Extended Data Fig. 6: Dendritically localized reporters reveal the effects of 5′ UTRs on the downstream translation in an activity-dependent manner.
From: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding
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.
