Abstract
Common genetic contributions to autism spectrum disorder (ASD) reside in risk gene variants that individually have minimal effect sizes. As environmental factors that perturb neurodevelopment also underlie idiopathic ASD, it is crucial to identify altered regulators that can orchestrate multiple ASD risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1–4 (CPEB1–4) regulate the translation of specific mRNAs by modulating their poly(A)-tails and thereby participate in embryonic development and synaptic plasticity. Here we find that CPEB4 binds transcripts of most high-confidence ASD risk genes. The brains of individuals with idiopathic ASD show imbalances in CPEB4 transcript isoforms that result from decreased inclusion of a neuron-specific microexon. In addition, 9% of the transcriptome shows reduced poly(A)-tail length. Notably, this percentage is much higher for high-confidence ASD risk genes, correlating with reduced expression of the protein products of ASD risk genes. An equivalent imbalance in CPEB4 transcript isoforms in mice mimics the changes in mRNA polyadenylation and protein expression of ASD risk genes and induces ASD-like neuroanatomical, electrophysiological and behavioural phenotypes. Together, these data identify CPEB4 as a regulator of ASD risk genes.
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References
Hallmayer, J. et al. Genetic heritability and shared environmental factors among twin pairs with autism. Arch. Gen. Psychiatry 68, 1095–1102 (2011).
Sandin, S. et al. The familial risk of autism. J. Am. Med. Assoc. 311, 1770–1777 (2014).
De Rubeis, S. & Buxbaum, J. D. Genetics and genomics of autism spectrum disorder: embracing complexity. Hum. Mol. Genet. 24 (R1), R24–R31 (2015).
Kim, Y. S. & Leventhal, B. L. Genetic epidemiology and insights into interactive genetic and environmental effects in autism spectrum disorders. Biol. Psychiatry 77, 66–74 (2015).
Sztainberg, Y. & Zoghbi, H. Y. Lessons learned from studying syndromic autism spectrum disorders. Nat. Neurosci. 19, 1408–1417 (2016).
Gaugler, T. et al. Most genetic risk for autism resides with common variation. Nat. Genet. 46, 881–885 (2014).
Geschwind, D. H. & State, M. W. Gene hunting in autism spectrum disorder: on the path to precision medicine. Lancet 14, 1109–1120 (2015).
Willsey, A. J. & State, M. W. Autism spectrum disorders: from genes to neurobiology. Curr. Opin. Neurobiol. 30, 92–99 (2015).
Ivshina, M., Lasko, P. & Richter, J. D. Cytoplasmic polyadenylation element binding proteins in development, health, and disease. Annu. Rev. Cell Dev. Biol. 30, 393–415 (2014).
Sarkissian, M., Mendez, R. & Richter, J. D. Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3. Genes Dev. 18, 48–61 (2004).
Si, K. et al. A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in aplysia. Cell 115, 893–904 (2003).
Fioriti, L. et al. The persistence of hippocampal-based memory requires protein synthesis mediated by the prion-like protein CPEB3. Neuron 86, 1433–1448 (2015).
Udagawa, T. et al. Genetic and acute CPEB1 depletion ameliorate fragile X pathophysiology. Nat. Med. 19, 1473–1477 (2013).
Sultana, R. et al. Identification of a novel gene on chromosome 7q11.2 interrupted by a translocation breakpoint in a pair of autistic twins. Genomics 80, 129–134 (2002).
Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011).
Parikshak, N. N. et al. Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism. Nature 540, 423–427 (2016).
Iossifov, I. et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature 515, 216–221 (2014).
De Rubeis, S. et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature 515, 209–215 (2014).
Takata, A. et al. Integrative analyses of de novo mutations provide deeper biological insights into autism spectrum disorder. Cell Reports 22, 734–747 (2018).
Calderone, V. et al. Sequential functions of CPEB1 and CPEB4 regulate pathologic expression of vascular endothelial growth factor and angiogenesis in chronic liver disease. Gastroenterology 150, 982–997 (2016).
Igea, A. & Méndez, R. Meiosis requires a translational positive loop where CPEB1 ensues its replacement by CPEB4. EMBO J. 29, 2182–2193 (2010).
