Extended Data Fig. 9: Activity-dependent eIF4G2 regulation by uORFs is specific to dendritic mRNAs with eIF4G2 binding sites in their 5′ UTRs.
From: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding
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.
