Shigeoka T , Koppers M , Wong HH , Lin JQ , Cagnetta R , Dwivedy A , de Freitas Nascimento J , van Tartwijk FW , Ströhl F , Cioni JM , Schaeffer J , Carrington M , Kaminski CF , Jung H , Harris WA , Holt CE .

Wellcome Trust , MR/K015850/1 Medical Research Council , MR/K02292X/1 Medical Research Council , 322817 European Research Council, 203249/Z/16/Z Wellcome Trust

	Figure 1. RP-Coding mRNAs Harbor a Common Loop Structure-Forming Sequence Motif in the 5′ UTR(A) Enrichment of GO terms in the Xenopus laevis RGC axon transcriptome.(B) Relative abundance (FPKM) of translated mRNAs coding for RPs (red) and other proteins (gray) in the mouse RGC axon (y axis) and retina (x axis), obtained by the Axon-TRAP system in vivo. The histogram (lower) shows the distribution of the ratio of abundance of 2 consecutive stages (refinement [P7.5]/branching [P0.5]). p value: Kolmogorov-Smirnov test between RP coding mRNAs and non-RP mRNAs.(C) Relative position of the CUIC and RNA-secondary structure of 5′ UTRs of mouse RP-coding mRNAs. Each bar represents 5′ UTR sequences and is colored by the predicted secondary structure. The position of CUIC is aligned at center (0 nt) and the x axis indicates the distance from the CUIC motif.(D) Average fraction of double-stranded nucleotides around the CUIC motif in 5′ UTRs of all CUIC-containing mouse genes and RPs (moving average, 7-nt window).(E) The ranking of RBPs that specifically bind to the CUIC motif of RP-coding mRNAs. The heatmap color indicates the specificity score calculated by the formula in the upper panel. The histogram (left) shows the average of the specificity score of each RBP for all CUIC-containing RP mRNAs. Red dots mark eIF3 components, and blue dots mark TIA1 and TIAL1.(F) UCSC Genome browser view of CLIP clusters of eIF3 components on 2 RP-coding mRNAs.
	Figure 2. CUIC Motif Is Involved with Alternative 5′ End and Netrin-1-Stimulated Translation of Axonal RP mRNAs(A) Plot showing the difference of the position of 5′ terminal of CUIC-containing mRNAs between the axon and the whole embryo. The x axis indicates the relative position of 5′ terminal compared to CUIC.(B) Diagram showing 2 isoforms of the CUIC-containing mRNA with alternative 5′ ends and the 5′ TOP mRNA.(C) Diagram (left), gel image (middle), and Sanger sequencing result (right) of 5′ RACE products of Rps4x/eS4 mRNAs in axon and eye samples.(D and E) Representative images (D) and quantification of quantitative immunofluorescence (QIF) (E) for Rps14/uS11 (n = 42, control; 59, CHX; 66, Netrin-1; 64, Netrin-1 + CHX) and Rps4x/eS4 (n = 59, control; 50, CHX; 70, Netrin-1; 44, Netrin-1 + CHX) in RGC growth cones with or without Netrin-1 (5 min)/cycloheximide (CHX) treatment (bars, average with 95% confidence interval [CI], Mann-Whitney U test compared to the control, ∗∗∗p ≤ 0.001, ∗p ≤ 0.05). Scale bar, 5 μm.(F and G) Plots (F) and representative images (G) of relative fluorescence recovery of Venus reporter constructs after photobleaching (error bar, SEM) in RGC growth cones. ∗∗∗p < 0.0001; 2-way ANOVA comparing full-length UTRs (n = 8) with Del-motif (n = 12). Scale bar, 2 μm.(H) Moving average (20 s window) of the count of detected translation events per unit area per second with the Netrin-1 stimulation in single-molecule translation imaging. p value: Mann-Whitney U test between full-length UTRs (n = 11) and Del-motif (n = 12) using the total count in each growth cone.
	Figure 3. Structural Positions of RPs Encoded by Axonally Localized mRNAs Are Surface Biased(A) Formula of interface-index (upper) and a partial structure of the ribosome, generated with PyMOL, showing the relation between RP position and interface-indices.(B) Human 80S ribosome structure, generated with PyMOL, showing the position of all RPs that are classified based on the interface-index.(C) Ranking of abundance (average FPKM) of RPs coding mRNAs (n = 2) and interface-index scores of RPs (more blue = higher, more red = lower).
