Autism-like phenotype and risk gene-RNA deadenylation by CPEB4 mis-splicing

Common genetic contributions to autism spectrum disorder (ASD) reside in risk-gene variants that individually have minimal effect-sizes. Since neurodevelopment-perturbing environmental factors also underlie idiopathic-ASD, it is crucial to identify altered regulators able to orchestrate multiple ASD-risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1-4 (CPEB1-4) regulate translation of specific mRNAs by modulating their poly(A)-tail and participate in embryonic development and synaptic plasticity. Here we find that CPEB4 binds transcripts of most high-confidence ASD genes. Idiopathic-ASD brains show CPEB4 transcript isoform imbalance due to decreased inclusion of a neuronal-specific microexon together with a new molecular signature of global poly(A)-tail shortening that remarkably impacts high-confidence ASD-risk genes with concomitant reduction of their protein levels. Equivalent CPEB4 transcript isoform imbalance in mice mimics the mRNA-polyadenylation and protein level changes of ASD genes and induces ASD-like neuroanatomical, electrophysiological and behavioral phenotypes. Altogether, these data unravel CPEB4 as a novel regulator of ASD-risk genes.

At the protein level, only CPEB4 was significantly altered in idiopathic ASD brains but, strikingly, it was decreased despite increased transcript levels ( Fig. 2b and Extended Data Fig. 2b-c). This suggests that CPEB4 is heavily post-transcriptionally regulated, fitting the autoamplification loop20,21 via CPEB4 binding to its own transcript (Supplementary Table  1a), in line with the multiple CPE sequences in its 3' UTR21. The decreased levels of CPEB4 in ASD occur particularly in young individuals (≤35 years-old), where protein levels were higher compared to older individuals in control (CTRL) samples (Fig. 2b).
Splicing alterations15,22, particularly of microexons23, have been reported in ASD. We thus looked for potential splicing alterations in mRNAs of the different CPEBs in our published16 rMATS-analysis of cortical RNA-seq data. Only CPEB4 showed significant splicing alterations in ASD samples, involving different combinations of two consecutive alternatively spliced exons (Extended Data Fig. 2d): the 51-nt exon 3 and the 24-nt neuronalspecific microexon (exon 4) (Fig. 2c). To specifically investigate changes in the level of inclusion of these exons, we re-analyzed the RNA-seq data using vast-tools24. This revealed significantly less inclusion of exon 4 in ASD brains (ΔPSI=-7.6, Extended data Fig. 2e) which, parallel to the decreased CPEB4 protein levels, essentially occurs in individuals under 35 years old (ΔPSI=-8.8) (Fig. 2d). Interestingly, this microexon encodes the 8-amino acid B region25, which adds potential motifs for posttranslational modifications, such as phosphorylation by AKT, S6K, PKA or PKC (NETPHOS 2.0)25 (Fig. 2c). In contrast, a tendency to more exon 3 inclusion was found in individuals under 35 (Fig. 2d).
Isoform-specific exon-junction RNA-seq reads were used to estimate the relative abundance of the four possible isoforms resulting from alternative splicing of exons 3 and 4 (Full length-CPEB4, CPEB4Δ3, CPEB4Δ4 and CPEB4Δ3Δ4). This revealed a significant increase of the CPEB4Δ4 transcript in ASD (Extended Data Fig. 2f). RT-PCR with primers that simultaneously amplify the four isoforms confirmed the increase in Δ4 transcripts (CPEB4Δ4 and CPEB4Δ3Δ4) to the expense of Ex4+ isoforms (FL-CPEB4 and CPEB4Δ3) in young ASD individuals ( Fig. 2e-f), a pattern that was further validated using conventional and digital-droplet ( Fig. 2g-h and Extended Data Fig. 2g-i) absolute qRT-PCR. Overall, these data demonstrate an increased Δ4/Ex4+ transcript ratio of CPEB4 in ASD, concomitantly with slightly increased total RNA and decreased total protein levels.

