Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A native function for RAN translation and CGG repeats in regulating fragile X protein synthesis

Nature Neuroscience volume 23, pages 386–397 (2020)Cite this article

Subjects

Abstract

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5′-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: CGG RAN translation impedes FMRP translation in neurons.
Fig. 2: mGluR-dependent FMRP synthesis requires RAN translation and the CGG repeat.
Fig. 3: RAN-targeting ASOs increase FMRP and suppress CGG repeat toxicity.
Fig. 4: CGG RAN ASOs alter FMRP translation dynamics in human neurons.
Fig. 5: RAN ASO increases FMRP in UFM neurons.
Fig. 6: CGG RAN ASO enhances survival in expanded repeat human neurons.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 2–5 and Extended Data Figs. 1, 5, 7 and 9 are provided with the paper.

References

  1. Paulson, H. in Handbook of Clinical Neurology, vol. 147 (eds Geschwind, D. H. et al.) 105–123 (Elsevier, 2018).

  2. Hannan, A. J. Tandem repeats mediating genetic plasticity in health and disease. Nat. Rev. Genet. 19, 286–298 (2018).

    Article  CAS  PubMed  Google Scholar 

  3. Todd, P. K. et al. CGG repeat-associated translation mediates neurodegeneration in fragile X tremor ataxia syndrome. Neuron 78, 440–455 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Zu, T. et al. Non-ATG-initiated translation directed by microsatellite expansions. Proc. Natl Acad. Sci. USA 108, 260–265 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Verkerk, A. J. et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65, 905–914 (1991).

    Article  CAS  PubMed  Google Scholar 

  6. Hagerman, R. J. et al. Fragile X syndrome. Nat. Rev. Dis. Primers 3, 17065 (2017).

    Article  PubMed  Google Scholar 

  7. Berry-Kravis, E. et al. Fragile X-associated tremor/ataxia syndrome: clinical features, genetics, and testing guidelines. Movement Dis. 22, 2018–2030 (2007). quiz 2140.

    Article  PubMed  Google Scholar 

  8. Greco, C. M. et al. Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers. Brain 125, 1760–1771 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Tassone, F. et al. Fragile X males with unmethylated, full mutation trinucleotide repeat expansions have elevated levels of FMR1 messenger RNA. Am. J. Med. Genet. 94, 232–236 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Feng, Y. et al. Translational suppression by trinucleotide repeat expansion at FMR1. Science 268, 731–734 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Pretto, D. et al. Clinical and molecular implications of mosaicism in FMR1 full mutations. Front. Genet. 5, 318 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Santa Maria, L. et al. FXTAS in an unmethylated mosaic male with fragile X syndrome from Chile. Clin. Genet. 86, 378–382 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Kearse, M. G. et al. CGG repeat-associated non-AUG translation utilizes a cap-dependent scanning mechanism of initiation to produce toxic proteins. Mol. Cell 62, 314–322 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sellier, C. et al. Translation of expanded CGG repeats into FMRpolyG is pathogenic and may contribute to fragile X tremor ataxia syndrome. Neuron 93, 331–347 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Krans, A., Skariah, G., Zhang, Y., Bayly, B. & Todd, P. K. Neuropathology of RAN translation proteins in fragile X-associated tremor/ataxia syndrome. Acta Neuropathol. Commun. 7, 152 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Gemayel, R., Vinces, M. D., Legendre, M. & Verstrepen, K. J. Variable tandem repeats accelerate evolution of coding and regulatory sequences. Annu. Rev. Genet. 44, 445–477 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Eichler, E. E. et al. Evolution of the cryptic FMR1 CGG repeat. Nat. Genet. 11, 301–308 (1995).

