Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

Abstract

Learning and memory require activity-induced changes in dendritic translation, but which mRNAs are involved and how they are regulated are unclear. In this study, to monitor how depolarization impacts local dendritic biology, we employed a dendritically targeted proximity labeling approach followed by crosslinking immunoprecipitation, ribosome profiling and mass spectrometry. Depolarization of primary cortical neurons with KCl or the glutamate agonist DHPG caused rapid reprogramming of dendritic protein expression, where changes in dendritic mRNAs and proteins are weakly correlated. For a subset of pre-localized messages, depolarization increased the translation of upstream open reading frames (uORFs) and their downstream coding sequences, enabling localized production of proteins involved in long-term potentiation, cell signaling and energy metabolism. This activity-dependent translation was accompanied by the phosphorylation and recruitment of the non-canonical translation initiation factor eIF4G2, and the translated uORFs were sufficient to confer depolarization-induced, eIF4G2-dependent translational control. These studies uncovered an unanticipated mechanism by which activity-dependent uORF translational control by eIF4G2 couples activity to local dendritic remodeling.

Fig. 1. TurboID is a robust methodology to isolate the molecular composition of the postsynaptic compartment in primary cortical neurons.

a, The implementation of TurboID in primary cortical neurons. b, Doxycycline (Dox) induction of TurboID-PSD95 (Flag) and its biotinylation pattern (streptavidin) in the presence and absence of biotin shown by western blots. c, IF to detect TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 after 30 min of biotin incubation. DAPI (blue) marker for nuclei, Flag (red) stain for TurboID and streptavidin (cyan) to demarcate the biotinylated proteins. Magnification, ×40. d, Diagram of neuronal activation workflow with TurboID labeling: neurons are first silenced with TTX (sodium channel blocker) and DL-AP5 (NMDA receptor antagonist) to standardize activity levels in culture; then, activated with KCl (dep) for 1 h or by DGPH for 10 min; and, finally, incubated with the same silencers and biotin to induce biotinylation and allow for recovery (30 min for KCl and 20 min for DHPG). Colors: salmon (resting); burgundy (depolarized by KCl); cyan (DHPG). e, Distribution of streptavidin signal (cyan) in Pan-TurboID-transduced or TurboID-PSD95-transduced activated neurons. DAPI (blue) marker for nuclei. Magnification, ×20. f, Fura-2 AM staining (left) and quantification (right) in resting and KCl-depolarized neurons. Each circle represents information from one field (data are mean ± s.d., 15 fields total from three biological replicates). Significance was derived from the biological replicates and calculated using the two-tailed, unpaired Student’s t-test. Scale bars, 50 μm. Source data

Fig. 2. TurboID combined with CLIP and MS reveals compartment-specific changes of RNAs and proteins in resting and KCl-depolarized neurons.

a, Comparison of PL-CLIP with other dendritic RNA-seq datasets (Methods; FDR < 0.05). b, GSEA on PL-CLIP-enriched transcripts in resting neurons (FDR < 0.05). c, Examples of PL-CLIP-enriched RNAs in resting and KCl-depolarized neurons. Expression (cpm) is plotted as log₂ (n = 4 biologically independent samples, values from PL-CLIP). Colors: salmon (resting); burgundy (depolarized). Significance was calculated using the two-tailed, paired Student’s t-test. P values: NS (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. P values: Shank1 (rest = 0.0033, dep = 0.42), Rpl24 (rest = 0.0061, dep = 0.0047), Map1b (rest = 0.0035, dep = 0.18), Syn1 (rest = 0.027, dep = 0.18), Tamm41 (rest = 0.0064, dep = 0.24), Uqcrc1 (rest = 0.0024, dep = 0.019) and Frmd6 (rest = 0.019, dep = 0.00081). d, GSEA on dendritically enriched RNAs upon depolarization (differential PL-CLIP: depolarized minus resting) (FDR < 0.05). e, Volcano plots showing proteins enriched and de-enriched (by log₂ fold change (FC)) in dendrites according to resting (449 enriched; 835 de-enriched), depolarized (658 enriched; 594 de-enriched) and differential (depolarized minus resting) (808 enriched; 609 de-enriched) PL-MS (n = 5 biologically independent samples). Significance was calculated using the two-tailed, paired (resting and depolarized) and unpaired (differential) Student’s t-test. Multiple testing correction was performed using the Benjamini–Hochberg method (a,b,d). NES, normalized enrichment score.