Xiong, H.Y. et al. The human splicing code reveals new insights into the genetic determinants of disease. Science 347, 1254806 (2015).
Irimia, M. et al. A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell 159, 1511–1523 (2014).
Tapial, J. et al. An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms. Genome Res. 27, 1759–1768 (2017).
Theis, M., Si, K. & Kandel, E. R. Two previously undescribed members of the mouse CPEB family of genes and their inducible expression in the principal cell layers of the hippocampus. Proc. Natl Acad. Sci. USA 100, 9602–9607 (2003).
Yamasue, H. & Domes, G. Oxytocin and autism spectrum disorders. Curr. Top. Behav. Neurosci. 35, 449–465 (2018).
Hu, W., Yuan, B. & Lodish, H. F. Cpeb4-mediated translational regulatory circuitry controls terminal erythroid differentiation. Dev. Cell 30, 660–672 (2014).
Li, Y. I., Sanchez-Pulido, L., Haerty, W. & Ponting, C. P. RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts. Genome Res. 25, 1–13 (2015).
Pedrotti, S. et al. The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function. Hum. Mol. Genet. 24, 2360–2374 (2015).
Chen, J. A., Peñagarikano, O., Belgard, T. G., Swarup, V. & Geschwind, D. H. The emerging picture of autism spectrum disorder: genetics and pathology. Annu. Rev. Pathol. 10, 111–144 (2015).
Rabaneda, L. G., Robles-Lanuza, E., Nieto-González, J. L. & Scholl, F. G. Neurexin dysfunction in adult neurons results in autistic-like behavior in mice. Cell Reports 8, 338–346 (2014).
Kalkbrenner, A. E., Schmidt, R. J. & Penlesky, A. C. Environmental chemical exposures and autism spectrum disorders: a review of the epidemiological evidence. Curr. Probl. Pediatr. Adolesc. Health Care 44, 277–318 (2014).
Maeyama, K. et al. Congenital cytomegalovirus infection in children with autism spectrum disorder: systematic review and meta-analysis. J. Autism Dev. Disord. 48, 1483–1491 (2018).
Batra, R. et al. RNA-binding protein CPEB1 remodels host and viral RNA landscapes. Nat. Struct. Mol. Biol. 23, 1101–1110 (2016).
Mangiarini, L. et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493–506 (1996).
Shin, J., Salameh, J. S. & Richter, J. D. Impaired neurodevelopment by the low complexity domain of CPEB4 reveals a convergent pathway with neurodegeneration. Sci. Rep. 6, 29395 (2016).
Mayford, M., et al. Control of memory formation through regulated expression of a CaMKII transgene. Science 274, 1678–1683 (1996).
Irizarry, R. A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31, e15 (2003).
Yates, A. et al. Ensembl 2016. Nucleic Acids Res. 44, D710–D716 (2016).
Piqué, M., López, J. M., Foissac, S., Guigó, R. & Méndez, R. A combinatorial code for CPE-mediated translational control. Cell 132, 434–448 (2008).
Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).
Cajigas, I. J. et al. The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging. Neuron 74, 453–466 (2012).
Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Hansen, K. D., Irizarry, R. A. & Wu, Z. Removing technical variability in RNA-seq data using conditional quantile normalization. Biostatistics 13, 204–216 (2012).
Shen, S. et al. MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-seq data. Nucleic Acids Res. 40, e61 (2012).
Belloc, E. & Méndez, R. A deadenylation negative feedback mechanism governs meiotic metaphase arrest. Nature 452, 1017–1021 (2008).
R Development Core Team. R: A language and environment for statistical computing (R Foundation for Statistical Computing, 2014).
Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).
Irizarry, R. A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).
Gentleman, R. C., Carey, V. J., Huber, W., Irizarry, R. & Dudoit, S. Bioinformatics and Computational Biology Solutions Using R and Bioconductor (Springer, New York, 2005).
Eklund, A. C. & Szallasi, Z. Correction of technical bias in clinical microarray data improves concordance with known biological information. Genome Biol. 9, R26 (2008).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protocols 4, 44–57 (2009).
