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Dynamic Axonal Translation in Developing and Mature Visual Circuits

View Article: PubMed Central - PubMed

ABSTRACT

Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.

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Cis-Regulatory Elements for Axonal Translation, Related to Figure 7(A) Lists of sequence motifs enriched in 5′UTRs, 3′UTRs and alternative exons of axon-enriched mRNAs / exons.(B) An example of axon-enriched motifs. The scatterplot compares the normalized mRNA levels (log2(FPKM)) between the axonal (y axis) and the retinal (x axis) translatome at stage P0.5 for genes with (red dots) and without (black dots) the motif. The density plot shows the distribution of log2 (axon (FPKM) / retina (FPKM)).(C) GO enrichment analysis for entire genome containing axon-specific sequence motifs (K: G or T; R: A or G; Y: C or T; M: A or C; R: A or G; and H A or C or T) and their relative efficiency in axonal mRNA translation measured by fluorescence recovery after photobleaching (FRAP) of motif-containing reporter constructs (myr-d2EGFP). Several axon-specific motifs were able to promote mRNA translation in the growth cone relative to a control myr-d2EGFP construct without a UTR. Statistical significance of FRAP compared to the no-UTR control was tested across all time-points (0-10mins) using a two-way ANOVA (from the top bar, n = 16, 5, 5, 7, 8, 7, 3, 8, 8, 8, 5, 8, and 6, respectively). For representative purposes, the mean fluorescence recovery at 10 min post-photobleaching is shown. Error bars represent SEM. ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to no-UTR control.
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figs7: Cis-Regulatory Elements for Axonal Translation, Related to Figure 7(A) Lists of sequence motifs enriched in 5′UTRs, 3′UTRs and alternative exons of axon-enriched mRNAs / exons.(B) An example of axon-enriched motifs. The scatterplot compares the normalized mRNA levels (log2(FPKM)) between the axonal (y axis) and the retinal (x axis) translatome at stage P0.5 for genes with (red dots) and without (black dots) the motif. The density plot shows the distribution of log2 (axon (FPKM) / retina (FPKM)).(C) GO enrichment analysis for entire genome containing axon-specific sequence motifs (K: G or T; R: A or G; Y: C or T; M: A or C; R: A or G; and H A or C or T) and their relative efficiency in axonal mRNA translation measured by fluorescence recovery after photobleaching (FRAP) of motif-containing reporter constructs (myr-d2EGFP). Several axon-specific motifs were able to promote mRNA translation in the growth cone relative to a control myr-d2EGFP construct without a UTR. Statistical significance of FRAP compared to the no-UTR control was tested across all time-points (0-10mins) using a two-way ANOVA (from the top bar, n = 16, 5, 5, 7, 8, 7, 3, 8, 8, 8, 5, 8, and 6, respectively). For representative purposes, the mean fluorescence recovery at 10 min post-photobleaching is shown. Error bars represent SEM. ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to no-UTR control.

Mentions: We next investigated whether axon-specific exons might contain “generalizable” motifs responsible for axonal mRNA translation. We searched for common sequence elements that are enriched in axon-specific alternative exons (Figure 7C) and in the 5′ and 3′ UTRs in constitutive exons (Figures S7A and S7B) of axon-enriched mRNAs (Figure 3A). To understand the potential function of identified sequence elements, we searched for genes that contain these elements in the entire mouse genome. Remarkably, the element-containing genes generally encode regulators of axon and synapse function (Figures 7C and S7C). Strikingly, five of six motifs identified from alternative exons and five of twelve motifs in constitutive exons of axon-enriched mRNAs showed a significant FRAP signal at 10 min, indicative of increased axonal mRNA translation of a reporter mRNA when incorporated in the 5′ or 3′ UTR as in Figure 7B (Figures 7C and S7C). These results suggest the potential links between the sequence elements and axonal mRNA translation and thus provide further insight into the mechanisms underlying the selective and dynamic nature of the axonal mRNA translation.


Dynamic Axonal Translation in Developing and Mature Visual Circuits
Cis-Regulatory Elements for Axonal Translation, Related to Figure 7(A) Lists of sequence motifs enriched in 5′UTRs, 3′UTRs and alternative exons of axon-enriched mRNAs / exons.(B) An example of axon-enriched motifs. The scatterplot compares the normalized mRNA levels (log2(FPKM)) between the axonal (y axis) and the retinal (x axis) translatome at stage P0.5 for genes with (red dots) and without (black dots) the motif. The density plot shows the distribution of log2 (axon (FPKM) / retina (FPKM)).(C) GO enrichment analysis for entire genome containing axon-specific sequence motifs (K: G or T; R: A or G; Y: C or T; M: A or C; R: A or G; and H A or C or T) and their relative efficiency in axonal mRNA translation measured by fluorescence recovery after photobleaching (FRAP) of motif-containing reporter constructs (myr-d2EGFP). Several axon-specific motifs were able to promote mRNA translation in the growth cone relative to a control myr-d2EGFP construct without a UTR. Statistical significance of FRAP compared to the no-UTR control was tested across all time-points (0-10mins) using a two-way ANOVA (from the top bar, n = 16, 5, 5, 7, 8, 7, 3, 8, 8, 8, 5, 8, and 6, respectively). For representative purposes, the mean fluorescence recovery at 10 min post-photobleaching is shown. Error bars represent SEM. ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to no-UTR control.
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figs7: Cis-Regulatory Elements for Axonal Translation, Related to Figure 7(A) Lists of sequence motifs enriched in 5′UTRs, 3′UTRs and alternative exons of axon-enriched mRNAs / exons.(B) An example of axon-enriched motifs. The scatterplot compares the normalized mRNA levels (log2(FPKM)) between the axonal (y axis) and the retinal (x axis) translatome at stage P0.5 for genes with (red dots) and without (black dots) the motif. The density plot shows the distribution of log2 (axon (FPKM) / retina (FPKM)).(C) GO enrichment analysis for entire genome containing axon-specific sequence motifs (K: G or T; R: A or G; Y: C or T; M: A or C; R: A or G; and H A or C or T) and their relative efficiency in axonal mRNA translation measured by fluorescence recovery after photobleaching (FRAP) of motif-containing reporter constructs (myr-d2EGFP). Several axon-specific motifs were able to promote mRNA translation in the growth cone relative to a control myr-d2EGFP construct without a UTR. Statistical significance of FRAP compared to the no-UTR control was tested across all time-points (0-10mins) using a two-way ANOVA (from the top bar, n = 16, 5, 5, 7, 8, 7, 3, 8, 8, 8, 5, 8, and 6, respectively). For representative purposes, the mean fluorescence recovery at 10 min post-photobleaching is shown. Error bars represent SEM. ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to no-UTR control.
Mentions: We next investigated whether axon-specific exons might contain “generalizable” motifs responsible for axonal mRNA translation. We searched for common sequence elements that are enriched in axon-specific alternative exons (Figure 7C) and in the 5′ and 3′ UTRs in constitutive exons (Figures S7A and S7B) of axon-enriched mRNAs (Figure 3A). To understand the potential function of identified sequence elements, we searched for genes that contain these elements in the entire mouse genome. Remarkably, the element-containing genes generally encode regulators of axon and synapse function (Figures 7C and S7C). Strikingly, five of six motifs identified from alternative exons and five of twelve motifs in constitutive exons of axon-enriched mRNAs showed a significant FRAP signal at 10 min, indicative of increased axonal mRNA translation of a reporter mRNA when incorporated in the 5′ or 3′ UTR as in Figure 7B (Figures 7C and S7C). These results suggest the potential links between the sequence elements and axonal mRNA translation and thus provide further insight into the mechanisms underlying the selective and dynamic nature of the axonal mRNA translation.

View Article: PubMed Central - PubMed

ABSTRACT

Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in&nbsp;vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in&nbsp;vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in&nbsp;vivo.

No MeSH data available.


Related in: MedlinePlus