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Aberrant splicing exonizes C9orf72 repeat expansion in ALS/FTD

Nature Neuroscience volume 28, pages 2034–2043 (2025)Cite this article

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Abstract

A nucleotide repeat expansion (NRE) (GGGGCC)n within the first annotated intron of the C9orf72 (C9) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While previous studies have shown that C9 NRE produces several toxic dipeptide repeat (DPR) proteins, the mechanism by which an intronic RNA segment can access the cytoplasmic translation machinery remains unclear. By selectively capturing and sequencing NRE-containing RNAs (NRE-capture-seq) from patient-derived fibroblasts and neurons, we found that, in contrast to previous models, C9 NRE is retained as part of an extended exon 1 due to the usage of various downstream alternative 5′ splice sites. These aberrant splice isoforms accumulate in C9-ALS/FTD brains, and their production is promoted by serine/arginine-rich splicing factor 1 (SRSF1). Antisense oligonucleotides targeting either SRSF1 or the aberrant C9 splice isoforms reduced the levels of DPR. Together, our findings revealed a crucial role of aberrant splicing in the biogenesis of NRE-containing RNAs and demonstrated potential therapeutic strategies to target these pathogenic transcripts.

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Fig. 1: ASO-based capture of NRE-containing transcripts.
Fig. 2: Exonization of C9 NRE by downstream 5′ splice site usage.
Fig. 3: Nucleocytoplasmic distribution of C9 NRE-exonized RNAs.
Fig. 4: C9 aberrant splicing in patient-derived MNs.
Fig. 5: C9 aberrant splicing in ALS/FTD brains.
Fig. 6: Repeat-length-dependent activation of cryptic splice sites by C9 NRE.
Fig. 7: SRSF1 promotes C9 aberrant splicing and nuclear export.

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Data availability

Total RNA-seq and NRE-capture-seq data are available in Gene Expression Omnibus (GEO) under accession number GSE247790. Raw data (FASTQ) and aligned BAM files of postmortem tissue RNA-seq data are available through Target ALS (https://dataengine.targetals.org/). RNA-seq data from neuronal nuclei with and without TDP-43 are available in GEO under accession number GSE126543. Source data are provided with this paper.

Code availability

Custom codes used in this study are available upon request.

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Acknowledgements

We thank S. Ferguson, W. Gilbert, P. Gopal, A. Horwich, J. Humphrey, A. Isaacs, Z. McEachin, P. Miura, K. Neugebauer, J. Steitz, S. Strittmatter, C. Thoreen, P. Todd and members of the Guo lab for helpful discussions and comments on the manuscript. Postmortem tissue bulk RNA-seq data were generated and shared by the New York Genome Center for Genomics of Neurodegenerative Diseases and the Target ALS Human Postmortem Tissue Core. This work was supported by the National Institutes of Health (NIH grants DP2 GM132930, R35 GM152208) and a McKnight Neurobiology of Brain Disorders Award (all to J.U.G.). High-performance computing at the Yale Center for Genome Analysis is supported by NIH (grant S10OD030363-01A1). S.Y. and D.W. were supported by training grant T32 NS041228 from the National Institute of Neurological Disorders and Stroke. J.M.S.S., M.A., T.A. and J.D.P. were supported by the Yale Alzheimer’s Disease Research Center (NIH grant P30 AG066508). J.U.G. is a New York Stem Cell Foundation-Robertson Investigator.

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Authors and Affiliations

  1. Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA

    Suzhou Yang, Denethi Wijegunawardana, Juliana M. S. Salgado, Tanina Arab, Manasi Agrawal, Jeffrey Zhou & Junjie U. Guo

  2. Interdepartmental Neuroscience Program, Yale University, New Haven, CT, USA

    Suzhou Yang, Denethi Wijegunawardana & Junjie U. Guo

  3. Neurobiology of Disease Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA

    Udit Sheth, Austin M. Veire & Tania F. Gendron

  4. Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA

    Udit Sheth, Austin M. Veire & Tania F. Gendron

  5. Department of Neurology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA

    João D. Pereira

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Contributions

S.Y. and J.U.G. conceived the study and wrote the manuscript with input from all authors. S.Y. conducted most experiments and analyzed the results. D.W. conducted RT-qPCR analysis in iPSCs and MNs. U.S. and A.M.V. performed DPR immunoassays under the supervision of T.G. J.M.S.S., M.A., T.A. and J.D.P. performed iPSC culture and MN differentiation. J.Z. assisted in reporter experiments.

Corresponding author

Correspondence to Junjie U. Guo.

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Yale University has filed a patent application based on this work. J.U.G. is a consultant for Corsalex, which was not involved in this project. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Reproducibility of cytoplasmic NRE-capture-seq between biological replicates.

Quantification of normalized read counts per gene from NRE-capture-seq. Left, comparison between two C9 NRE− fibroblast cell lines. Right, comparison between two C9 NRE+ fibroblast cell lines. C9orF72 is indicated in red. Pearson’s correlation coefficients (r) are shown.