Lucas, J. J. et al. Decreased nuclear β-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3β conditional transgenic mice. EMBO J. 20, 27–39 (2001).
Risher, W. C., Ustunkaya, T., Singh Alvarado, J. & Eroglu, C. Rapid Golgi analysis method for efficient and unbiased classification of dendritic spines. PLoS ONE 9, e107591 (2014).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
Peça, J. et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437–442 (2011).
Ting, J. T., Daigle, T. L., Chen, Q. & Feng, G. Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol. Biol. 1183, 221–242 (2014).
Zhao, S. et al. Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8, 745–752 (2011).
Guzman, S. J., Schlögl, A. & Schmidt-Hieber, C. Stimfit: quantifying electrophysiological data with Python. Front. Neuroinform. 8, 16 (2014).
Acknowledgements
This work was supported by grants: ISCIII-CiberNed-PI2013/09 & -PI2015-2/06 (J.J.L., R.F.-C.), FEDER-PI14/00125 & -PI17/00199 (P.N.), MINECO-SAF2012-34177 & -SAF2015-65371-R (J.J.L.), FEDER-BFU2014-54122-P (R.M.), -BFU2014-55076-P (M.I.), -BFU2016-76050-P (R.F.-C.), -SEV-2012-0208 to CRG by European Union FEDER (M.I.); NIMH 5R37 MH060233, 5R01 MH09714 and 5R01 MH100027 (D.H.G.); Junta de Andalucía-P12-CTS-2232 & -CTS-600 (R.F.-C.); Generalitat de Catalunya-2014/SGR/143 (P.N.); ERC-StG-LS2-637591 (M.I.); and from Fundación Botín-Banco Santander/Santander Universities Global Division, Fundación BBVA, and Fundación Ramón Areces. A.P. was recipient of a MICINN FPI-fellowship; N.N.P. was supported by the NRSA F30 MH099886, UCLA Medical Scientist Training Program and V.S. by a Larry Hillblom Postdoctoral Fellowship. We thank the computing facilities of Extremadura Research Centre for Advanced Technologies (CETA-CIEMAT/Government of Spain), which is funded by ERDF. Tissue, biological specimens or data used in this research were obtained from the Autism BrainNet (formerly the Autism Tissue Program), which is sponsored by the Simons Foundation, and the University of Maryland Brain and Tissue Bank (a component of the NIH NeuroBioBank). We thank the patients and families who participated in the tissue donation programs; R. García-Escudero for bioinformatics advice; M. Lucas for technical assistance; and the following core facilities: CBMSO-Genomics & Massive Sequencing, CBMSO-Animal Facility, CBMSO-Confocal Microscopy, CBMSO-CNB Mouse Transgenesis, CNB-Proteomics, IRB-Functional Genomic and IRB-Bioinformatics/Biostatistics.
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Nature thanks R. Kelleher, N. Sonenberg and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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A.P. contributed to study design and was involved in all assays and data collection. H.A. and E.B. contributed to RIP and PolyU chromatography experiments and analysed data from doxycycline-treated mice. M.S.-G., S.P. and I.H.H. performed western blotting and qRT–PCR analysis. J.L.N.-G. performed electrophysiological recordings. A.R. optimized microarray processing. V.S., A.E., J.I.D.-H., N.N.P. and M.I. performed bioinformatics analyses. R.F.-C., P.N., O.P., M.I., R.M. and D.H.G. made intellectual contributions to experimental design and discussion. D.H.G., R.M. and J.J.L. directed the study and designed experiments. J.J.L. wrote the paper with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Enrichment in ASD risk genes among CPEB1–4 binding transcripts whose poly(A)-tail was shortened in an HD mouse model with altered CPEBs.
