Local translation of RhoA regulates growth cone collapse

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Abstract

Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily functions to repel axons from inappropriate targets, induces cytoskeletal rearrangements that result in growth cone collapse1. These effects require intra-axonal messenger RNA translation. Here we show that transcripts for RhoA, a small guanosine triphosphatase (GTPase) that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3′ untranslated region (UTR). Sema3A induces intra-axonal translation of RhoA mRNA, and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a new mechanism for the regulation of RhoA signalling.

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Figure 1: RhoA mRNA is localized in axons and growth cones.
Figure 2: The RhoA 3′UTR contains an axonal targeting element.
Figure 3: Sema3A induces RhoA translation.
Figure 4: Sema3A activates translation of a RhoA reporter.
Figure 5: Axonal RhoA translation mediates Sema3A signalling.

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Acknowledgements

We thank S. Schlesinger for Sindbis plasmids and N. O'Connor for advice on 3D deconvolution and axonal volume calculation. This work is supported by the National Institute of Mental Health (S.R.J.), the National Alliance for Autism Research (S.R.J.), the Charles A. Dana foundation and the Medical Scientist Training Program (E.Z.M.). A.J. is supported by D. Johnston (Baylor Medical College).

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Correspondence to Samie R. Jaffrey.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure Legends

This file contains legends for Supplementary Figures S1-S9.

Supplementary Table S1

This table contains oligonucleotide sequences used in in situ hybridization and RT-PCR.

Supplementary Figure S1

Effects of protein synthesis inhibitors on growth cone collapse in severed DRG axons.

Supplementary Figure S2

In situ hybridization against RhoA and other transcripts in DRG axons.

Supplementary Figure S3

Isolation of DRG axons using a modified Boyden chamber.

Supplementary Figure S4

Quantification of in situ hybridization signals in axons and cell bodies.

Supplementary Figure S5

Effect of increased NGF concentration on growth cone collapse.

Supplementary Figure S6

Effects of vehicle and anisomycin treatment on the RhoA reporter.

Supplementary Figure S7

Alignment of the 3′UTRs of RhoA mRNA from different species.

Supplementary Figure S8

Inhibition of growth cone collapse by C. botulinum C3 exoenzyme.

Supplementary Figure S9

Infectivity and expression levels of Sindbis-IRES virions.

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