Extended Data Fig. 2 Replication and validation of cytoplasmic NRE-capture-seq.

(a) Read coverages of FB503 and FB506 cytoplasmic RNA inputs (top two tracks) and NRE-captured RNAs (bottom two tracks). (b) Schematics of RT-qPCR primers used in (c) and (d). (c) RT-qPCR quantification of the enrichment of aberrant Ex1c-Ex2 splice junction by NRE-capture, comparing C9 NRE+ and NRE− samples. (d) RT-qPCR quantification of siRNA knockdown efficiency in C9 NRE+ fibroblasts. siNT, non-targeting siRNA. siEx2 1 and 2, two siRNAs targeting C9orF72 exon 2. Data represent mean ± s.d. n = 2 biological replicates.

Extended Data Fig. 3 Replication of nuclear NRE-RAP-seq in FB506 fibroblasts.

Read coverage of FB506 nuclear RNA input (top) and NRE-captured RNAs (bottom). Top 10 splice junctions within and near intron 1 detected in NRE-capture-seq results are drawn. RPM, reads per million.

Extended Data Fig. 4 C9 aberrant splicing in C9 NRE+ iPSCs and MNs.

(a) Representative image of neuronal marker immunofluorescence. Scale bars, 20 µm. (b) RT-qPCR quantification of the Ex1c–Ex2 splice junction (left) and the Ex2–Ex3 splice junction (right), comparing C9 NRE+ and C9 NRE− iPSCs. (c) RT-qPCR quantification of the NRE-flanking region (left) and the Ex1c–Ex2 splice junction (right) in NRE-captured RNAs, comparing C9 NRE+ and C9 NRE− MNs, normalized to the value of C9 NRE− MN sample. (d) RT-qPCR quantification of ASO knockdown efficiency in C9 NRE+ fibroblasts. NT, nontargeting ASO. Ex1c–Ex2, ASO targeting Ex1c–Ex2 splice junction. Data represent mean ± s.d. n = 2 biological replicates.

Extended Data Fig. 5 C9 aberrant splicing in C9 NRE+ neuronal nuclei with and without TDP-43.

Normalized counts of reads containing Ex1b–Ex2, Ex1c–Ex2, and Ex1d–Ex2 splice junctions, comparing neuronal nuclei samples with and without loss of TDP-43. RNA-seq data (GSE126543) of ref. 43. The lower hinge, midline, and upper hinge of each box correspond to the first quartile, median, and third quartile, respectively. Upper and lower whiskers extend from the hinge to the maximal or minimal values, respectively, no further than 1.5 times the inter-quartile range. P values, two-tailed ratio t tests.

Extended Data Fig. 6 Impact of cryptic splicing on luciferase activities of repeat-containing reporters.

(a) Normalized activities of dual-luciferase from in vitro transcribed reporters with no NRE and (GGGGCC)33 insert. Data represent mean ± s.d. n = 7 biological replicates. P value, two-tailed ratio t test. (b) Normalized activities of dual-luciferase from original reporter and J1-mutated reporter (top, mutation shown in orange) with no NRE and (GGGGCC)33 insert. n = 3 biological replicates. P values, two-tailed ratio t tests.

Extended Data Fig. 7 Validation of SRSF1 knockdown.

(a) Western blot of SRSF1 in C9 NRE+ fibroblasts transfected with non-targeting (NT) siRNAs and a pool of four siRNAs targeting SRSF1 (S1). (b) RT-PCR quantification of known SRSF1 splicing targets in C9 NRE+ fibroblasts transfected with non-targeting (NT) siRNAs and a pool of four siRNAs targeting SRSF1 (S1). Data represent mean ± s.d. n = 3 biological replicates. P values, two-tailed ratio t tests.

Source data

Extended Data Fig. 8 Effect of SRSF1 knockdown on endogenous C9 NRE and repeat-containing reporter mRNAs.

(a) RT-qPCR quantification of the relative abundance of NRE-flanking region and Ex1c–Ex2 isoform after SRSF1 knockdown in C9 NRE− fibroblasts. Data represent mean ± s.d. n = 3 biological replicates. P values, two-tailed ratio t tests. (b) RT-qPCR quantification of the nucleocytoplasmic distribution of J1-mutated (GGGGCC)33 reporter transcripts (top, mutation shown in orange) in siRNA-treated HEK293T cells. S1, a pool of siRNAs targeting SRSF1 transcripts. Data represent mean ± s.d. n = 3 biological replicates. P values, two-tailed ratio t tests.

Supplementary information

Source data

Source Data Extended Data Fig. 7

Unprocessed western blot and agarose gel images.

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Yang, S., Wijegunawardana, D., Sheth, U. et al. Aberrant splicing exonizes C9orf72 repeat expansion in ALS/FTD. Nat Neurosci 28, 2034–2043 (2025). https://doi.org/10.1038/s41593-025-02039-5

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