a, Experimental design of RIP from wild-type and HD mice (with altered CPEB1 and CPEB4, see Methods). b, Percentage of CPE sequences in the 3′ UTR of total genome, brain genes and CPEB1 and CPEB4 binders from the RIP experiment. c, Percentage of CPEB1- or CPEB4-only binders with shortened (red), lengthened (blue) or unaltered (purple) poly(A) tails. d, Symbol and gene names of CPEB4 binders in wild-type striatum with the most shortened poly(A)-tails (FC ≤ –3.0) in HD mice. The last column indicates whether they are also CPEB1 binders (Y, yes; N, no). High-confidence ASD risk genes (SFARI categories 1–3) are highlighted in pink. e, FC enrichment of high-confidence ASD genes (SFARI categories 1–3 and 1–2) in CPEB4 binders whose poly(A) tails were shortened in HD mice (FC ≤ –3.0). f, Heat maps of CPEB4/CPEB1 binders in SFARI ASD genes or with intellectual disability genes removed (ASD only). g, As in f, for weighted gene co-expression network analysis (WGCNA) modules involved in ASD. h, FC enrichment of percentage of CPE sequences and CPEB4 binders of ASD genes (SFARI categories 1–2, n = 63) versus total genome stratified by 5′ UTR, 3′ UTR, CDS or gDNA length and ratio of neuronal-to-glial expression. b, e–g, One-sided Fisher’s exact test. c, Pearson’s chi-squared test. h, Statistical details for simulations in Methods section. **P < 0.01, ***P < 0.001.
Extended Data Fig. 2 mRNA and protein levels of CPEBs in cortex of individuals with idiopathic ASD and features of CPEB4 mis-splicing.
a, CPEB1–CPEB3 mRNA expression levels according to RNA-seq data (n = 63 for control, n = 43 for ASD). b, c, CPEB1–3 protein levels (b; n = 10) and CPEB4 protein levels (c; n = 20 for control and n = 19 for ASD). d, Diagram representing the alternative splicing of CPEB4 by rMATS. PSI is shown under each event (n = 81 for control and n = 82 ASD cortical prefrontal and temporal samples). e, CPEB4 exon 4 inclusion level (PSI) in all (left) and over-35-year-old (right) individuals. f, Percentage of each CPEB4 splicing isoform by VAST-TOOLS analysis of isoform-specific exon–exon junctions (EEJs). g, h, Percentage of each CPEB4 splicing isoform by digital-droplet PCR (g) and absolute qRT–PCR (h). i, Δ4/Ex4+ CPEB4 isoform ratio in cortex from individuals with idiopathic ASD (n = 11) and control individuals (n = 10) under 35 years old. For gel source data, see Supplementary Fig. 1. a, d–g, i, Two-sided Mann–Whitney–Wilcoxon test. b, h, Two-sided unpaired t-test. Box plots: centre line shows median; box shows 25th and 75th percentiles; whiskers show minimum and maximum values. Bar graphs show mean ± s.e.m. *P < 0.05, **P < 0.01.
Extended Data Fig. 3 Supplementary data for global poly(A)-alterations and protein levels in brains from individuals with idiopathic ASD.
a, Experimental design. b, Poly(A) changes in CPEB4 binders. c, Gene counts histogram from GO analysis (KEGG pathways) of genes with poly(A) tail changes. d, Frequency distribution of FCs of poly(A) alteration of total genes (black) and ASD genes (SFARI categories 1–2, pink). e, Percentage of genes with shortened (red), lengthened (blue) or unaltered (purple) poly(A)-tail length in the whole transcriptome and ASD genes (SFARI categories 4–1) patient-by-patient. f, FC enrichment of brain-, oligodendrocytic-, astrocytic-, neuronal-, synaptic- and ASD-specific genes (SFARI categories 1–2) with shortened poly(A)-tails compared to total genome. g, FC enrichment of ASD (SFARI categories 1–2) genes shortened in ASD human versus total genome stratified by 5′ UTR, 3′ UTR, CDS and gDNA length and by ratio or neuronal-to-glial expression. h, Hire-PAT assay of PTEN poly(A)-tail in control and ASD cases (n = 3). i, Protein levels of neuronal and astrocytic specific genes in cortex from individuals with idiopathic ASD and control individuals (n = 7). For gel source data, see Supplementary Fig. 1. b, f, One-sided Fisher’s exact test. c, FDR Benjamini–Hochberg. d, Two-sided Mann–Whitney–Wilcoxon test. f, P values of genes with shortened poly(A) in each group with respect to ASD genes. g, Statistical details in simulations in Methods. h, i, Two-sided unpaired t-test. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 4 Poly(A) changes in CPEB4-deficient mice.