    Article  CAS  PubMed  Google Scholar 

  18. Collins, S. C. et al. Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males. Am. J Med. Genet. A 152A, 2512–2520 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protoc. 7, 1534–1550 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hinnebusch, A. G., Ivanov, I. P. & Sonenberg, N. Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science 352, 1413–1416 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chen, L. S., Tassone, F., Sahota, P. & Hagerman, P. J. The (CGG)n repeat element within the 5′ untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter. Hum. Mol. Genet. 12, 3067–3074 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Ludwig, A. L., Hershey, J. W. & Hagerman, P. J. Initiation of translation of the FMR1 mRNA occurs predominantly through 5′-end-dependent ribosomal scanning. J. Mol. Biol. 407, 21–34 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hou, L. et al. Dynamic translational and proteasomal regulation of fragile X mental retardation protein controls mGluR-dependent long-term depression. Neuron 51, 441–454 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Nalavadi, V. C., Muddashetty, R. S., Gross, C. & Bassell, G. J. Dephosphorylation-induced ubiquitination and degradation of FMRP in dendrites: a role in immediate early mGluR-stimulated translation. J. Neurosci. 32, 2582–2587 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Weiler, I. J. et al. Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation. Proc. Natl Acad. Sci. USA 94, 5395–5400 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bear, M. F., Huber, K. M. & Warren, S. T. The mGluR theory of fragile X mental retardation. Trends Neurosci. 27, 370–377 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Todd, P. K., Mack, K. J. & Malter, J. S. The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95. Proc. Natl Acad. Sci. USA 100, 14374–14378 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Iliff, A. J. et al. Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in fragile X premutation mice. Hum. Mol. Genet. 22, 1180–1192 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Huber, K. M., Gallagher, S. M., Warren, S. T. & Bear, M. F. Altered synaptic plasticity in a mouse model of fragile X mental retardation. Proc. Natl Acad. Sci. USA 99, 7746–7750 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Suhl, J. A. et al. A 3′ untranslated region variant in FMR1 eliminates neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR. Proc. Natl Acad. Sci. USA 112, E6553–E6561 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Muslimov, I. A., Patel, M. V., Rose, A. & Tiedge, H. Spatial code recognition in neuronal RNA targeting: role of RNA-hnRNP A2 interactions. J. Cell Biol. 194, 441–457 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Green, K. M. et al. RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response. Nat. Commun. 8, 2005 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Disney, M. D. et al. A small molecule that targets r(CGG)(exp) and improves defects in fragile X-associated tremor ataxia syndrome. ACS Chem. Biol. 7, 1711–1718 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yang, W. Y. et al. Small molecule recognition and tools to study modulation of r(CGG)(exp) in fragile X-associated tremor ataxia syndrome. ACS Chem. Biol. 11, 2456–2465 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Di Prisco, G. V. et al. Translational control of mGluR-dependent long-term depression and object-place learning by eIF2alpha. Nat. Neurosci. 17, 1073–1082 (2014).

    Article  PubMed  CAS  Google Scholar 

  38. Sidrauski, C., McGeachy, A. M., Ingolia, N. T. & Walter, P. The small molecule ISRIB reverses the effects of eIF2alpha phosphorylation on translation and stress granule assembly. eLife 4, e05033 (2015).

    Article  PubMed Central  CAS  Google Scholar 

  39. Cheng, W. et al. C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2alpha phosphorylation. Nat. Commun. 9, 51 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Liang, X. H. et al. Antisense oligonucleotides targeting translation inhibitory elements in 5′ UTRs can selectively increase protein levels. Nucleic Acids Res. 45, 9528–9546 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Liang, X. H. et al. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat. Biotechnol. 34, 875–880 (2016).

    Article  CAS  PubMed  Google Scholar 

  42. Tabet, R. et al. CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts. Nat. Commun. 9, 152 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Linsalata, A. E. et al. DDX3X and specific initiation factors modulate FMR1 repeat-associated non-AUG-initiated translation. EMBO Rep. 20, e47498 (2019).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  44. Burman, R. W., Popovich, B. W., Jacky, P. B. & Turker, M. S. Fully expanded FMR1 CGG repeats exhibit a length- and differentiation-dependent instability in cell hybrids that is independent of DNA methylation. Hum. Mol. Genet. 8, 2293–2302 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Tabrizi, S. J. et al. Targeting huntingtin expression in patients with Huntington’s disease. N. Engl. J. Med. 380, 2307–2316 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Haenfler, J. et al. Targeted reactivation of FMR1 transcription in fragile X syndrome embryonic stem cells. Front. Mol. Neurosci. 11, 282 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992 e976 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Finkel, R. S. et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet 388, 3017–3026 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Sutton, M. A. et al. Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis. Cell 125, 785–799 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Ifrim, M. F., Williams, K. R. & Bassell, G. J. Single-molecule imaging of PSD-95 mRNA translation in dendrites and its dysregulation in a mouse model of fragile X syndrome. J. Neurosci. 35, 7116–7130 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Tatavarty, V. et al. Single-molecule imaging of translational output from individual RNA granules in neurons. Mol. Biol. Cell 23, 918–929 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Barbarese, E. et al. Conditional knockout of tumor overexpressed gene in mouse neurons affects RNA granule assembly, granule translation, LTP and short term habituation. PLoS ONE 8, e69989 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006).

