Fig. 3: TurboID-mediated ribosome profiling reveals activity-dependent increase in ribosome occupancy and uORF usage in 5′ UTRs of dendritic mRNAs.
From: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding
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.