Fig. 3. TurboID-mediated ribosome profiling reveals activity-dependent increase in ribosome occupancy and uORF usage in 5′ UTRs of dendritic mRNAs.

a, Schematic for PL-Ribo-seq. TurboID-PSD95 labels dendritic ribosomes in primary cortical neurons. RPL10A western blot showing the input and streptavidin pulldown fractions from TurboID-PSD95-expressing neurons that are incubated with (+biotin) and without (−biotin) biotin for 30 min. Percentages refer to the volumetric percentage loaded on the gel. b, In TurboID-PSD95-transduced neurons, biotinylation (streptavidin, cyan) can be detected only in dendrites, even though RPL10A (red) stains across the whole neuron, as detected by IF. DAPI (blue) marker for nuclei. Magnification, ×20. Scale bar, 50 μm. c, Streptavidin pulldown and input RPKM values (log₂) for each gene are plotted for Pan-TurboID (left) and TurboID-PSD95 (right) PL-Ribo-seq. Pan-TurboID pulldown and input correlation indicates that Pan-TurboID represents information from the whole neuron. d, GSEA on dendritically translated RNAs by resting PL-Ribo-seq (FDR < 0.05). Multiple testing correction was performed using the Benjamini–Hochberg method. e, Examples of RNAs that are translationally upregulated in dendrites in response to depolarization by PL-Ribo-seq (n = 3 biologically independent samples, values from PL-Ribo-seq). Dendritic log₂ enrichment of translation for each RNA shown in resting and depolarized conditions. Significance was calculated using the two-tailed, unpaired Student’s t-test. Data are presented as mean ± s.d. P values: NS (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. P values: Spkh1 = 0.00046, Rgs14 = 0.011, Ghrl = 7.12 × 10⁻⁸, Apobec1 = 0.0075, Igf2bp1 = 0.00053, Pml = 0.0023, Eif1ad3 = 0.0022, P2rx7 = 0.024 and Timm23 = 0.0052. f, Examples of gene sets (n indicates number of genes detected in each gene set) that are translationally upregulated in dendrites with neuronal depolarization and that correspond to examples of RNAs from e. Significance for box plots was determined by the two-sided Wilcoxon signed-rank test. g, Ribosome occupancy in 5′ UTRs of all detected RNAs (n = 14,684 in rest; n = 14,770 in dep) in the whole neuron (input) and in dendrites (pulldown) detected by PL-Ribo-seq from TurboID-PSD95-transduced neurons. RPKM changes for each region between resting and depolarized conditions were tested using the permutation t-test. The P values were then adjusted with Bonferroni correction. h, Dendritic translation of 5′ UTRs with uORFs identified by ORF-RATER in resting and depolarized PL-Ribo-seq. Rest: n of all 5′ UTRs = 10,818; uORFs = 967. Dep: n of all 5′ UTRs = 10,718; uORFs = 859. Significance for box plots was determined by the one-sided Wilcoxon signed-rank test. i, Examples of RNAs with uORFs in their 5′ UTRs. PL-Ribo-seq reads (log₂) in 5′ UTRs and CDS from the pulldowns (pd) of TurboID-PSD95-transduced neurons are shown for resting (top) and depolarized (bottom) dendrites. j, GSEA performed on translationally upregulated and downregulated dendritic RNAs with increased 5′ UTR translation in response to depolarization (P < 0.01). P values were derived using the ‘fgsea’ package and the adaptive multi-level split Monte Carlo method. Colors: salmon (resting); burgundy (depolarized). Box plots show lower and upper hinges corresponding to the first and third quartiles (representing the 25th and 75th percentiles, respectively). Whiskers extend from the hinge to the 1.5 × interquartile range. The center line indicates the median. FC, fold change; NES, normalized enrichment score. Source data

Fig. 4. uORFs in the 5′ UTRs of dendritic mRNAs regulate downstream CDS translation upon neuronal activation.