    Article  CAS  PubMed  Google Scholar 

  54. Choi, H. M. et al. Mapping a multiplexed zoo of mRNA expression. Development 143, 3632–3637 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Huss, D. et al. Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction. Cold Spring Harbor Protoc. 2015, 259–268 (2015).

    Article  Google Scholar 

  56. Choi, H. M. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Saluto, A. et al. An enhanced polymerase chain reaction assay to detect pre- and full mutation alleles of the fragile X mental retardation 1 gene. J. Mol. Diagn. 7, 605–612 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Shi, Y., Kirwan, P. & Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836–1846 (2012).

    Article  CAS  PubMed  Google Scholar 

  59. Barmada, S. J. et al. Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1. Proc. Natl Acad. Sci. USA 112, 7821–7826 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R. & Finkbeiner, S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank P.K.T. and M.A.S.’s laboratory members and A. Goldstrohm for helpful discussions, commentary and technical advice. C. Pearson provided TC43-97 fibroblasts originally generated by M. Turker44. Compound 1A was a gift from M. Disney35. This work was funded by the NIH (grant nos. R01NS099280 and R01NS086810), and philanthropic donations to P.K.T. C.M.R was supported by the National Institute of Neurological Disorders and Stroke (NIN.DS, grant no. F31 NS090883-03). M.G.K was supported by grant no. F32NS089124 and is now supported by grant no. K99GM.126064. J.M.H. was funded by grant no. T32NS007222 and a post-doctoral fellowship from FRAXA. S.J.B. and B.N.F. were supported by the NIH (grant nos. R01-NS097542 and 1P30AG053760). S.E.W. was supported by the NIH (grant no. T-32-NS076401). M.I. and G.B. were supported by the NINDS (grant no. 5P30NS05507708). M.R.G. was supported by the NIH (grant no. T32NS007222). M.A.S. was supported by the Michigan Discovery Fund. F.R. and P.J.-N. were supported by Ionis Pharmaceuticals, who also funded reagent generation. The iPSC work was supported by the University of Michigan Human Stem Cell and Genome Editing Core.

Author information

Author notes

  1. These authors contributed equally: Caitlin M. Rodriguez, Shannon E. Wright.

Authors and Affiliations

  1. Department of Neurology, University of Michigan, Ann Arbor, MI, USA

    Caitlin M. Rodriguez, Shannon E. Wright, Michael G. Kearse, Jill M. Haenfler, Brittany N. Flores, Yu Liu, Mary R. Glineburg, Amy Krans, Jack M. Parent, Sami J. Barmada & Peter K. Todd