	Figure 4. Axonally Synthesized RPs Co-localize with Ribosome-Containing Granules(A) Experimental workflow and a diagram describing the positional relation of each protein/probe in the rRNA-FUNCAT-PLA experiments.(B and C) Representative images (B) and plots for the number of PLA puncta in each condition (C) detected in the axon (bars, average with 95% CI, Mann-Whitney U test, ∗∗∗p ≤ 0.001). Scale bar, 5 μm.(D) Experimental workflow of the Venus + Cy5-UTP FRAP experiments (upper). Live imaging of Cy5-UTP (magenta) and UTR-Rps4x-Venus fusion or UTR-Venus (green) reporter before and 0, 5, and 10 min after photobleaching of the Venus (green) fluorescence. The yellow arrowheads indicate the sites of co-localization, and the white lines indicate the outlines of axons. Scale bar, 2 μm.(E) Plot showing Pearson’s correlation coefficient of pixel intensities between Cy5-UTP signals and recovered Venus signals (Venus-Rps4x [n = 8] or Venus-only [n = 12]). Bars, average with SEM, Mann-Whitney U test, ∗p ≤ 0.05.
	Figure 5. Axonally Synthesized RPs Physically Interact with the Ribosome in a Nucleolus-Independent Manner(A) Experimental strategy of axon purification, SILAC labeling, and axonal ribosome purification.(B) Diagram and gel image of RT-PCR detection of mature 18S rRNA and pre-rRNA.(C) Cumulative percentage of the relative position of labeled peptides detected in axonal (cyan) and eye (red) ribosome samples.(D) List of RPs whose labeled peptides were detected in the axonal ribosomes. The heatmap shows the translation levels of RPs in 3 different developmental stages of in vivo mouse RGC axons.(E) Human 80S ribosome structure with indication of the RPs whose labeled peptides were detected in the axonal ribosomes. The colors of the squares represent the interface-index of each labeled RP (see Figure 3).(F) Immunostaining of ribosomal assembly factors and a secondary antibody-only control in cultured Xenopus laevis RGC axons. Scale bar, 5 μm.
	Figure 6. Locally Synthesized Rps4x/eS4 Is Required to Maintain Ribosome Function in Axons(A) Diagram (left) and image of RGC axons after FITC-morpholino introduction in a microfluidic chamber.(B–F) Images (B, D, and E) and QIF plots (bars, average, 95% CI, and distribution of normalized levels) (C and F) of Rps3a/eS1, Rps4x/eS4, Rpl17/uL22, and puromycin immunostaining in axons treated with control morpholinos (Cont.) or with morpholinos against rps3a (eS1) (B and C) or against rps4x (eS4) (D–F), with Welch t test (∗p = 0.030, C; 0.015, F; ∗∗∗p = 0.0004; n.s., not significant).(G) qRT-PCR quantification, normalized to control MO, of 18S rRNA in axonal samples treated with control or rps4x (eS4) morpholinos (n.s., not significant in Mann-Whitney U test).
	Figure 7. Axonal Translation of Rps4x/eS4 Is Crucial for Axon Branching In Vivo(A) Experimental workflow (upper) for eye electroporation and live image acquisition of axon branches/arbors. Dual promoter constructs (lower) used for rescue conditions.(B) Lateral view of a single in vivo RGC axon in the tectum with color-coded images of axon shaft (white), primary (red), secondary (blue), and tertiary (yellow) branches.(C and D) Average, 95% CI, and distribution of total branch length per axon (C) and axon complexity index (ACI; see Figure S7B for formula) (D) in the embryos electroporated with the morpholino/rescue constructs (1-way ANOVA with 2-stage step-up method of Benjamini, Krieger, and Yekutiei multiple comparisons test); n = 21 (Cont. MO), 47 (MO), 21 (MO + WT), 25 (MO + del-5′ UTR), and 37 (MO + del-CUIC).(E–G) Experimental workflow for each knockdown (KD) experiment (left) and quantification (right) of in vivo axon branching in control MO− and rps4x MO+ axons after eye electroporation (whole-cell KD) (n = 12, Cont.; 20, Rps4x) (E) and tectum electroporation at stages 41–43 (axonal KD) (middle, n = 14, Cont.; 19, Rps4x) (F), and at stages 35–38 (tectum KD) (lower, n = 10, Cont.; 15, Rps4x) (G). Images (middle panel) show a merged overlay of 3 time points (0, 5, and 10 min in blue, red, and green, respectively). Line graphs (right) show the number of added/removed branches/filopodia (paired and unpaired t test, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not significant).

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