a, b, Construct design and CPEB4 protein levels of CPEB4 KOGT/+ mice (a; n = 7) and CPEB4 KO mice (b; n = 3). LCD isoform. c, d, Percentage of transcripts with poly(A)-tail changes in CPEB4 KOGT/+ (c) and CPEB4 KO (d) cortex and striatal samples (n = 2), in whole transcriptome and in ASD gene lists. e, Comparison of genes with poly(A) changes between CPEB4 KOGT/+ and CPEB4 KO mice. f, Comparison of genes with poly(A) changes between ASD cases and CPEB4-deficient mice. g, h, FC enrichment of brain-, oligodendrocyte-, astrocyte-, neuron-, synapse- and ASD-specific genes (SFARI categories 1–2) with lengthened poly(A)-tails with respect to total transcriptome in CPEB4 KOGT/+ mice (g) and CPEB4 KO mice (h). For gel source data, see Supplementary Fig. 1. a, Two-sided unpaired t-test. c, d, One-sided Fisher’s exact test, P values of ASD transcripts with lengthened poly(A) versus total. e, f, Hypergeometric test. g, h, One-sided Fisher’s exact test, P values of genes with lengthened poly(A) in each group with respect to ASD genes. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 5 Supplementary characterization of TgCPEB4Δ4 mice.
a, Breeding protocol to obtain TgCPEB4Δ4 mice. Number of mice and percentages of births observed and expected for the four experimental genotypes. b, Kaplan–Meier curve for cumulative survival (continuous line) and probability of developing cranial dysmorphology (dashed line) (n = 44 for control, n = 39 for TgCPEB4Δ4 mice). c, Evolution of body weight (grams). Males (continuous line), n = 25 controls, n = 9 TgCPEB4Δ4 mice. Females (dashed line), n = 26 control, n = 7 TgCPEB4Δ4 mice. d, β-GAL nuclear staining in forebrain neurons from 1.5-month-old controls (n = 6) and TgCPEB4Δ4 mice (n = 4). LV, lateral ventricle. Scale bars, 250 μm. e, Striatal CPEB4 immunohistochemistry shows cytoplasm pattern in control (n = 6), no staining in CPEB4 KO (n = 2) and overexpressing neurons in TgCPEB4Δ4 mice (n = 4). Scale bars, 50 μm. f, g, Protein (f) and mRNA (g) expression levels of CPEB1–4 in forebrain at embryonic day 18 (n = 3) and cortex at 1.5 months (n = 6), 1 year (n = 4) and 2 years (n = 5) of control and TgCPEB4Δ4 mice. For gel source data, see Supplementary Fig. 1. a, Pearson’s chi-squared test. c, f, g, Two-sided unpaired t-test. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 6 Supplementary data showing global poly(A)-alterations and protein levels in TgCPEB4Δ4 mice.
a, Comparison of genes with poly(A) changes in the same or the opposite direction between humans with ASD and TgCPEB4Δ4 mice. b, FC enrichment of brain-, oligodendrocyte-, astrocyte-, neuron-, synapse- and ASD-specific (SFARI categories 1–2) genes with shortened poly(A)-tails with respect to total genome in TgCPEB4Δ4 mice. c, FC enrichment of ASD (SFARI categories 1–2, n = 62) genes shortened in TgCPEB4Δ4 mice and lengthened in CPEB4 KOGT/+ and CPEB4 KO mice versus total genome stratified by 5′ UTR, 3′ UTR, CDS and gDNA length, and ratio of neuronal-to-glial expression. d, Protein levels in striatum of 1.5-month-old control and TgCPEB4Δ4 mice (n = 7). e, Hire-PAT assay of Auts2 poly(A)-tail in control and TgCPEB4Δ4 mice (n = 3). f, Protein levels of neuron- and astrocyte-specific genes in cortex of control and TgCPEB4Δ4 mice (n = 7). For gel source data, see Supplementary Fig. 1. a, Hypergeometric test. b, One-sided Fisher’s exact test, P values of genes with shortened poly(A) in each group with respect to ASD genes. c, Statistical details in simulations in Methods. d–f, Two-sided unpaired t-test. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 7 TgCPEB4Δ4/CPEB4-KOGT/+ mice but not CPEB4 KOGT/+ mice show ASD gene protein changes.