  2. Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA

    Caitlin M. Rodriguez & Shannon E. Wright

  3. Department of Biological Chemistry and Pharmacology, Center for RNA Biology, Ohio State University, Columbus, OH, USA

    Michael G. Kearse

  4. Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI, USA

    Brittany N. Flores

  5. Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA

    Marius F. Ifrim & Gary J. Bassell

  6. VA Ann Arbor Healthcare System, Ann Arbor, MI, USA

    Amy Krans, Jack M. Parent & Peter K. Todd

  7. Ionis Pharmaceuticals, Carlsbad, CA, USA

    Paymaan Jafar-Nejad & Frank Rigo

  8. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA

    Michael A. Sutton

Authors
  1. Caitlin M. Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shannon E. Wright
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael G. Kearse
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jill M. Haenfler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brittany N. Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marius F. Ifrim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mary R. Glineburg
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Amy Krans
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Paymaan Jafar-Nejad
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Michael A. Sutton
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Gary J. Bassell
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jack M. Parent
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Frank Rigo
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sami J. Barmada
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Peter K. Todd
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.M.R., F.R. and P.K.T. conceived the project. C.M.R., S.E.W. and P.K.T. designed the experiments. C.M.R. performed cell and rodent neuron-based assays, immunoblots and imaging. S.E.W. helped with this. M.G.K. generated initial constructs and performed in vitro assays. Y.L. and J.M.P. derived the iPSCs. C.M.R., J.M.H., Y.L. and S.E.W. characterized iPSCs and derived neurons. B.N.F. and S.J.B. performed rodent neuronal toxicity experiments. S.E.W. performed iPSC-derived neuronal survival experiments and DHPG assays. C.M.R., M.I. and G.J.B. designed and performed live Venus imaging in neurons. M.R.G. optimized HCR assay for nLuc. A.K. aided with reporter design and cloning, and developed and characterized the FMRpoly(G) antibody. P.K.T., C.M.R. and F.R. designed the ASOs. P.J-N. and F.R. supplied the ASOs and aided in data interpretation. M.A.S. provided critical equipment and intellectual input. C.M.R., S.E.W. and P.K.T. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Peter K. Todd.

Ethics declarations

Competing interests

P.K.T. served as a consultant with Denali Therapeutics and has licensed technology through the University of Michigan to Denali. P.K.T., C.M.R., F.R. and P.J. hold a patent on ASOs targeting CGG RAN. F.R. and P.J. are paid employees of Ionis Pharmaceuticals. All other authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Impact of CGG RAN translation on FMRP reporter synthesis.

A, Left: in vitro translated (CGG)n FMRP-nluc reporter mRNAs harboring different CGG repeat sizes. ‡‡‡: FMRP-nanoluciferase-3X FLAG protein. ‡: N-terminal extension of FMRP from RAN initiation at the ACG codon in the (polyarginine) 0-frame. ‡ is detected at up to 18 repeats, but is attenuated at normal repeat sizes as previously described . ‡‡: N-terminal extension of FMRP from initiation in the 0RAN, downstream of the repeat. The ‡‡ product is only detectable in vitro, and is not detectable in cells. Right: Luciferase activity of in vitro translated FMRP-nLuc reporters (n=3; 0 vs 18: p=0.000000000000033; 18 vs 28: p=000001109; 28 vs 45: p=0.00234; 28 vs 57: p=0.0000518; 28 vs 69: p=0.0004997; 28 vs 100: p=0.001371). B, Luciferase activity from FMRP reporter in vitro after replacement of (CGG)25 repeat with unstructured (GAA)25 repeat (n=3; p=0.4940). C, Schematic of RAN translation reporters. D, Left: luciferase activity showing relative levels of FMRP, +1 RAN, and 0-frame RAN reporters at 25 and 100 repeats in HEK293 cells (n=3; CGG25: FMRP vs +1 RAN p=0.00002612, FMRP vs 0-frame RAN p=0.00001338; CGG100: FMRP vs +1 RAN p=0.0009, FMRP vs 0-frame RAN p=0.0003). E, Top: Immunoblot of (CGG)n FMRP-nLuc reporters in HEK293T cells with indicated mutations. Bottom: luciferase activity from reporters translated in vitro (RRL). +1-AUG represents insertion of AUG in place of +1 ACG RAN initiation codon. RAN initiation sites in the (CGG)n FMRP-nLuc reporters were mutated to preclude initiation in the 0-frame (0-AAA), the +1 reading frame (+1-AAA), or both (0/+1-AAA) (n=3; CGG25: WT vs 0-AAA p=0.0001, WT vs +1-AAA p=0.1977, WT vs 0/1-AAA p=0.0001; CGG100: WT vs 0-AAA p=0.0001, WT vs +1-AAA p=0.4437, WT vs 0/1-AAA p=0.0001). F, RT-qPCR to nLuc mRNA from SH-SY5Y cells expressing the indicated FMRP reporters with 100 repeats (n=3; p=0.4940). G, Flag immunocytochemistry for (CGG)100 FMRP-nLuc reporters in rat neurons co-expressing mCherry (red) to fill the cell. H, Quantification of Flag signal for the WT (CGG)100 reporter (n=23) and for the 0/+1-AAA (CGG)100 reporter (n=21), where “n” is the mean CTCF signal from 5 neurons (p=0.0258). Panel A: One-way ANOVA with multiple comparisons. Panels D, E: One-way ANOVA with multiple comparisons, within repeat groups. Panel B, F, H: two sided Student’s t-test. n.s.=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Graphs are mean ± S.E.M.