a, Dendritic GFP reporter with myristoylation (myr) and LDLR-C-terminal (LDLRct) sequences and its localization by Flag (green) IF. DAPI (blue) marker for nuclei; MAP2 (red) marker for dendrites. Magnification, ×20. Scale bar, 50 µm. b, Flag western blot for input (in) and pulldown (pd) from TurboID-PSD95-transduced neurons. c, Coverage of PL-Ribo-seq pulldown (log₂) for Kcnj9 uORF and main ORF from resting and depolarized (dep) TurboID-PSD95-transduced neurons. d, Myristoylated, myristoylated with Kcnj9-5′ UTR, myristoylated with LDLRct and myristoylated with LDLRct and Kcnj9-5′ UTR reporters in resting and depolarized neurons. Myristoylation and LDLRct sequences are in the CDS. GFP fold changes (dep/rest) from Flag (GFP) and β-Actin western blots are calculated as: [Flag^Dep / β-Actin^Dep] / [Flag^Rest / β-Actin^Rest]. All are compared to myrGFP negative control (n = 3). The center line is at mean. e,f, GFP fold changes of dendritic reporters with 5′ UTRs housing uORFs with increased translation upon depolarization (5′ UTR-up) and translationally upregulated (CDS-up) (e) or downregulated (CDS-down) (f) CDS. Negative controls: myrGFP, scrambled-Efcab-, -Rspo3-, -Immp1l- and -Spink10-5′ UTRs. All are compared to myrGFP (n = 4). P values: Efcab1-scr = 0.16; Rspo3-scr = 0.59; Cmc4 = 0.013; Efcab1 = 0.0067; Katnb1l = 0.021; Lrrc51 = 0.0038; Nsun3 = 0.00011; Polg = 0.89; Rspo3 = 0.0086; Slc25a19 = 0.011; Zfp286 = 0.0089; Immp1l-scr = 0.089; Spink10-scr = 0.063; Cnep1r1 = 0.0072; Cptp = 0.0021; Ikzf5 = 0.00029; Immp1l = 0.00041; Mphosph9 = 0.00067; Mtif2 = 0.00077; Spink10 = 0.0032; and Vrk3 = 0.00031. g,h, Western blots of Mphosph9 and Kcnj9 CDS translation with and without their 5′ UTRs (g) are quantified by normalizing Flag expression in each condition to β-Actin levels (h). In the same plot, also shown are the qPCRs of reporters, normalized as described for the western blots (n = 3). Protein changes of CDS with 5′ UTRs are indicated by red asterisks. P values: Mphosph9 (protein = 0.13; RNA = 0.0017), 5′ UTR + Mphosph (protein = 0.0014; RNA = 0.066), Kcnj9 (protein = 0.49; RNA = 0.091), 5′ UTR + Kcnj9 (protein =0.00031; RNA = 0.34). i,j, IF images (i) and quantifications (j) of MPHOSPH9 (downregulated) and KCNJ9 (upregulated) with MAP2 (dendrites) and DAPI (nuclei) in resting and depolarized neurons (n = 3). Magnification, ×20. Scale bars, 25 µm. All data are presented as mean ± s.d. Significance was calculated by two-tailed paired (d–f) and unpaired (h,j) Student’s t-test. P values: NS (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples. Source data

Fig. 5. eIF4G2 is required to upregulate dendritic mRNA translation upon activation.

a, Dendritic mRNAs with increased ribosome occupancy in their 5′ UTRs upon depolarization are divided into two groups: translationally upregulated (CDS-up) and downregulated (CDS-down) in their CDS. Enriched mRNA binding protein (RBP) sites in the 5′ UTRs of these groups are shown in the heatmap using motifs from RBPmap. P values were determined using hypergeometric testing. b, GSEA performed on differential (depolarized minus resting) log₂-ranked eIF4G2 CLIP target mRNAs bound in their 5′ UTRs. P values were derived using the ‘fgsea’ package and the adaptive multi-level split Monte Carlo method. NES, normalized enrichment score. c, mRNAs with increased eIF4G2 binding in their 5′ UTRs upon depolarization are determined by eIF4G2 CLIP and referred to as eIF4G2-bound. CDS translation of all detected mRNAs in the differential PL-Ribo-seq (depolarized (dep) minus resting (rest)) (all, n = 16,759) and the eIF4G2-bound group (n = 1,420) is compared. d, Nsun3-5′ UTR reporters: wild-type (Wt) harbors eIF4G2 binding sites (vertical bars); eIF4G2++, two more eIF4G2 binding sites added; eIF4G2−, endogenous eIF4G2 binding sites scrambled. uORF mutant Nsun3-5′ UTR reporters: Start mut (start codon mutated); Elong (stop codon inserted after start); Stop (stop codon mutated). Fold changes are quantified from western blots of Flag and β-Actin as in Fig. 4d, and significance was calculated by comparing to Wt (n = 3). P values: Start mut = 0.0022, eIF4G2++ = 0.59, eIF4G2− = 0.0023, Elong = 0.0010 and Stop = 0.0034. e, Western blot quantifications of Flag and β-Actin of Nsun3-5′ UTR reporters in non-targeting (−) or eIF4G2 siRNA-treated (+) conditions in resting and depolarized neurons are quantified as in Fig. 4d (n = 4). Box plot whiskers extend to minimum and maximum, with the center line at median. Significance was calculated using the two-tailed, unpaired Student’s t-test. f, Subset of eIF4G2-bound mRNAs that are translationally upregulated (n = 321) are shown in Wt (all, n = 16,759) and eIF4G2 knockdown (all, n = 17,547) PL-Ribo-seq data, in both conditions compared to all detected mRNAs in the corresponding dep-rest PL-Ribo-seq. g, Dendritic translational regulation of the Wt Nsun3-5′ UTR reporter is tested in resting and depolarized Wt and eIF4G2 knockdown (Kd) neurons, in which eIF4G2 levels are rescued by a dendritically localized Wt or phospho-mutant version of eIF4G2. Fold changes from western blots of Flag and β-Actin are quantified as in Fig. 4d. Significance was calculated by comparing to Wt (n = 3). P values: Kd = 0.0090, Kd+Wt = 0.39, Kd+Mut1 = 0.00055, Kd+Mut2 = 0.66, Kd+Mut3 = 0.52 and Kd+Mut4 = 0.041. c,f, Significance was calculated using the two-sided Kolmogorov–Smirnov test. Box plots show lower and upper hinges corresponding to 25th and 75th percentiles. Whiskers extend from the hinge to the 1.5 × interquartile range. The center line indicates the median. d,g, Significance was calculated using the two-tailed, paired Student’s t-test. All data are presented as mean ± s.d. P values: NS (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Fig. 6. Model for activity-dependent eIF4G2:uORF-mediated translational control in dendrites.

