On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons

Toshiaki Shigeoka; Max Koppers; Hovy Ho-Wai Wong; Julie Qiaojin Lin; Roberta Cagnetta; Asha Dwivedy; Janaina de Freitas Nascimento; Francesca W. van Tartwijk; Florian Ströhl; Jean-Michel Cioni; Julia Schaeffer; Mark Carrington; Clemens F. Kaminski; Hosung Jung; William A. Harris; Christine E. Holt

doi:10.1016/j.celrep.2019.11.025

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons

Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, Julie Qiaojin Lin, Roberta Cagnetta, Asha Dwivedy, Janaina de Freitas Nascimento, Francesca W. van Tartwijk, Florian Ströhl, Jean-Michel Cioni, Julia Schaeffer, Mark Carrington, Clemens F. Kaminski, Hosung Jung, William A. Harris, Christine E. Holt

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

© 2019 The AuthorsRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons. © 2019 The AuthorsLocal protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching.

Original language	English
Pages (from-to)	3605-3619.e10
Journal	Cell Reports
Volume	29
Issue number	11
DOIs	https://doi.org/10.1016/j.celrep.2019.11.025
Publication status	Published - 10 Dec 2019
Externally published	Yes

Funding

We thank J.K. Mooslehner for technical assistance. We also thank Mike Deery for invaluable expertise and the MS/MS analysis and Jeroen Krijgsveld and Christian Frese for helpful discussions. We are grateful to Simone Pellegrino and Julie Aspden for useful PyMOL software discussions, Caia Duncan for assistance with polysome fractionations, and Erin Schuman’s group for helpful discussions on ribosome purification. This work was supported by Wellcome Trust grants 085314/Z/08/Z and 203249/Z/16/Z to C.E.H. and 100329/Z/12/Z to W.A.H.; European Research Council Advanced Grant ( 322817 ) to C.E.H.; Champalimaud Vision Award to C.E.H.; and the Netherlands Organization for Scientific Research ( NWO Rubicon 019.161LW.033 ) to M.K. C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (EPSRC) (grants EP/L015889/1 and EP/H018301/1 ); the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z ); the UK Medical Research Council (MRC) (grants MR/K015850/1 and MR/K02292X/1 ); and Infinitus (China) . We thank J.K. Mooslehner for technical assistance. We also thank Mike Deery for invaluable expertise and the MS/MS analysis and Jeroen Krijgsveld and Christian Frese for helpful discussions. We are grateful to Simone Pellegrino and Julie Aspden for useful PyMOL software discussions, Caia Duncan for assistance with polysome fractionations, and Erin Schuman's group for helpful discussions on ribosome purification. This work was supported by Wellcome Trust grants 085314/Z/08/Z and 203249/Z/16/Z to C.E.H. and 100329/Z/12/Z to W.A.H.; European Research Council Advanced Grant (322817) to C.E.H.; Champalimaud Vision Award to C.E.H.; and the Netherlands Organization for Scientific Research (NWO Rubicon 019.161LW.033) to M.K. C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (EPSRC) (grants EP/L015889/1 and EP/H018301/1); the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z); the UK Medical Research Council (MRC) (grants MR/K015850/1 and MR/K02292X/1); and Infinitus (China). T.S. and C.E.H. designed the experiments. T.S. wrote the manuscript with support from M.K. C.E.H. W.A.H. H.H.-W.W. and R.C. H.H.-W.W. performed the in vivo knockdown and branching analysis. J.Q.L. performed FRAP. J.Q.L. F.W.v.T. F.S. and C.F.K. performed the single-molecule translation imaging. J.Q.L. performed the FUNCAT-rRNA proximity ligation assay. J.Q.L. T.S. M.K. and R.C. performed the in vitro knockdown. T.S. M.K. J.-M.C. A.D. and R.C. carried out the in vitro retinal cultures and SILAC. M.K. T.S. J.d.F.N. and M.C. performed the fractionation/cushioning of the axon samples. R.C. and H.J. analyzed Netrin-1-induced axonal translation. J.Q.L. M.K. and R.C. performed the immunostaining. T.S. M.K. and J.S. performed the qRT-PCR. T.S. performed the 5? RACE. M.K. T.S. J.Q.L. and R.C. performed the biochemical experiments. T.S. performed the bioinformatics analysis. T.S. and C.E.H. supervised the project. The authors declare no competing interests.

Funders	Funder number
NWO Rubicon	Rubicon 019.161LW.033
Netherlands Organization for Scientific Research
SILAC
UK Medical Research Council
Wellcome Trust	085314/Z/08/Z, 100329/Z/12/Z, 203249/Z/16/Z
Seventh Framework Programme	322817
Medical Research Council	MR/K015850/1, MR/K02292X/1
Engineering and Physical Sciences Research Council	EP/H018301/1, 089703/Z/09/Z, 3-3249/Z/16/Z, EP/L015889/1
Research Councils UK
European Research Council
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	019.161LW.033

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Shigeoka, T., Koppers, M., Wong, H. H.-W., Lin, J. Q., Cagnetta, R., Dwivedy, A., de Freitas Nascimento, J., van Tartwijk, F. W., Ströhl, F., Cioni, J.-M., Schaeffer, J., Carrington, M., Kaminski, C. F., Jung, H., Harris, W. A., & Holt, C. E. (2019). On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons. Cell Reports, 29(11), 3605-3619.e10. https://doi.org/10.1016/j.celrep.2019.11.025

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title = "On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons",

abstract = "{\textcopyright} 2019 The AuthorsRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons. {\textcopyright} 2019 The AuthorsLocal protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching.",

author = "Toshiaki Shigeoka and Max Koppers and Wong, {Hovy Ho-Wai} and Lin, {Julie Qiaojin} and Roberta Cagnetta and Asha Dwivedy and {de Freitas Nascimento}, Janaina and {van Tartwijk}, {Francesca W.} and Florian Str{\"o}hl and Jean-Michel Cioni and Julia Schaeffer and Mark Carrington and Kaminski, {Clemens F.} and Hosung Jung and Harris, {William A.} and Holt, {Christine E.}",

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month = dec,

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doi = "10.1016/j.celrep.2019.11.025",

language = "English",

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Shigeoka, T, Koppers, M, Wong, HH-W, Lin, JQ, Cagnetta, R, Dwivedy, A, de Freitas Nascimento, J, van Tartwijk, FW, Ströhl, F, Cioni, J-M, Schaeffer, J, Carrington, M, Kaminski, CF, Jung, H, Harris, WA & Holt, CE 2019, 'On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons', Cell Reports, vol. 29, no. 11, pp. 3605-3619.e10. https://doi.org/10.1016/j.celrep.2019.11.025

TY - JOUR

T1 - On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons

AU - Shigeoka, Toshiaki

AU - Koppers, Max

AU - Wong, Hovy Ho-Wai

AU - Lin, Julie Qiaojin

AU - Cagnetta, Roberta

AU - Dwivedy, Asha

AU - de Freitas Nascimento, Janaina

AU - van Tartwijk, Francesca W.

AU - Ströhl, Florian

AU - Cioni, Jean-Michel

AU - Schaeffer, Julia

AU - Carrington, Mark

AU - Kaminski, Clemens F.

AU - Jung, Hosung

AU - Harris, William A.

AU - Holt, Christine E.

PY - 2019/12/10

Y1 - 2019/12/10

N2 - © 2019 The AuthorsRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons. © 2019 The AuthorsLocal protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching.

AB - © 2019 The AuthorsRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons. © 2019 The AuthorsLocal protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching.

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U2 - 10.1016/j.celrep.2019.11.025

DO - 10.1016/j.celrep.2019.11.025

M3 - Article

SN - 2211-1247

VL - 29

SP - 3605-3619.e10

JO - Cell Reports

JF - Cell Reports

IS - 11

ER -

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons

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