Source data

Extended Data Fig. 2 Live tracking of CGG RAN translation in neuronal dendrites.

A, Left: Venus with and without (∆AUG) an AUG initiation codon serve as a positive and negative translation control. Right: Venus fluorescent proteins with the AUG deleted were inserted after the 5′ leader of FMR1, in the +1 reading frame to serve as a reporter for +1CGG RAN. B, Live imaging of mature rat hippocampal neurons expressing indicated Venus reporters (green) and mCherry (red). C, Quantification of Venus reporter signals with 32 repeats (n=11) or 90 repeats (n=14) (p=0.0002). D, Single molecule imaging of CGG RAN translation in distal neuronal processes expressing indicated +1CGG RAN-Venus reporters after photo bleaching. Green dots represent individual translation events. E, Quantification of CGG RAN events in processes (n=3; p=0.2355). Panels C,E: Two sided Student’s t-test. n.s.=not significant, ***p<0.001. Graphs are mean ± S.E.M.

Extended Data Fig. 3 FMR1 reporter mRNA trafficking in neuronal dendrites.

A, Mouse embryonic fibroblasts were transfected with +1 (CGG)100 RAN-nLuc reporters or mock transfected were probed for nLuc RNA by Hybridization Chain Reaction (HCR) and co-stained for FLAG. The specificity of the probes for nLuc is illustrated by the presence of signal (red) in transfected cells only. B, Left: HCR of nLuc mRNA combined with ICC to co-transfected mApple verifies dendritic export of reporter RNAs. Right: Quantification of dendritic nLuc reporter mRNA; AUG-nLuc (n=16), 0 repeat (n=16), 20 repeats (n=18), 90 repeats (n=20) (AUG vs 0: p=0.1447; AUG vs 20: p=0.0373; AUG vs 90: p=0.0044). One-way ANOVA with correction for multiple comparisons. Box extends to 25th/75th percentiles with a line at mean and whiskers indicate 95% CI.

Extended Data Fig. 4 Modifiers of mGluR dependent FMRP reporter synthesis.

A, Schematic of the AUG (+1) CGGn FMRP-nLuc-3′ UTR reporter, which drives translation in the +1 RAN reading frame and reports for FMRP. B, relative nLuc values in neurons transfected with CGG25 or CGG100 FMRP-nLuc-3′ UTR reporters with or without a +1 AUG mutant 5′ leader region (n=3; CGG20: p=0.0002; CGG100: p=0.0036). C, Relative nLuc values in neurons transfected with CGG25 FMRP-nLuc-3′ UTR reporters: WT (n=12), +1 AUG/Mock (n=12), +1 AUG/DHPG (n=11) (WT/Mock vs +1AUG/mock: p=0.005; WT/Mock vs +1AUG/DHPG: p=0.4504). D, Rat hippocampal neurons were transfected with a nLuc construct for the C9Orf72 G4C2 hexanucleotide repeat in the Glycine/Alanine reading frame (n=6; p=0.8642). E, Hippocampal neurons were treated with 1A at 20 hours post-transfection. Quantification of nLuc expression following 3 hours of 1A treatment is represented for each indicated reporter (n=3; AUG/Vehicle vs AUG/1A: p=0.8038; RAN/Vehicle vs RAN/1A: p=0.0437; FMRP/Vehicle vs FMRP/1A: p=0.5728). F, Rat hippocampal neurons were treated with ISRIB 6 hours before treatment with vehicle or DHPG (Mock/Mock: n=18, Mock/DHPG n=18, ISRIB/Mock n=17, ISRIB/DHPG n=18; Mock/Mock vs Mock/DHPG: p=0.0342, Mock/DHPG vs ISRIB/DHPG: p=0.0678, ISRIB/Mock vs ISRIB/DHPG: p=0.0058). Panel B, C, F: One-way ANOVA with multiple comparisons. Panel D, E: Two sided Student’s t-test. n.s.=not significant, *p<0.05, **p<0.01, ***p<0.001. Graph is mean ± S.E.M.

Extended Data Fig. 5 CGG RAN ASOs in human cell lines.