A subset of dendritically enriched mRNAs, including those with roles in signaling and mitochondrial functions, is translationally silent in resting postsynaptic sites. We demonstrate that, after neuronal activation and calcium influx, eIF4G2 is phosphorylated at threonine (T507), enabling direct binding to select 5′ UTRs (Fig. 5g) to upregulate the translation of the downstream CDSs. Translation of uORFs from these 5′ UTRs is enhanced even though overall dendritic translation is suppressed (Fig. 3h and Extended Data Figs. 5e and 6c). eIF4G2 binding then allows efficient scanning of ribosomes into the downstream CDSs, enabling the rapid production of dendritic proteins that are needed for synaptic plasticity and energy homeostasis.

Extended Data Fig. 1. Labeling by TurboID and activation by KCl or DHPG are used to study resting and activated dendritic molecular profiles in primary cortical neurons.

a, Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b, TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate (n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c, IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d, IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e, IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f, Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right (n = 3); relative levels are not significant by two-tailed, paired Student’s t-test. g, Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h, Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs (n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i, Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) (n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j, Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO₂)) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP (n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO₂ = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO₂ = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO₂ = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO₂ = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO₂ = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO₂ = 0.024). k, Quantitative PCR (qPCR) results shown for immediate early genes, Arc, Fos and Jun. The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state (n = 3). l, Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted (n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t-test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m, Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t-test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples. Source data

Extended Data Fig. 2. PL-CLIP identifies dendritic RNAs.

a, The expression of neuronal (mouse CA1 pyramidal and developing forebrain) and non-neuronal (oligodendrocytes, astrocytes, glial cells, microglial cells, endothelial cells and mural cells) transcripts are assessed among RNAs detected by PL-CLIP. Significance was determined by the two-sided Wilcoxon signed-rank test. P values: Neuron and CA1 pyramidal < 2.2e⁻¹⁶, Oligodendrocyte = 0.00013, Astrocyte = 1.71e⁻⁸, Glia < 2.2e⁻¹⁶, Microglia = 2.77e⁻¹², Endothelial = 0.21, Mural = 0.00043. b, Principal component analysis (PCA) shown for Pan-TurboID and TurboID-PSD95 inputs (left) and streptavidin pulldowns (right) in resting and depolarized PL-CLIP (n = 4 biologically independent samples). Inputs separate only by neuronal state but not by TurboID; whereas, pulldowns are differentiated by state and TurboID (localization). c, Comparison of PL-CLIP-enriched RNAs with previously published dendritic and axonal^– RNAs (FDR < 0.05). Multiple testing correction was performed using the Benjamini-Hochberg method. d, Cumulative distribution function (CDF) plot of PL-CLIP RNAs, dendrite-enriched/present RNAs (left) and FMRP CA1 targets (right) from. FC: fold change. Significance was calculated by the two-sided Wilcoxon signed-rank test. P values: Dendrite present and enriched < 2.2e⁻¹⁶, CA1 FMRP targets = 0.00059, Dendritic FMRP targets = 3.69e⁻¹⁰, FMRP cell body targets < 2.2e⁻¹⁶, FMRP synaptic targets = 2.23e⁻¹³. e, Examples of dendritically-enriched RNAs that encode chromatin, Golgi and protein complex localization-related proteins (n = 4, values from PL-CLIP). P values: Cbx = 0.049, Fanca = 0.026, Foxo3 = 0.012, Rad50 = 0.0022, Pde9a = 0.019, Abca1 = 0.032, Hap1 = 0.035, Nptxr = 0.022. Significance was calculated using the two-tailed, paired Student’s t-test. f, Left: Dendritic enrichment of Chop, Ire1 and Atf4 levels in resting and depolarized neurons. Dendritic enrichment (log₂) is calculated by normalizing the transcript levels in TurboID-PSD95 to those in Pan-TurboID. P values: Chop = 0.038, Ire1 = 0.47, Atf4 = 0.99. Right: Total Chop, Ire1 and Atf4 expression levels (log₂ cpm) are shown in the inputs from resting and depolarized Pan-TurboID-transduced neurons (n = 4, values from PL-CLIP). P values: Chop = 0.14, Ire1 = 0.95, Atf4 = 0.052. Significance was calculated using the two-tailed, unpaired Student’s t-test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Extended Data Fig. 3. PL-CLIP identifies properties of dendritic RNAs in resting and depolarized neurons.