a, Breeding protocol to obtain TgCPEB4Δ4/CPEB4-KOGT/+ mice. b, CPEB4 protein levels in cortex of control 1.5-month-old (n = 16), CPEB4 KOGT/+ (n = 8), TgCPEB4Δ4 (n = 11) and TgCPEB4Δ4/CPEB4-KOGT/+ mice (n = 5). c, Percentage of CPEB4 splicing isoforms and Δ4/Ex4+ ratio in cortex of control, CPEB4 KOGT/+, TgCPEB4Δ4 and TgCPEB4Δ4/CPEB4-KOGT/+ mice (n = 3) by PCR with primers annealing to exons 2 and 5. d, f, Protein levels of ASD genes in control (n = 8) and TgCPEB4Δ4/CPEB4-KOGT/+ mice (n = 6) (d) and control and CPEB4 KOGT/+ mice (n = 7) (f). e, Protein levels of neuron- and astrocyte-specific genes in cortex of control (n = 8) and TgCPEB4Δ4/CPEB4-KOGT/+ mice (n = 6). For gel source data, see Supplementary Fig. 1. b, One-way ANOVA followed by Games–Howell post hoc test. c, One-way ANOVA followed by Tukey’s post hoc test. d–f, Two-sided unpaired t-test. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 8 TgCPEB4Δ4/CPEB4-KOGT/+ mice, but not CPEB4 KOGT/+ mice, show anatomical and behavioural alterations.
a, Brain weight in 6-week-old control (n = 45), CPEB4 KOGT/+ (n = 25), TgCPEB4Δ4 (n = 13) and TgCPEB4Δ4/CPEB4-KOGT/+ (n = 6) mice and evolution of body weight of control (n = 74), CPEB4 KOGT/+ (n = 27), TgCPEB4Δ4 (n = 18) and TgCPEB4Δ4/CPEB4-KOGT/+ (n = 6) mice. b, Immunohistochemistry against anti-cleaved caspase-3 in cortex (n = 3 slices from six controls and six TgCPEB4Δ4 mice). Scale bars, 250 μm. c, Striatal neuronal cell density in control (n = 19) and TgCPEB4Δ4 mice (n = 5). d, Spine density (spines per μm) in cortical layers II/III of pyramidal neurons in CPEB4 KOGT/+ mice (n = 5 cells from three controls, and n = 5 cells from four CPEB4 KOGT/+ mice). e, Amplitude (pA) and frequency (Hz) of mEPSCs recorded from pyramidal neurons of the somatosensory cortex, in CPEB4 KOGT/+ mice (n = 13 cells from five controls, and n = 17 cells from six CPEB4 KOGT/+ mice). f, Ultrasonic calls of pups during 5 min after separation from their mothers as mean of data from postnatal days 6 and 12 in control (n = 36), CPEB4 KOGT/+ (n = 22), TgCPEB4Δ4 (n = 17) and TgCPEB4Δ4/CPEB4-KOGT/+ (n = 4) pups. g, Stereotypical running represented as distance travelled (cm) in the periphery in the open field test in control (n = 74), CPEB4 KOGT/+ (n = 25), TgCPEB4Δ4 (n = 19) and TgCPEB4Δ4/CPEB4-KOGT/+ (n = 6) mice. h, Time interacting with empty cage or an unfamiliar mouse during 10 min for control (n = 40), CPEB4 KOGT/+ (n = 24), TgCPEB4Δ4 (n = 11) and TgCPEB4Δ4/CPEB4-KOGT/+ (n = 4) mice. a, One-way ANOVA followed by Games–Howell post hoc test. b, Two-sided Mann–Whitney–Wilcoxon test. c, e, Two-sided unpaired t-test. f, g, Kruskal–Wallis one-way ANOVA. h, Two-sided Wilcoxon signed-rank test. Data are mean ± s.e.m. n.s., non-significant, *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 9 Effect on ASD-like behaviours of doxycycline-mediated temporal regulation of transgene expression in TgCPEB4Δ4 mice.