A, Schematic of other tested non-cleaving RAN blocking ASOs. Colored bars overlap the corresponding FMR1 5′ leader sequence and start sites; 0 frame ACG (orange), +1 frame ACG (+1RAN ASO-1, purple(18nt) or blue(16nt)) and +1 frame GUG (+1RAN ASO-2, maroon). B, Effect of +1RAN ASO-16 nucleotide on endogenous FMRP expression (0nM vs 25nM: p=0.0021; 0nM vs 75nM: p=0.0404; 0nM vs 100nM: p=0.2021). C, Effect of +0 RAN ASO (18nt) on endogenous FMRP expression (0nM vs 25nM: p=0.9999; 0nM vs 75nM: p=0.9997; 0nM vs 100nM: p=0.9580). D, Effect of Control ASO on endogenous FMRP expression (0nM vs 25nM: p=0.8183; 0nM vs 75nM: p=0.1780; 0nM vs 100nM: p=0.8486). E, Effect of combinatorial treatment with (+1RAN ASO-1 and +1RAN ASO-2 on endogenous FMRP expression at indicated doses (0nM vs 50nM: p=0.6231; 0nM vs 75nM: p=0.0127; 0nM vs 100nM: p=0.0171). F, Impact of +1RAN ASO-1 transfection into patient derived fibroblasts (0nM vs 50nM: p=0.2795; 0nM vs 75nM: p=0.0336; 0nM vs 100nM: p=0.5035). G, Representative immunoblot of FMRP expression after treatment with +1RAN ASO-1 (technical replicates of main Fig. 4a) in transfected control iPSCs. For all experiments, n=3, replicated in 3 independent experiments. For all graphs: One-way ANOVA with a Fisher’s LSD test for dose dependency. n.s.=not significant, *p<0.05, **p<0.01. Graphs are mean ± S.E.M.

Source data

Extended Data Fig. 6 FMRpolyG expression in human cells and control human neurons.

A, Immunocytochemistry against FMRpolyG on Control ((CGG)23), FXTAS ((CGG)100-117), and FXS ((CGG)931-940, fully methylated) patient derived lymphoblasts. B, Rater-blinded quantification of FMRpolyG staining expressed as a ratio to pre-immune serum at the same concentration (μg/mL) on the same cells. Values are expressed relative to the FXS line, which does not express the FMR1 transcript (Control n=50, FXTAS n=133, FXS n=102; p=0.0000000000005). C, Immunocytochemistry to FMRpolyG (red) in mature control human neurons (TUJ1-positive (green)) treated with +1RAN ASO-1 or Control ASO treatment. D, Quantification of FMRpolyG signal with +1RAN ASO-1 (n=90) or Control ASO (n=69) treatment, where “n” is the mean CTCF signal from 5 neurons (p=0.0485). Panel B: Kruskal Wallis test with post-hoc two sided Mann Whitney U tests. Panel D: Two sided unpaired Student t-test. *p<0.05, ****p<0.0001. Box extends to 25th/75th percentiles with a line at mean and whiskers indicate 95% CI. Marked dots are only shown for values outside the 95% CI.

Extended Data Fig. 7 RAN ASOs block mGluR-dependent FMRP translation in human neurons.

A, Immunocytochemistry for mGluR5 in control human iPSC-derived neurons. B, mGluR mRNA expression as quantified by RT-PCR in Control iPSCs vs. neurons at day 49 of differentiation (n=2). C, Impact of 5 minutes of 50μΜ DHPG on calcium transients in human iPSC derived neurons. Graph represents number of neurons with active calcium transients compared to total number of neurons tracked over two independent neuronal cultures (n=2). D, Time course of DHPG effect on FMRP levels in human neurons (0 min: n=20, 5 min: n=35, 30 min: n=34, 60 min: n=46; 0 vs 5 min: p=0.2972, 0 vs 30 min: p=0.0158, 0 vs 60 min: p=0.0072). E, Left: Immunoblot from Control human neurons treated with +1RAN ASO-1 with or without DHPG treatment. Right: Quantification of FMRP expression in human neurons treated with DHPG after pretreatment with +1RAN ASO-1 relative to Control ASO treated neurons (n=3; p=0.0110). F, Representative western of FMRP expression after DHPG in control human neurons pretreated with the indicated ASOs and then treated with vehicle or DHPG. G, PKR has a 30-nucleotide CGG repeat in its 5′ leader. Endogenous expression of PKR was assessed after vehicle or DHPG treatment in iPSC-derived control human neurons. Left: representative immunoblot to PKR after indicated treatments. Right: Quantification of PKR expression by immunoblot in response to DHPG treatment (n=4; p=0.9696). Panel B, C, E, G: Two sided unpaired student t-test. Panel D: One-way ANOVA with multiple comparisons and post-hoc LSD. *p<0.05, **p<0.01. Graphs are mean ± S.E.M (±SD for B and C).