a, Length and (b) GC content of resting and depolarized PL-CLIP-enriched dendritic RNAs and all detected RNAs in cortical neurons (‘All’, n = 17,654) are compared. Colors: salmon (resting, n = 2,788); burgundy (depolarized, n = 3,727). Significance was calculated by the two-sided Wilcoxon signed-rank test. Box plots show lower and upper hinges corresponding to the first and third quartiles (representing 25th and 75th percentile, respectively). Whiskers extend from the hinge to the 1.5x interquartile range. The center line indicates the median. P values: all significance values reported are < 2.2e-16 except for Transcript length (Rest vs. Dep = 4.18e-15), CDS (Rest vs. Dep = 0.081), 5′UTR (All vs. Rest = 2.10e-8; All vs. Dep = 4.22e-10; Rest vs. Dep = 0.93), 3′UTR (All vs. Rest = 0.15). c, RNA fluorescence in situ hybridization (RNA-FISH) combined with MAP2 IF to show dendrites and dendritically de-enriched (Snca) and enriched (Kmt2d, Map2 and Dlg4) RNAs by resting PL-CLIP. Images are quantified on the right (n = 3). Significance was derived from biological replicates, showing the center line at median and using the two-tailed, paired Student’s t-test. d, RNA-FISH combined with IF showing dendritic (MAP2-green) localization of Rapgef4 (red) and Ppp1r9b (white) in resting and depolarized neurons, (e) quantified for soma and dendrites. 4 fields of images per replicate for n = 2 presented as mean ±s.d. All images are taken at ×40 magnification, except for Dlg4, which was taken at ×60. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. Scale bars, 100 μm. n indicates the number of biologically independent samples.

Extended Data Fig. 4. PL-MS identifies the dendritic proteome and its relation to local RNA levels.

a, PCA plot showing five replicates of PL-MS from Pan-TurboID and TurboID-PSD95-transduced resting and depolarized neurons. Minus biotin counterpart of each sample is subtracted for each dot on the plot. b, Comparison of PL-MS-enriched proteome with previously published dataset (FDR < 0.05). c, TurboID-PSD95-enriched proteome vs. Distler et al. data (FDR < 0.05). Multiple testing correction was performed using the Benjamini-Hochberg method. d, Pearson correlation of PL-MS and PL-CLIP on dendritic RNAs in resting neurons. e, Shown are the distributions of PL-MS-enriched dendritic proteome from resting (rest), depolarized (dep) and differential (dep minus rest) data in ranked resting PL-CLIP using a GSEA-based strategy (FDR < 0.05) (Methods). Multiple testing correction was performed using the Benjamini-Hochberg method in the fgsea package. P adjusted: Rest = 0.0026, Dep = 0.18, Dep-Rest = 0.0027. P values: ** <0.01. f, Pearson correlation of PL-MS and PL-CLIP on dendritic RNAs in depolarized neurons. (d,f) The reported P values are two-sided.

Extended Data Fig. 5. PL-Ribo-seq accurately identifies locally translated RNAs and their relation to local protein levels in dendrites.

a, Percent RPL10A recovered from pulldown samples from Pan-TurboID or TurboID-PSD95-transduced neurons was calculated by dividing the pulldown band (100% volume loaded) value by that of the input band (10% volume loaded) (n = 3). The center line is at median. Significance was calculated using the two-tailed, paired Student’s t-test. b, Quality metrics of ribosome profiling data on the streptavidin pulldown fraction of TurboID-PSD95 in resting neurons shown on the top row. The coverage in the CDS and UTRs are shown. Similar metrics are shown for the input fraction in the bottom two rows: the read length distribution, percentage of P-sites in the CDS and UTRs along with the length of each region, the P-site signal in each reading frame and the P-site coverage from the start and stop codons. c, PCA shown for Pan-TurboID and TurboID-PSD95 input (left) and streptavidin pulldown (right) fractions in resting and depolarized neurons for three replicates of PL-Ribo-seq. d, Comparison of TurboID-PSD95-enriched translated RNAs in resting neurons with previously published sets of dendritic and axonal translatomes using a GSEA-based strategy. (FDR < 0.05). e, Ribosome profiling RPKM read distribution in the coding sequences (CDS) of all detected RNAs (n of Rest = 18,855, n of Dep = 18,887) in inputs and streptavidin pulldowns from Pan-TurboID and TurboID-PSD95-transduced resting and depolarized neurons. RPKM changes for each region between resting and depolarized conditions were tested using the permutation t-test. The P values were then adjusted with Bonferroni correction. Box plots show lower and upper hinges corresponding to the first and third quartiles (representing 25th and 75th percentile, respectively). Whiskers extend from the hinge to the 1.5x interquartile range. The center line indicates the median. f, Puromycin incorporation in resting and depolarized neurons shown by IF using a puromycin-specific antibody. DAPI (blue) marker for nuclei, red for puromycin. Magnification, ×20. Corrected fluorescence per cell was calculated by counting 45 cells (n = 3). Significance was derived from the biological replicate averages and calculated using the two-tailed, unpaired Student’s t-test. The center line is at median. Scale bars, 50 μm. g, Pearson correlation of PL-MS and PL-Ribo-seq on dendritic RNAs in resting neurons. h, Shown are the distributions of PL-MS-enriched dendritic proteome from resting (rest), depolarized (dep) and differential (dep minus rest) data in ranked resting PL-Ribo-seq using a GSEA-based strategy (FDR < 0.05). P adjusted: Rest = 3.70e⁻⁹, Dep = 0.0019, Dep-Rest = 0.69. i, Pearson correlation of PL-MS and PL-Ribo-seq on dendritic RNAs in depolarized neurons. j, Shown are the distributions of PL-MS-enriched dendritic proteome from resting, depolarized and differential data in ranked differential PL-Ribo-seq using a GSEA-based strategy (FDR < 0.05). P adjusted = Rest = 0.00083, Dep = 1.60e⁻¹³, Dep-Rest = 6.95e⁻¹⁶. P values: ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples. (d,h,j) Multiple testing correction was performed using the Benjamini-Hochberg method. (g,i) The reported P values are two-sided. Source data