a–d, TgCPEB4Δ4 mice with transgene expression starting at the age of 3 weeks (OFF/ON-TgCPEB4Δ4 mice) do not display ASD-like behavioural phenotypes. a, β-GAL nuclear staining in forebrain neurons and CPEB4 immunohistochemistry in 3-month-old control and TgCPEB4Δ4 mice (n = 3). b, Evolution of body weight (grams) of male (n = 29 controls, n = 11 OFF/ON-TgCPEB4Δ4) and female mice (n = 29 control, n = 10 OFF/ON-TgCPEB4Δ4). No premature death or cranial dysmorphology was observed in OFF/ON-TgCPEB4Δ4 mice. c, Total distance travelled by control (n = 9) and OFF/ON-TgCPEB4Δ4 (n = 7) mice and percentage of their distance in the periphery and in the centre in open field test. d, Time interacting with either an empty cage, an unfamiliar mouse or without any interaction during 10 min for control (n = 12) and OFF/ON-TgCPEB4Δ4 mice (n = 5). e–h, Silencing transgene expression in TgCPEB4Δ4 mice that have expressed the transgene during embryonic development does not revert ASD-like behaviours (ON/OFF-TgCPEB4Δ4 mice). e, Kaplan–Meier curve for cumulative survival (solid line) and percentage of mice developing cranial dysmorphology (dashed line), n = 21 for controls, n = 16 for ON/OFF-TgCPEB4Δ4 mice. f, Evolution of body weight (grams) in male (n = 19 controls and n = 10 ON/OFF-TgCPEB4Δ4) and female mice (n = 12 control and n = 6 ON/OFF-TgCPEB4Δ4). g, Total distance travelled by control (n = 16) and ON/OFF-TgCPEB4Δ4 (n = 10) mice and percentage of their distance in the periphery and in the centre in open field test. h, Time interacting with either an empty cage, an unfamiliar mouse or without any interaction during 10 min for control (n = 20) and ON/OFF-TgCPEB4Δ4 mice (n = 13). b–d, f, Two-sided unpaired t-test. g, h, Two-sided Mann–Whitney-Wilcoxon test. h, Two-sided Wilcoxon signed-rank test. Data are mean ± s.e.m. n.s., non-significant, *P < 0.05, **P < 0.01, ***P < 0.001.
Supplementary information
Supplementary Figure 1
This file contains the uncropped blots.
Supplementary Table 1
Results from CPEB1 and CPEB4 RIP experiment. a, (Columns a-c), Probe, symbol and gene name. (Columns d-e), Fold enrichment (FC) of mRNA immunoprecipitated with CPEB1 and (Columns g-h), CPEB4 proteins in St of WT and HD mice (FC > 1.75 highlighted in green). (Columns f and i), CPEB1 and/or CPEB4 positive (+) binder genes. (Column j), Transcripts recognized by CPEB1 and/or CPEB4 (CPEB1 only, CPEB4 only and Common). b, Transcripts with polyA-tail shortened in HD mice (FC < -1.5) and CPEB4 binders in WT Striatum. (Columns a-d), Number, probe, symbol and gene name. (Column e), Indicates if they are also CPEB1 binders (Y: yes, N: no).
Supplementary Table 2
Lists of highly validated ASD genes. (Columns a-b), Gene symbol and gene name. (Columns c-d), SFARI category (cat. 1-4) and gene syndromic in SFARI (https://gene.sfari.org/autdb/GS_Home.do) in July 2017 (Column e), ASD only gene list, where genes linked to intellectual disability have been removed. (Columns f-g), De Rubeis et al., 2014 (FDR<0.05) and Iossifov et al., 2014 positive genes (ASD39 list). (Column h), Takata et al. 2018 list (PBH < 0.05).
