Fig. 4: Transport of mRNAs encoding ribosomal proteins is reduced in the axon of BORC-subunit-KO i3Neurons. | Nature Neuroscience

Fig. 4: Transport of mRNAs encoding ribosomal proteins is reduced in the axon of BORC-subunit-KO i3Neurons.

From: Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration

Fig. 4: Transport of mRNAs encoding ribosomal proteins is reduced in the axon of BORC-subunit-KO i3Neurons.

a, Constructs used for RNA visualization. A construct encoding HaloTags fused to the PP7 coat protein was stably co-expressed with the coding sequencing (CDS) of the gene of interest followed by its 3′ UTR, fused to PP7 RNA stem-loop repeats in WT, BORCS5-KO or BORCS7-KO i3Neurons. b,d, Kymographs of RPS7 (b) or RPS27A (d) i3Neurons co-expressing the constructs described in a were transduced with LAMP1-mNeonGreen (LAMP1-NG) and incubated with the fluorescent Halo substrate JF646 to image axonal lysosome-related vesicle and mRNA movement, respectively. Axons were imaged live for 60 s. Kymographs were generated from Supplementary Videos 1 and 2. Single-color images are represented in inverted grayscale. See Extended Data Fig. 6 for negative control of JF646 staining. c,e, Quantification of co-moving LAMP1-NG and RPS7 (c) or RPS27A (e) mRNA tracks. Values are the mean ± s.d. of ~22 kymographs from ~22 neurons per condition and are expressed as the percentage of the indicated marker that co-moves with LAMP1-NG. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. BORCS5 KO versus WT ***P < 0.001 and BORCS7 KO versus WT ***P < 0.001 for both c and e. f, Scheme of a neuronal spheroid. Axons were imaged live for 30 s, and kymographs were generated from the videos. Lines with negative or positive slopes represent anterograde or retrograde movement, respectively. g, Kymographs of axonal mRNA movement in i3Neurons co-expressing combinations of RPS7-PP7 or RPS27A-PP7 repeats and HaloTag-PP7 coat constructs were generated from spheroids. h, Quantification of anterograde, retrograde and static RPS7 or RPS27A mRNA tracks. Values are the mean ± s.d. from ~20 kymographs from ~20 neurons per condition and are expressed as the percentage of the indicated marker in anterograde, retrograde or static particles. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. P values relative to WT: RPS7 anterograde, BORCS5 KO ***P < 0.001, BORCS7 KO ***P < 0.001; retrograde, BORCS5 KO ***P < 0.001, BORCS7 KO ***P < 0.001; static, BORCS5 KO *P = 0.023, BORCS7 KO *P = 0.023. RPS27A anterograde, BORCS5 KO **P = 0.004, BORCS7 KO **P = 0.004; retrograde, BORCS5 KO ***P < 0.001, BORCS7 KO ***P < 0.001; static, BORCS5 KO **P = 0.003, BORCS7 KO **P = 0.003.

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