Extended Data Fig. 6. Dendritically localized reporters reveal the effects of 5′ UTRs on the downstream translation in an activity-dependent manner.

a, Shown is the Pearson correlation of differential (depolarized minus resting) PL-Ribo-seq counts (log₂ RPKM) from 5′UTR and CDS across all transcripts detected by dendritic ribosome profiling. Correlation coefficient, R, and the two-sided P value are shown in the upper right corner. b, Results of ORF-RATER analysis on all detected ORFs (top) and start codon usage (bottom). c, Dendritic translation of uORFs that are identified by ORF-RATER in DHPG-activated PL-Ribo-seq (n of all 5′UTRs = 10,313; uORFs = 881). Significance was calculated by the one-sided Wilcoxon signed-rank test. Box plots show lower and upper hinges corresponding to the first and third quartiles (representing 25th and 75th percentile, respectively). Whiskers extend from the hinge to the 1.5x interquartile range. The center line indicates the median. d, Different dendritic localization signals, Camk2a-3′UTR and BC1, are tested by streptavidin pulldowns from TurboID-PSD95 expressing cortical neurons. e, FISH and IF performed on superfolder GFP localized with BC1, Camk2a-3′UTR and myr-LDRct localization signals. DAPI shown for nuclei. For FISH and IF, GFP RNA and protein (by Flag antibody) are targeted, respectively. Magnification, ×20. Scale bars, 25 µm. f, ORF-RATER ribosome coverage in the 5′UTR and CDS of Kcnj9, with a non-cognate start codon in its uORF, GTG. Harr: harringtonine-treated; Chx: cycloheximide-treated; Unt: untreated neurons. g, Shown are western blots that are quantified in Fig. 4e and f. GFP protein detected by Flag, and β-Actin used as loading control. h, GFP fold changes of dendritic reporters with Cmc4, Lrrc51 and Nsun3 5′UTRs are quantified by Flag and β-Actin western blots and qPCRs upon neuronal activation by DHPG (n = 3 biologically independent samples). All data are presented as mean ±s.d. Significance was calculated using the two-tailed, unpaired Student’s t-test. P values: Cmc4 (protein = 0.00016; RNA = 0.64), Lrrc51 (protein = 0.0010; RNA = 0.98), Nsun3 (protein = 0.0027; RNA = 0.42). i, Larger fields of IFs for MPHOSPH9 (downregulated) and KCNJ9 (upregulated) in resting and depolarized (dep) neurons in support of Fig. 4i. Magnification, ×20. Scale bars, 25 µm. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. Source data

Extended Data Fig. 7. eIF4G2 binding increases in the 5′ UTRs of dendritic mRNAs upon depolarization and is associated with their increased translation.

