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Dynamic Axonal Translation in Developing and Mature Visual Circuits

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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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Alternative Splicing Generates High Isoform Diversity in Axons(A) Percentage of genes with alternative events from all axonally translated genes. Alternative events are classified into five different classes depicted in the left panel.(B) Scatter and density plots for the distribution of percentage spliced in (Ψ) values between the retina (x axis) and the axon (y axis).(C) Model for biased distribution of Ψ values in the axon. The comparison of two isoforms suggests that one of two isoforms is predominant in the axon.(D and E) The sequence reads on Acot7 and Stx3 loci visualized with integrative genomics viewer (IGV). The histograms show the depth of the reads displayed at each locus. The retinal isoforms are detected only in the retinal translatome, whereas the axonal isoforms are detected both in the axonal and retinal translatomes.See also Figure S6.
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fig6: Alternative Splicing Generates High Isoform Diversity in Axons(A) Percentage of genes with alternative events from all axonally translated genes. Alternative events are classified into five different classes depicted in the left panel.(B) Scatter and density plots for the distribution of percentage spliced in (Ψ) values between the retina (x axis) and the axon (y axis).(C) Model for biased distribution of Ψ values in the axon. The comparison of two isoforms suggests that one of two isoforms is predominant in the axon.(D and E) The sequence reads on Acot7 and Stx3 loci visualized with integrative genomics viewer (IGV). The histograms show the depth of the reads displayed at each locus. The retinal isoforms are detected only in the retinal translatome, whereas the axonal isoforms are detected both in the axonal and retinal translatomes.See also Figure S6.

Mentions: Post-transcriptional RNA processing events, including alternative splicing, are widely used to control gene expression in neurons. To assess whether these regulate local mRNA translation, we analyzed the mRNA isoforms on mapped sequence reads using MISO software (Katz et al., 2010). Intriguingly, the axonal translatome showed more-extensive diversity of mRNA isoforms than the somal translatome (Figure 6A). To address the possibility of isoform-specific axonal translation, we selected 164 alternative events that produce two isoforms both in the axonal and retinal translatomes. Then, we calculated the “percentage spliced in” (PSI or Ψ) values, which represent the fraction of the longer isoform (Katz et al., 2010). Ψ retina was uniformly distributed (0 < Ψ < 1), indicating that there is no clear bias in translational efficacy (Figures 6B and 6C). However, Ψ axon was biased to the two extremes (i.e., Ψ = 0 or Ψ = 1), indicating that only one of the two isoforms is selectively translated in the axon (Figures 6B and 6C). Notable examples are Acot7, an acyl-coenzyme A (CoA) thioesterase gene required for lipid biosynthesis and neuron survival (Ellis et al., 2013); Syntaxin 3 (Stx3), a SNARE component gene; and Clta, a clathrin light chain A gene, which show clear axon-specific usage of first, last, and internal exons, respectively (Figures 6D, 6E, and S6A). Intriguingly, axon-specific isoforms of Acot7 and Stx3 encode proteins with slightly different amino acids at the N and C termini, respectively (see gene models in Figures 6D and 6E), suggesting that alternative splicing may couple axon-specific protein isoforms with a unique sequence tag in the UTR.


Dynamic Axonal Translation in Developing and Mature Visual Circuits
Alternative Splicing Generates High Isoform Diversity in Axons(A) Percentage of genes with alternative events from all axonally translated genes. Alternative events are classified into five different classes depicted in the left panel.(B) Scatter and density plots for the distribution of percentage spliced in (Ψ) values between the retina (x axis) and the axon (y axis).(C) Model for biased distribution of Ψ values in the axon. The comparison of two isoforms suggests that one of two isoforms is predominant in the axon.(D and E) The sequence reads on Acot7 and Stx3 loci visualized with integrative genomics viewer (IGV). The histograms show the depth of the reads displayed at each locus. The retinal isoforms are detected only in the retinal translatome, whereas the axonal isoforms are detected both in the axonal and retinal translatomes.See also Figure S6.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4930487&req=5

fig6: Alternative Splicing Generates High Isoform Diversity in Axons(A) Percentage of genes with alternative events from all axonally translated genes. Alternative events are classified into five different classes depicted in the left panel.(B) Scatter and density plots for the distribution of percentage spliced in (Ψ) values between the retina (x axis) and the axon (y axis).(C) Model for biased distribution of Ψ values in the axon. The comparison of two isoforms suggests that one of two isoforms is predominant in the axon.(D and E) The sequence reads on Acot7 and Stx3 loci visualized with integrative genomics viewer (IGV). The histograms show the depth of the reads displayed at each locus. The retinal isoforms are detected only in the retinal translatome, whereas the axonal isoforms are detected both in the axonal and retinal translatomes.See also Figure S6.
Mentions: Post-transcriptional RNA processing events, including alternative splicing, are widely used to control gene expression in neurons. To assess whether these regulate local mRNA translation, we analyzed the mRNA isoforms on mapped sequence reads using MISO software (Katz et al., 2010). Intriguingly, the axonal translatome showed more-extensive diversity of mRNA isoforms than the somal translatome (Figure 6A). To address the possibility of isoform-specific axonal translation, we selected 164 alternative events that produce two isoforms both in the axonal and retinal translatomes. Then, we calculated the “percentage spliced in” (PSI or Ψ) values, which represent the fraction of the longer isoform (Katz et al., 2010). Ψ retina was uniformly distributed (0 < Ψ < 1), indicating that there is no clear bias in translational efficacy (Figures 6B and 6C). However, Ψ axon was biased to the two extremes (i.e., Ψ = 0 or Ψ = 1), indicating that only one of the two isoforms is selectively translated in the axon (Figures 6B and 6C). Notable examples are Acot7, an acyl-coenzyme A (CoA) thioesterase gene required for lipid biosynthesis and neuron survival (Ellis et al., 2013); Syntaxin 3 (Stx3), a SNARE component gene; and Clta, a clathrin light chain A gene, which show clear axon-specific usage of first, last, and internal exons, respectively (Figures 6D, 6E, and S6A). Intriguingly, axon-specific isoforms of Acot7 and Stx3 encode proteins with slightly different amino acids at the N and C termini, respectively (see gene models in Figures 6D and 6E), suggesting that alternative splicing may couple axon-specific protein isoforms with a unique sequence tag in the UTR.

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