Supplementary Table 3
Results of poly(U) chromatography in human prefrontal Cx (CTRL vs. ASD patients). (Columns a-d), Probe, gene symbol, gene name and chromosomal location. (Columns e-ae), Individual samples expression in input, wash and eluted fractions. (Columns af-ag), CTRL and ASD mean. (Columns ah-aj), Log2 fold change, fold change of poly(A) tail length in CTRL vs. ASD patients and P-values. Shortened with respect to CTRL: FC of poly(A) tail length negative and P<0.05. Lengthened with respect to CTRL: FC positive and P<0.05. CTRL (n = 5) and ASD patients (n = 6), statistical details in method section.
Supplementary Table 4
Results of poly(U) chromatography in mouse Cx-St (Control vs. TgCPEB4∆4, WT vs. CPEB4 KOGT/+ and CPEB4 KO). (Columns a-d), Probe, gene symbol, gene name and chromosomal location. (Columns e-v), Individual samples expression in input, wash and eluted fractions from Control and TgCPEB4∆4 mice. (Columns w-x), Control and TgCPEB4∆4 mice mean, log2 fold change, fold change of poly(A) tail length in control vs. TgCPEB4∆4 mice and P-values. (Columns ab-as), Individual samples expression in input, wash and eluted fractions from WT, CPEB4 KOGT/+ and CPEB4 KO mice. (Columns at-av), WT, CPEB4 KOGT/+ and CPEB4 KO mean. (Columns aw-ay), WT vs CPEB4 KOGT/+; log2 fold change, fold change of poly(A) tail length and P-values. (Columns az-bb), WT vs. CPEB4 KO; log2 fold change, fold change of poly(A)-tail length and P-values. Shortened with respect to control: FC of poly(A) tail length negative and P<0.05. Lengthened with respect to control: FC positive and P<0.05. Control (n = 3) vs. TgCPEB4∆4 (n = 3), WT vs. CPEB4 KOGT/+ and CPEB4 KO (n = 2), statistical details in methods section.
Supplementary Table 5
Enrichment of ASD genes. (Column a), Control gene groups: total genome, brain, neuronal and synaptic-enriched. (Column b), Stratification test: 5’ UTR (+/- 75 nt), 3’ UTR (+/- 150 nt), CDS length (+/- 200 nt), genomic size (+/-2,000 bp) and ratio of Neuronal vs. Glial expression (+/- 0.1). (Column c-v), P-values and FC of percentage of CPEs, CPEB4 binders and genes with poly(A)-tail shortened and lengthened in ASD human, TgCPEB4Δ4, CPEB4 KOGT/+ and CPEB4 KO mice of ASD genes (cat. 1-2).
Supplementary Table 6
Metadata for human samples. (Columns a-p), BrainID, Sex, Age, PMI (hours), RIN (Bank), RIN of PolyA, Diagnosis, Detailed Diagnosis, Brain Bank, Brain Weight, pH, Primary Cause of Death, Seizures, Seizure notes, Medications, Comorbidity notes, (Columns q-s), Available samples, (Column q), Protein for WB analysis, (Columns r-s), mRNA for qRT-PCR and gene expression by RNAseq. (Columns t-s), Totals reads in Bam, Ba9 and Ba41 42 22, Splicing Alteration by RNAseq Ba9 and Ba41 42 22. (Columns v-y), Exon 3 and (Columns z-ac), Exon 4 vast-tools output and PSI Ba9 and Ba41 42 22.
Supplementary Table 7
Primers used for RT and PCR reactions. Primers used for RT-PCR (either digital-droplet PCR or real-time quantitative conventional PCR) and for high-resolution poly(A) tail (HIRE-PAT) analysis. (Column a), Human or mouse gene name and specific isoform amplified. (Column b-c), Forward o reverse primer and sequence.
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Parras, A., Anta, H., Santos-Galindo, M. et al. Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing. Nature 560, 441–446 (2018). https://doi.org/10.1038/s41586-018-0423-5
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DOI: https://doi.org/10.1038/s41586-018-0423-5
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