a, RBP sites in the 5′ UTRs of mRNAs enriched in resting (rest) and depolarized (dep) dendrites are shown in the heatmap using motifs from RBPmap. P values were determined using hypergeometric testing. The RBP sites enriched more upon depolarization are shown in the delta (dep minus rest) column. b, The most enriched binding motif of eIF4G2 in 5′ UTRs that are increasingly translated in dendrites upon depolarization by PL-Ribo-seq. eIF4G2 has 4 predicted motifs in RBPmap. c, The distribution of eIF4G2 CLIP peak counts in 3′ UTRs, 5′ UTRs and CDS for all 4 eIF4G2 binding motifs. d, Examples of eIF4G2 target mRNAs identified in resting eIF4G2 CLIP (peak heights, log₂); peaks in similar genomic locations were identified in human cell lines by eCLIP. Pcdh and Pafah1b1 harbor uORFs in their 5′ UTRs. e, The distribution of eIF4G2 CLIP peaks is calculated by combining the eIF4G2 peaks from resting and depolarized primary cortical neurons. f, mRNAs with 5′ UTRs that are increasingly bound by eIF4G2 upon depolarization as determined by eIF4G2 CLIP are referred to as eIF4G2-bound. All detected mRNAs in the differential (depolarized (dep) minus resting (rest)) PL-CLIP (All, n = 17,654) are compared to the eIF4G2-bound group (n = 1,413). g, Differential dendritic CDS translation of all mRNAs with increased ribosomes in their 5′ UTRs (All 5′UTR up, n = 1,920) and differential dendritic CDS translation of mRNAs with increased eIF4G2 binding and increased ribosome occupancy in their 5′ UTRs (5′UTR up + eIF4G2-bound, n = 238) are compared. The latter group is translated more in dendrites upon depolarization compared to the average of all mRNAs with increased ribosome occupancy in their 5′UTRs in dendrites. (f,g) Significance was calculated using the two-sided Kolmogorov-Smirnov test. Box plots show lower and upper hinges corresponding to the first and third quartiles (representing 25th and 75th percentile, respectively). Whiskers extend from the hinge to the 1.5x interquartile range. The center line indicates the median. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001.

Extended Data Fig. 8. Nsun3 is translationally upregulated in dendrites upon depolarization.

a, Shown on the left is the dendritic translation by PL-Ribo-seq (log₂) in the 5′ UTR, CDS and 3′ UTR of Nsun3 in resting and depolarized neurons. Shown on the right are the PL-CLIP expression values (log₂) of Nsun3 in the Pan-TurboID and TurboID-PSD95 pulldowns from resting and depolarized neurons (n = 3, values from PL-Ribo-seq). P values: PL-Ribo-seq (5′UTR = 0.019; CDS = 0.0035; 3′UTR = 0.64), PL-CLIP (Pan = 0.88, PSD95 = 0.045). b, Protein and RNA analyses of Nsun3-5′UTR reporters in resting (Dep-) and depolarized (Dep+) neurons. Wild type (Wt) Nsun3-5′UTR harbors eIF4G2 binding sites; eIF4G2++, additional eIF4G2 binding sites; eIF4G2-, endogenous eIF4G2 binding sites scrambled. uORF mutants: Start mut (start codon mutated); Elong (stop codon inserted after start); Stop (stop codon mutated). Shown on the left are the Flag and β-Actin western blots that are quantified in Fig. 5d. Shown on the right are the qPCR quantifications (quantified as in Fig. 4d) for each Nsun3 reporter (n = 3). Significance was calculated by comparing to the Wt and using the two-tailed, paired Student’s t-test. P values: Start mut = 0.0098, eIF4G2 + + = 0.26, eIF4G2- = 0.014, Elong = 0.048, Stop = 0.0026. c, Nascent translation (by proximity ligation assay, PLA) and (d) total protein (by IF) levels of NSUN3 are shown (n = 2) plotted as floating box plots from max to min with center lines at mean). Magnification, ×20. Scale bars, 25 µm. e, Western blots of Flag and β-Actin of Nsun3-5′UTR reporters in non-targeting (−) or eIF4G2 siRNA-treated (+) conditions in resting and depolarized neurons for Fig. 5e. Western blot of eIF4G2 and the quantifications of the knockdowns are shown (n = 3). (a,e) Significance was calculated using the two-tailed, unpaired Student’s t-test. (a,b,e) All data are presented as mean ±s.d. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples. Source data

Extended Data Fig. 9. Activity-dependent eIF4G2 regulation by uORFs is specific to dendritic mRNAs with eIF4G2 binding sites in their 5′ UTRs.

a, Shown on the left is the dendritic translation by PL-Ribo-seq (log₂) in the 5′ UTR, CDS and 3′ UTR of Mtf1 in resting and depolarized neurons (n = 3). Shown on the right are the PL-CLIP expression values (log₂) of Mtf1 in the Pan-TurboID and TurboID-PSD95 pulldowns from resting and depolarized neurons (n = 4). P values: PL-Ribo-seq (5′UTR = 0.0079; CDS = 0.00014; 3′UTR = 0.39), PL-CLIP (Pan = 0.74; PSD95 = 0.87). b, Reporter constructs with wild type (Wt) Mtf1 5′UTR, which have eIF4G2 binding sites (black vertical bars), and with Mtf1 5′UTR with mutations in its uORF. ATG, uORF start codon mutated to ATG; Start, uORF start codon mutated to GAG; Scr, uORF sequence scrambled with start and stop codons unchanged; eIF4G2-, eIF4G2 binding sites scrambled; Insert, distance between the uORF and GFP increased by randomized 15 nucleotide insertion; Elong, stop codon inserted after the start codon in the uORF; Stop, stop codon in the uORF is mutated to prevent uORF termination. The quantifications of the GFP protein and mRNA fold changes by western blots and qPCRs, respectively, are calculated and shown as in Fig. 4d (n = 3). P values: protein (Atg = 0.0091; Start = 0.00028; Scr = 0.0026; eIF4G2- = 0.0012; Insert= 0.029; Elong = 0.00073; Stop = 0.00046), RNA (Atg = 0.19; Start = 0.0092; Scr = 0.044; eIF4G2- = 0.43; Insert = 0.49; Elong = 0.065; Stop = 0.37). (c, d) Similar to (a) and (b), PL-Ribo-seq and PL-CLIP data for Zfp64 and the constructs with Zfp64 5′UTR (n = 3) are shown. P values: PL-Ribo-seq (5′UTR = 0.011; CDS = 0.040; 3′UTR = 0.66), PL-CLIP (Pan = 0.18; PSD95 = 0.0069). Zfp64 5′UTR does not have eIF4G2 binding sites. For Zfp64, a variant version of the uORF, where it is duplicated, is included (2x) as well as Start and Insert mutants designed as in (b). P values: protein (2x = 0.047; Start = 0.00039; Insert = 0.0049), RNA (2x = 0.016; Start = 0.043; Insert = 0.019). (e, f) Similar to (a) and (b), PL-Ribo-seq and PL-CLIP data for Katnbl1 and the constructs with Katnbl1 5′UTR (n = 3) are shown. P values: PL-Ribo-seq (5′UTR = 0.013; CDS = 0.0037; 3′UTR = 0.38), PL-CLIP (Pan = 0.86; PSD95 = 0.68). Since Katnbl1 uORF has two overlapping ORFs, separate start codon mutations (St 1 and St 2) as well as a double mutant (1 + 2) are included. Deletion (Del) mutant: the distance between the uORF and GFP start codon is decreased by 30 nucleotides. Katnbl1 5′UTR does not have eIF4G2 binding sites. P values: protein (St 1 = 0.0029; St 2 = 0.0057; 1 + 2 = 0.0062; Del = 0.0061), RNA (St 1 = 0.35; St 2 = 0.13; 1 + 2 = 0.085; Del = 0.022). (g-i) Nascent translation and total protein levels using PLA and IF, respectively, shown for G) MTF1, H) ZFP64 and I) KATNBL1. Magnification, ×20. Quantifications are shown on the right (n = 2) plotted as floating box plots from max to min with center lines at mean. j, Western blots showing GFP translation (Flag) from constructs with wild type and eIF4G2- Mtf1 5′UTR, Zfp64 5′UTR and Katnbl1 5′UTR with (siRNA+) and without eIF4G2 (siRNA-) knockdown. β-Actin used as loading control; eIF4G2 shown to indicate knockdowns. k, Flag and β-Actin western blots from (j) are quantified as in Fig. 4d. Significance was calculated between siRNA- and siRNA+ conditions (n = 3). The center lines for box plots are at mean. P values: Mtf1 = 0.0013, eIF4G2- Mtf1 = 0.55, Zfp64 = 0.72, Katnbl1 = 0.65. (a,c,e,k) Significance was calculated using the two-tailed, unpaired Student’s t-test. (b,d,f) All data are presented as mean ±s.d. Significance was calculated with respect to Wt, using the two-tailed, paired Student’s t-test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples. Source data

Extended Data Fig. 10. Translational upregulation of dendritic downstream ORFs by eIF4G2 is mediated by its local calcium-influx-induced phosphorylation.

a, CDF plots of dendritic translation of eIF4G2-bound transcripts that harbor uORFs in wild type and eIF4G2 knockdown conditions. Significance was calculated by the two-sided Wilcoxon signed-rank test. b, Dendritic enrichment (log₂) of eIF4G2 is shown using the resting and depolarized PL-MS data (n = 5, values from PL-MS data). Significance was calculated using the two-tailed, unpaired Student’s t-test. Box plot whiskers extend from min to max, with the center line at median. c, Nascent translation of Nsun3 and Slc25a19, examples of nuclear-encoded mitochondria-related RNAs, is shown by PLA (red) in the presence (EGTA-) and upon depletion (EGTA+) of calcium in resting and KCl-depolarized neurons (n = 2). DAPI (blue, marker for nuclei). d, Shown are the Flag and β-Actin western blots that are quantified in Fig. 5g. eIF4G2 western blot shows endogenous eIF4G2 (lower bands), which is reduced by siRNA knockdowns (Kd), as well as the localized wild type and phospho-mutant eIF4G2 variants (upper bands, higher molecular weight due to the inclusion of dendritic localization signals). Fold changes are shown as in Fig. 5g but here significance was calculated with respect to the knockdown, using the two-tailed, paired Student’s t-test (n = 3). (c,d) All data are presented as mean ±s.d. n indicates the number of biologically independent samples. Source data