Source data

Extended Data Fig. 8 Characterization of unmethylated Fragile X full mutation iPSC line TC43-97.

A, Detection of three pluripotency markers—OCT-3/4, Nanog, and SSEA4—in the TC43-97 iPSCs confirms successful reprogramming of the fibroblast line. B, Cytogenetic analysis of cells in metaphase revealed an apparently normal male karyotype of TC43-97 iPSCs. C, Methylation sensitive qPCR of FMR1 promoter demonstrates lack of DNA methylation in TC43-97 iPSCs. Methylation levels were calculated relative to the FX hESC condition (n=3). D, Quantification of immunoblots to FMRP in the TC43-97 iPSCs relative to Control (n=3; p=0.0088). Panel D: Two sided Student’s t-test, **p<0.01. Graphs are mean ± S.E.M.

Extended Data Fig. 9 +1RAN ASO effects in TC43-97 neurons.

A, RT-PCR from indicated iPSC lines 24 hours after transfection with indicated ASOs: Control/Control ASO (n=4), Control/+1RAN ASO-1 (n=4), TC43-97/Control ASO (n=7), TC43-97/+1RAN ASO-1 (n=6, p=0.0522). B, Representative western blot showing relative FMRP expression in both Control and TC43-97 neurons treated with increasing doses of +1RAN ASO-1. Each lane is quantified relative to GAPDH and as a percent of the untreated control neurons. C, Soma (DAPI, blue) and processes (Tuj1, red) of differentiated neurons are TUJ1 positive in both control and TC43-97 neurons. D, Representative FMRP immunoblot from TC43-97 neurons treated with DHPG and indicated ASOs. E, Quantification of FMRP fluorescence by immunocytochemistry in human neurons treated as indicated, normalized to untreated Control ASO neurons quantified in parallel. Average fluorescence was binned for every 5 neurons consecutively analyzed to represent an individual data point. (Control ASO/Mock: n=7, Control ASO/DHPG: n=5, +1RAN ASO-1/Mock: n=6, +1RAN ASO-1/DHPG: n=5; Control ASO/Mock vs +1RAN ASO-1/Mock: p=0.0163). F, Survival analysis of TC43-97 iPSC derived neurons treated with 150nM +1RAN ASO-1 (n=272) or Control ASO (n=310), independent experiment and neuronal derivation #2 (p=0.000148). G, Survival analysis of TC43-97 iPSC derived neurons treated with 150nM +1RAN ASO-1 (n=220) or Control ASO (n=190), independent experiment and neuronal derivation #3 (p=0.027). Survival is plotted as cumulative risk of death. Panel C: Two sided Student T-test. Panel E: Two-way ANOVA with post-hoc correction for multiple comparisons. Panels F, G: Cox proportional hazard analysis. n.s.= not significant. * p<0.05. ***p<0.001. Graph is mean ± S.E.M. Box extends to 25th/75th percentiles with a line at mean and whiskers indicate 95% CI.

Source data

Extended Data Fig. 10 Proposed Model for how RAN translation regulates FMRP synthesis.

CGG RAN regulates FMRP synthesis by limiting access of initiation complexes to the AUG initiation codon of FMRP in a repeat-dependent manner. II: mGluR activation bypasses CGG RAN, which allows for enhanced synthesis of FMRP. III: In the absence of CGG RAN or CGG repeat, steady-state FMRP synthesis increases but is decoupled from mGluR activation. IV: Non-cleaving RAN ASOs prevent CGG RAN initiation. This increases steady-state FMRP production, decreases FMRpolyG production and enhances neuronal survival.

Supplementary information

Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 9

Unprocessed western blots.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rodriguez, C.M., Wright, S.E., Kearse, M.G. et al. A native function for RAN translation and CGG repeats in regulating fragile X protein synthesis. Nat Neurosci 23, 386–397 (2020). https://doi.org/10.1038/s41593-020-0590-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41593-020-0590-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing