **p<0.01; ****p<0.0001, Kruskal-Wallis test, Dunn’s multiple comparisons test. See table S2. ( G ) Western blot analysis of ribosomal proteins, AMPA receptor subunits and known AMPA receptor interactors after IP of ribosomes or GluA1, with on-bead washes performed with or without magnesium. Blots are from one of three experimental replicates. Input: 0.25 ug of synaptic cushion (0.5% of IP input), IP: 50% of IP eluate. ( H ) Western blot analysis of PABP, ribosomal proteins and AMPA receptor subunits after IP of ribosomes or GluA1 with and without RNase I treatment. Blots are from one of three experimental replicates. Input: 0.5 ug of synaptic cushion (1% of IP input), IP: 50% of IP eluate. Asterisk indicates a possible glycosylated or partially proteolyzed form of GluA1. Abbreviations: hom., homogenate; syn.cush, synaptic fraction cushion; GOMF, Gene Ontology Molecular Function; IP, immunoprecipitation; mIgG, mouse IgG; rIgG, rabbit IgG; PABP, poly(A)-binding protein.

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Control, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Overview of synaptosome preparation with SynPER. Shown in bold are the fractions used in . ( B ) Synaptic proteins are enriched in SynPER synaptosomes. Shown are the relative intensities of six selected proteins in the tissue homogenate, cytosolic fraction, and synaptic fraction (before cushion ultracentrifugation). Nucleolin serves as a nuclear marker and CDK5 serves as a cytosolic marker. ( C ) Principal component analysis (PCA) of the log 2 protein intensities in the tissue homogenate, synaptic fraction cushion and IP eluate fractions. ( D ) Overall log 2 protein intensities per replicate from the tissue homogenate, synaptic fraction cushion and IP eluate fractions. ( E ) GO overrepresentation analysis for proteins in the ribosome IP and GluA1 IP eluate fractions. Shown are the top 10 enriched GOBP and GOCC terms. ( F ) Total protein stain images for the immunoblots in .

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Marker, Staining, Western Blot

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Confocal image of primary hippocampal neurons immunostained for MAP2 (magenta) and RPS11 (white). Scale bar, 20 µm. ( B ) Confocal images of dendrites immunolabeled for surface GluA1 (magenta), MAP2 (cyan) and RPL36A (green). Scale bar, 2 µm. ( C ) Overview of STED imaging analysis. Scale bar, 0.5 µm. ( D ) Probability density (area = 1) of target-normalized nearest-neighbor distances. Teal: surface GluA1 to RPS11 (normalized by RPS11 density). Purple: RPS11 to surface GluA1 (normalized by surface GluA1 density). Black line: CSR PDF. Excess density at small distances indicates closer-than-random proximity. (GluA1 to RPS11: n=1524 distances (puncta); RPS11 to GluA1: n=4254 distances (puncta). ( E ) Empirical cumulative distributions of the same normalized distances. Black line: CSR CDF. Curves left/above CSR indicate closer-than-random proximity. Dashed vertical line marks the CSR median (0.469). ( F, G ) Median NND per spine compared with simulation values in which surface GluA1 object coordinates were held fixed and RPS11 objects were randomly repositioned within the spine ROI (100 randomizations per spine). For each spine, the simulated value equals the median across its 100 simulated medians. ( F ) Left: paired dot plot of per-spine median surface GluA1 to RPS11 NNDs (exp. vs sim.); lines connect the same spine. Horizontal bars represent the group median across spines. ****p<0.0001, two-tailed Wilcoxon matched-pairs signed rank test. Right: ECDF of per-spine medians (exp. vs sim.). A left/upward shift of the experimental curve indicates shorter medians than the simulated null. ****p<0.0001, two-tailed Kolmogorov-Smirnov test. ( G ) Left: paired dot plot of per-spine median RPS11 to surface GluA1 NNDs (exp. vs sim.); lines connect the same spine. Horizontal bars represent the group median across spines. p=0.1263, two-tailed Wilcoxon matched-pairs signed rank test. Right: ECDF of per-spine medians (exp. vs sim.). ns=not significant (p=0.0141), two-tailed Kolmogorov-Smirnov test; n=323 spines from 100 dendritic segments from 23 cells from three independent cultures. Abbreviations: CSR, complete spatial randomness; PDF, probability density function; CDF, cumulative distribution function; NNDs, nearest-neighbor distances; exp., experimental; sim., simulated.

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Immunolabeling, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Overview of STED imaging analysis pipeline. ( B ) Relative frequency histograms of nearest-neighbor distances. GluA1 to RPS11: n=1524 distances (puncta); RPS11 to GluA1: n=4254 distances (puncta). ( C ) Fraction of total spines per replicate (top) or spines per cell (bottom) containing at least one ribosome located within 100 nm of surface GluA1. Error bars show mean ± SEM; n=323 spines from 100 dendritic segments from 23 cells from 3 independent cultures.

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Imaging

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Immunoprecipitation-ribosome profiling (IP-Ribo-Seq) experimental workflow. ( B ) Overlap of detected transcripts in synapse RNA-seq and synapse Ribo-seq datasets. ( C ) Volcano plot comparing translational levels of 6,889 transcripts between synaptic and cytosolic compartments (RPLP0 IP from synaptic fraction vs. RPLP0 IP from cytosolic fraction). Significantly enriched transcripts are highlighted (FDR ≤ 0.05 and |log 2 FC| ≥ log 2 (1.3)). ( D ) Functional segregation of transcripts differentially translated between synaptic and cytosolic compartments. Shown are the top 5 enriched GOCC terms for synapse- and cytosol-enriched transcripts. ( E ) Functional comparison of synapse-enriched transcripts with neuropil- and somata-enriched transcripts from . Top 15 GOCC terms are shown, ranked by enrichment score (mean log 2 fold enrichment). ( F ) Comparison of relative abundance ranks between synaptic and neuropil compartments. Paired dot plot showing footprint abundance ranks of synapse-enriched transcripts in the synaptic and neuropil translatomes. Transcripts with ≤5% rank change were classified as rank-stable. Transcripts with >40% upward or downward shifts from the neuropil to synaptic compartments were classified as rank-increased and rank-decreased, respectively. ( G ) Top 5 GOCC terms for rank-increased transcripts. ( H ) MA plot comparing the translational level of 7,365 transcripts between GluA1-associated ribosomes and the total synaptic ribosome pool. ( I ) Coverage profiles representing the average GluA1 IP (top) or Ribo IP (bottom) footprint coverage for candidate GluA1-associated ribosome ( Camk2a , Shank1 , Eef2 ) transcripts. The y-axis indicates the number of normalized reads. ( J ) Top 10 enriched GOCC terms for GluA1-vs RPLP0-enriched transcripts. The dashed line indicates -log10(FDR). ( K ) Schematic of a dendritic spine highlighting some of the transcripts preferentially translated by GluA1-associated ribosomes (rose) or the total synaptic ribosome pool (tan). Transcripts that are not enriched in either (i.e., translated in both) are labeled in gray.

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Immunoprecipitation, RNA Sequencing, Functional Assay, Comparison, Labeling

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Footprint read counts across sequential filtering steps: total, aligned, uniquely mapped, and UMI-deduplicated reads. Error bars show mean ± SEM. ( B ) Number of footprint reads (UMI-deduplicated) assigned to mRNA, rRNA, ncRNA, and mitochondrial genome-encoded transcripts. Error bars show mean ± SEM. ( C ) Percentage of footprints (UMI-deduplicated reads; mean ± SEM) mapping to various genomic features including the 5’UTR (untranslated region), CDS (coding sequence), 3’UTR and introns. ( D ) Heatmap of Pearson’s r correlations between biological replicates of the synaptic fraction cushion (input), RPLP0 IP, GluA1 IP, and isotype control footprint libraries (log 2 CPM).

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Sequencing, Control

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Multidimensional scaling (MDS) plot showing sample similarity based on the 1,000 most variable genes. ( B ) Distribution of footprint read lengths obtained in Ribo IP libraries. Error bars show mean ± SEM. ( C ) Distribution of footprint read lengths obtained in GluA1 IP libraries. Error bars show mean ± SEM. ( D ) Percentage of P-sites in each reading frame (0, +1, +2) across the 5’ UTR, CDS, and 3’ UTR, using 28-32 nucleotide footprints. Error bars show mean ± SEM. ( E ) Percentage of P-sites in each reading frame (0, +1, +2) across the 5’ UTR, CDS, and 3’ UTR, stratified for read length. ( F ) Metagene plots of P-site density centered on start and stop codons, with each nucleotide position showing the summed P-site count across transcripts. ( G ) MA plot showing all nuclear-encoded protein-coding transcripts, including non-IP-enriched transcripts and nuclear-encoded mitochondrial genes (background).

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques:

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Proteins associated with the GO terms ‘perinuclear region of cytoplasm’, ‘nuclear envelope’, ‘endoplasmic reticulum’, and ‘Golgi apparatus’ are not enriched in the synaptic fraction. ( B ) RNA-seq transcript read counts across sequential filtering steps: total, aligned and uniquely mapped. Error bars show mean ± SEM. ( C ) Number of uniquely-mapped reads assigned to mRNA, rRNA, ncRNA, and mitochondrial genome-encoded transcripts. Error bars show mean ± SEM. ( D ) GO overrepresentation analysis of the 12,283 actively translated synaptic mRNAs shown in , with all detected transcripts in the synaptic RNA-seq and Ribo-seq datasets used as background. Given the relative depletion of most ER-annotated proteins in the synaptic fraction , enrichment of membrane-related terms likely reflects synaptic translation of integral membrane proteins rather than contamination by rough ER-associated ribosomes. Similarly, terms such as ‘vesicle-mediated transport in synapse’, ‘Golgi vesicle transport’, and ‘Golgi apparatus subcompartment’ likely reflect active maintenance/translation of local secretory and trafficking organelles rather than contamination with perinuclear ER and canonical Golgi membranes. ( E ) Top 10 enriched GOBP terms for GluA1- vs RPLP0-enriched transcripts. The dashed line indicates -log10(FDR). ( F ) Top 10 enriched GOMF terms for GluA1- vs RPLP0-enriched transcripts. The dashed line indicates -log10(FDR).

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: RNA Sequencing, Membrane

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Approach for manipulating endogenous GluA1 surface levels (as developed by Kareemo et al. ). GluA1 surface trafficking was prevented by expressing, for 3.5 days, an ER-retained intrabody (via a C-terminal KDEL motif) that binds GluA1 and sequesters it in the ER. ( B ) Images of primary hippocampal dendrites expressing mNeon-tagged, ER-retained versions of either the GluA1 intrabody (GluA1-KDEL) or a control nanobody directed against a GFP epitope, and immunolabeled for surface GluA1, Homer1, and MAP2. Scale bar, 10 µm. ( C ) Quantification of GluA1 surface signal in control and GluA1-KDEL-expressing neurons. Error bars show mean ± SEM. ****p<0.0001, two-tailed unpaired t-test; n=45-46 neurons from 2 independent experiments. ( D ) Detection of nascent CaMKIIα in dendrites from control and GluA1-KDEL expressing neurons. Nascent CaMKIIα was labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 10 µm. ( E ) Quantification of nascent CaMKIIα in synaptic regions from a 50-80 μm dendritic segment from control and GluA1-KDEL expressing neurons. Error bars show mean ± SEM. *p<0.05, ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=23-56 dendritic segments from 12-56 neurons from 2-3 independent cultures.

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Expressing, Control, Immunolabeling, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Estimate of synapse abundance (synaptic area normalized to the total area) for 50-80 μm dendritic segments from control and GluA1-KDEL expressing neurons. Error bars show mean ± SEM. ns=not significant, two-tailed unpaired t-test; n=146-176 dendritic segments from 146-176 neurons from 2-3 independent cultures. ( B ) Detection of nascent Drebrin in dendrites from control and GluA1-KDEL expressing neurons. Nascent Drebrin was labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 10 µm. ( C ) Quantification of nascent Drebrin in postsynaptic regions from a 50-80 μm dendritic segment from control and GluA1-KDEL expressing neurons. Error bars show mean ± SEM. ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=22-48 dendritic segments from 22-48 neurons from 2 independent cultures.

Article Snippet: On DIV 18, cells were treated with 1 μM TTX for 30 min and GluA1 surface immunolabeling was performed live for 5 min at 37°C using a mouse extracellular GluA1 antibody (1:200; Merck, #MAB2263).

Techniques: Control, Expressing, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Schematic of immunoprecipitation-mass spectrometry (IP-MS) workflow. ( B ) Number of protein groups quantified per replicate from the tissue homogenate, synaptic fraction cushion and IP eluate fractions. ( C ) Distinct biochemical strategies (PL-MS and IP-MS) capture AMPA receptors as interactors of synaptic ribosomes. IP proteins were filtered based on uniqueness to the IP or >3-fold enrichment compared to the corresponding isotype control. Protein groups were collapsed to unique gene identifiers prior to overlap analysis. Values correspond to the number of unique gene identifiers. Right: Proteins found in the union of all three interactomes that are associated with the GO terms “AMPA glutamate receptor complex” or “Glutamate receptor signaling pathway”. ( D ) Relative intensities of six selected proteins that are enriched in the ribosome IP and GluA1 IP eluate fractions compared to the corresponding isotype control and are associated with the GO term “AMPA glutamate receptor complex”. Error bars show mean ± SEM. ( E ) GO overrepresentation analysis for proteins in the ribosome IP and GluA1 IP eluate fractions. Shown are the top 10 enriched GOMF terms. ( F ) Average log 2 protein intensities of ribosomal proteins (RPs) in the ribosome IP and GluA1 IP eluate fractions compared to the corresponding isotype control. Horizontal bars represent the median; **p<0.01; ****p<0.0001, Kruskal-Wallis test, Dunn’s multiple comparisons test. See table S2. ( G ) Western blot analysis of ribosomal proteins, AMPA receptor subunits and known AMPA receptor interactors after IP of ribosomes or GluA1, with on-bead washes performed with or without magnesium. Blots are from one of three experimental replicates. Input: 0.25 ug of synaptic cushion (0.5% of IP input), IP: 50% of IP eluate. ( H ) Western blot analysis of PABP, ribosomal proteins and AMPA receptor subunits after IP of ribosomes or GluA1 with and without RNase I treatment. Blots are from one of three experimental replicates. Input: 0.5 ug of synaptic cushion (1% of IP input), IP: 50% of IP eluate. Asterisk indicates a possible glycosylated or partially proteolyzed form of GluA1. Abbreviations: hom., homogenate; syn.cush, synaptic fraction cushion; GOMF, Gene Ontology Molecular Function; IP, immunoprecipitation; mIgG, mouse IgG; rIgG, rabbit IgG; PABP, poly(A)-binding protein.

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Control, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Overview of synaptosome preparation with SynPER. Shown in bold are the fractions used in . ( B ) Synaptic proteins are enriched in SynPER synaptosomes. Shown are the relative intensities of six selected proteins in the tissue homogenate, cytosolic fraction, and synaptic fraction (before cushion ultracentrifugation). Nucleolin serves as a nuclear marker and CDK5 serves as a cytosolic marker. ( C ) Principal component analysis (PCA) of the log 2 protein intensities in the tissue homogenate, synaptic fraction cushion and IP eluate fractions. ( D ) Overall log 2 protein intensities per replicate from the tissue homogenate, synaptic fraction cushion and IP eluate fractions. ( E ) GO overrepresentation analysis for proteins in the ribosome IP and GluA1 IP eluate fractions. Shown are the top 10 enriched GOBP and GOCC terms. ( F ) Total protein stain images for the immunoblots in .

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Marker, Staining, Western Blot

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Confocal image of primary hippocampal neurons immunostained for MAP2 (magenta) and RPS11 (white). Scale bar, 20 µm. ( B ) Confocal images of dendrites immunolabeled for surface GluA1 (magenta), MAP2 (cyan) and RPL36A (green). Scale bar, 2 µm. ( C ) Overview of STED imaging analysis. Scale bar, 0.5 µm. ( D ) Probability density (area = 1) of target-normalized nearest-neighbor distances. Teal: surface GluA1 to RPS11 (normalized by RPS11 density). Purple: RPS11 to surface GluA1 (normalized by surface GluA1 density). Black line: CSR PDF. Excess density at small distances indicates closer-than-random proximity. (GluA1 to RPS11: n=1524 distances (puncta); RPS11 to GluA1: n=4254 distances (puncta). ( E ) Empirical cumulative distributions of the same normalized distances. Black line: CSR CDF. Curves left/above CSR indicate closer-than-random proximity. Dashed vertical line marks the CSR median (0.469). ( F, G ) Median NND per spine compared with simulation values in which surface GluA1 object coordinates were held fixed and RPS11 objects were randomly repositioned within the spine ROI (100 randomizations per spine). For each spine, the simulated value equals the median across its 100 simulated medians. ( F ) Left: paired dot plot of per-spine median surface GluA1 to RPS11 NNDs (exp. vs sim.); lines connect the same spine. Horizontal bars represent the group median across spines. ****p<0.0001, two-tailed Wilcoxon matched-pairs signed rank test. Right: ECDF of per-spine medians (exp. vs sim.). A left/upward shift of the experimental curve indicates shorter medians than the simulated null. ****p<0.0001, two-tailed Kolmogorov-Smirnov test. ( G ) Left: paired dot plot of per-spine median RPS11 to surface GluA1 NNDs (exp. vs sim.); lines connect the same spine. Horizontal bars represent the group median across spines. p=0.1263, two-tailed Wilcoxon matched-pairs signed rank test. Right: ECDF of per-spine medians (exp. vs sim.). ns=not significant (p=0.0141), two-tailed Kolmogorov-Smirnov test; n=323 spines from 100 dendritic segments from 23 cells from three independent cultures. Abbreviations: CSR, complete spatial randomness; PDF, probability density function; CDF, cumulative distribution function; NNDs, nearest-neighbor distances; exp., experimental; sim., simulated.

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Immunolabeling, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Overview of STED imaging analysis pipeline. ( B ) Relative frequency histograms of nearest-neighbor distances. GluA1 to RPS11: n=1524 distances (puncta); RPS11 to GluA1: n=4254 distances (puncta). ( C ) Fraction of total spines per replicate (top) or spines per cell (bottom) containing at least one ribosome located within 100 nm of surface GluA1. Error bars show mean ± SEM; n=323 spines from 100 dendritic segments from 23 cells from 3 independent cultures.

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Imaging

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Immunoprecipitation-ribosome profiling (IP-Ribo-Seq) experimental workflow. ( B ) Overlap of detected transcripts in synapse RNA-seq and synapse Ribo-seq datasets. ( C ) Volcano plot comparing translational levels of 6,889 transcripts between synaptic and cytosolic compartments (RPLP0 IP from synaptic fraction vs. RPLP0 IP from cytosolic fraction). Significantly enriched transcripts are highlighted (FDR ≤ 0.05 and |log 2 FC| ≥ log 2 (1.3)). ( D ) Functional segregation of transcripts differentially translated between synaptic and cytosolic compartments. Shown are the top 5 enriched GOCC terms for synapse- and cytosol-enriched transcripts. ( E ) Functional comparison of synapse-enriched transcripts with neuropil- and somata-enriched transcripts from . Top 15 GOCC terms are shown, ranked by enrichment score (mean log 2 fold enrichment). ( F ) Comparison of relative abundance ranks between synaptic and neuropil compartments. Paired dot plot showing footprint abundance ranks of synapse-enriched transcripts in the synaptic and neuropil translatomes. Transcripts with ≤5% rank change were classified as rank-stable. Transcripts with >40% upward or downward shifts from the neuropil to synaptic compartments were classified as rank-increased and rank-decreased, respectively. ( G ) Top 5 GOCC terms for rank-increased transcripts. ( H ) MA plot comparing the translational level of 7,365 transcripts between GluA1-associated ribosomes and the total synaptic ribosome pool. ( I ) Coverage profiles representing the average GluA1 IP (top) or Ribo IP (bottom) footprint coverage for candidate GluA1-associated ribosome ( Camk2a , Shank1 , Eef2 ) transcripts. The y-axis indicates the number of normalized reads. ( J ) Top 10 enriched GOCC terms for GluA1-vs RPLP0-enriched transcripts. The dashed line indicates -log10(FDR). ( K ) Schematic of a dendritic spine highlighting some of the transcripts preferentially translated by GluA1-associated ribosomes (rose) or the total synaptic ribosome pool (tan). Transcripts that are not enriched in either (i.e., translated in both) are labeled in gray.

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Immunoprecipitation, RNA Sequencing, Functional Assay, Comparison, Labeling

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Footprint read counts across sequential filtering steps: total, aligned, uniquely mapped, and UMI-deduplicated reads. Error bars show mean ± SEM. ( B ) Number of footprint reads (UMI-deduplicated) assigned to mRNA, rRNA, ncRNA, and mitochondrial genome-encoded transcripts. Error bars show mean ± SEM. ( C ) Percentage of footprints (UMI-deduplicated reads; mean ± SEM) mapping to various genomic features including the 5’UTR (untranslated region), CDS (coding sequence), 3’UTR and introns. ( D ) Heatmap of Pearson’s r correlations between biological replicates of the synaptic fraction cushion (input), RPLP0 IP, GluA1 IP, and isotype control footprint libraries (log 2 CPM).

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Sequencing, Control

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Multidimensional scaling (MDS) plot showing sample similarity based on the 1,000 most variable genes. ( B ) Distribution of footprint read lengths obtained in Ribo IP libraries. Error bars show mean ± SEM. ( C ) Distribution of footprint read lengths obtained in GluA1 IP libraries. Error bars show mean ± SEM. ( D ) Percentage of P-sites in each reading frame (0, +1, +2) across the 5’ UTR, CDS, and 3’ UTR, using 28-32 nucleotide footprints. Error bars show mean ± SEM. ( E ) Percentage of P-sites in each reading frame (0, +1, +2) across the 5’ UTR, CDS, and 3’ UTR, stratified for read length. ( F ) Metagene plots of P-site density centered on start and stop codons, with each nucleotide position showing the summed P-site count across transcripts. ( G ) MA plot showing all nuclear-encoded protein-coding transcripts, including non-IP-enriched transcripts and nuclear-encoded mitochondrial genes (background).

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques:

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Proteins associated with the GO terms ‘perinuclear region of cytoplasm’, ‘nuclear envelope’, ‘endoplasmic reticulum’, and ‘Golgi apparatus’ are not enriched in the synaptic fraction. ( B ) RNA-seq transcript read counts across sequential filtering steps: total, aligned and uniquely mapped. Error bars show mean ± SEM. ( C ) Number of uniquely-mapped reads assigned to mRNA, rRNA, ncRNA, and mitochondrial genome-encoded transcripts. Error bars show mean ± SEM. ( D ) GO overrepresentation analysis of the 12,283 actively translated synaptic mRNAs shown in , with all detected transcripts in the synaptic RNA-seq and Ribo-seq datasets used as background. Given the relative depletion of most ER-annotated proteins in the synaptic fraction , enrichment of membrane-related terms likely reflects synaptic translation of integral membrane proteins rather than contamination by rough ER-associated ribosomes. Similarly, terms such as ‘vesicle-mediated transport in synapse’, ‘Golgi vesicle transport’, and ‘Golgi apparatus subcompartment’ likely reflect active maintenance/translation of local secretory and trafficking organelles rather than contamination with perinuclear ER and canonical Golgi membranes. ( E ) Top 10 enriched GOBP terms for GluA1- vs RPLP0-enriched transcripts. The dashed line indicates -log10(FDR). ( F ) Top 10 enriched GOMF terms for GluA1- vs RPLP0-enriched transcripts. The dashed line indicates -log10(FDR).

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: RNA Sequencing, Membrane

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Approach for manipulating endogenous GluA1 surface levels (as developed by Kareemo et al. ). GluA1 surface trafficking was prevented by expressing, for 3.5 days, an ER-retained intrabody (via a C-terminal KDEL motif) that binds GluA1 and sequesters it in the ER. ( B ) Images of primary hippocampal dendrites expressing mNeon-tagged, ER-retained versions of either the GluA1 intrabody (GluA1-KDEL) or a control nanobody directed against a GFP epitope, and immunolabeled for surface GluA1, Homer1, and MAP2. Scale bar, 10 µm. ( C ) Quantification of GluA1 surface signal in control and GluA1-KDEL-expressing neurons. Error bars show mean ± SEM. ****p<0.0001, two-tailed unpaired t-test; n=45-46 neurons from 2 independent experiments. ( D ) Detection of nascent CaMKIIα in dendrites from control and GluA1-KDEL expressing neurons. Nascent CaMKIIα was labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 10 µm. ( E ) Quantification of nascent CaMKIIα in synaptic regions from a 50-80 μm dendritic segment from control and GluA1-KDEL expressing neurons. Error bars show mean ± SEM. *p<0.05, ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=23-56 dendritic segments from 12-56 neurons from 2-3 independent cultures.

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Expressing, Control, Immunolabeling, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Estimate of synapse abundance (synaptic area normalized to the total area) for 50-80 μm dendritic segments from control and GluA1-KDEL expressing neurons. Error bars show mean ± SEM. ns=not significant, two-tailed unpaired t-test; n=146-176 dendritic segments from 146-176 neurons from 2-3 independent cultures. ( B ) Detection of nascent Drebrin in dendrites from control and GluA1-KDEL expressing neurons. Nascent Drebrin was labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 10 µm. ( C ) Quantification of nascent Drebrin in postsynaptic regions from a 50-80 μm dendritic segment from control and GluA1-KDEL expressing neurons. Error bars show mean ± SEM. ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=22-48 dendritic segments from 22-48 neurons from 2 independent cultures.

Article Snippet: For confocal imaging of RPL36A and surface GluA1, nontransduced DIV19 neurons were surface labeled for 10 min at 37°C using a rabbit extracellular GluA1 antibody (1:200; Merck, #ABN241) and stained for MAP2 (1:1000; Synaptic Systems, #188004) and RPL36A (1:500; Santa Cruz Biotechnology, #SC-100831).

Techniques: Control, Expressing, Two Tailed Test, Labeling