Deadenylation of ASD gene mRNAs in ASD brains
We then explored potential genome-wide poly(A)-tail length changes in mRNA from postmortem prefrontal Cx tissue of young controls and idiopathic ASD cases (Extended Data Fig. 3a). In ASD samples, 10.2% and 9.1% of transcripts showed poly(A)-tail lengthening and shortening, respectively ( Fig. 3a "Total" column and Supplementary Table 3). Remarkably, transcripts deadenylated in ASD brains were significantly enriched for CPEB4 binders (Extended Data Fig. 3b). Gene ontology analysis detected "oxytocin signalling pathway" -implicated in social behaviour and proposed to be therapeutically relevant to ASD26-as the most significantly enriched term (among deadenyated CPEB4-binding transcripts, Extended Data Fig. 3c). Interestingly, the SFARI ASD-risk genes globally showed a deadenylation signature ( Fig. 3a and Extended Data Fig. 3d-e). Strikingly, the poly(A)-tail shortening of ASD genes was progressively exacerbated with the increased confidence in causality as defined by SFARI curated gene categories (Fig. 3a). This was not a by-product of enrichment of brain, neuronal or synaptic specific transcripts, or biased 5'UTR, CDS and 3'UTR lengths, gene sizes and ratio of neuronal vs. glial expression (Extended Data Fig. 3f-g and Supplementary Table 5). As expected, CPEB4 binders were overrepresented among deadenylated SFARI cat. 1-3 genes (Fig. 3b), thus suggesting a role of CPEB4 in the observed deadenylation of ASD genes.
We next analysed the protein levels of CPEB4-bound ASD genes in Cx tissue of young idiopathic ASD cases (Fig. 3c). The four studied SFARI cat. 1-2 genes (PTEN, DYRK1A, FOXP1 and WAC) with significant poly(A) shortening (PTEN transcript deadenylation also validated by Hire-PAT, Extended Data Fig. 3h) showed significantly decreased protein levels in ASD brains despite unchanged transcript levels (Fig. 3d). A similar pattern was observed for tested SFARI cat. 3 genes showing a trend for deadenylation (AUTS2, RBFOX1 and ZBTB20). Furthermore, PCDH9 an SFARI cat. 4 gene displaying one of the most prominent and significant shortenings in poly(A)-tail length, also showed decreased protein levels despite increased transcript levels ( Fig. 3c-d). Unaltered protein levels of neuronal-and glial-specific genes such as CALB1, D2R, SNAP25, TUBB3 and IBA1, whose poly(A) tails were not changed in ASD (Extended Data Fig. 3i), rule out an underlying non-specific decrease in protein translation in ASD. Together, these results are consistent with Δ4 CPEB4 isoforms favouring deadenylation (and concomitant decreased protein levels) of target transcripts, including CPEB4 itself, and multiple ASD-risk genes.
Altogether, the presented human data indicate that ASD-risk genes are enriched in CPEcontaining and CPEB4-binder transcripts. Moreover, idiopathic ASD brains show (i) CPEB4 mis-splicing (exon 4 skipping) and reduced CPEB4 protein levels, (ii) a new molecular signature of mRNA deadenylation that remarkably impacts high-confidence ASD-risk genes and (iii) concomitant decreased protein levels of multiple CPEB4-target and deadenylated ASD-risk gene transcripts.

ASD-like poly(A) changes in TgCPEB4Δ4 mice
To determine if changes in CPEB4 splicing and/or protein levels can cause the observed changes in polyadenylation and translation of ASD-risk gene mRNAs, we used different mouse models to mimic the CPEB4 changes observed in idiopathic ASD brains. First, we used two models to emulate the decreased CPEB4 protein levels: a heterozygous CPEB4 KO GT /+ model27 showing partial reduction of CPEB4 protein with unaltered isoform ratios (Extended Data Fig. 4a) and a homozygous CPEB4 KO model20 showing full suppression of CPEB4 protein (Extended Data Fig. 4b). Both models showed similar changes in global transcript polyadenylation, but in the opposite direction to what was observed in ASD cases (Extended Data Fig. 4c-f and Supplementary Table 4). CPEB4-deficient mice showed prominent and significant poly(A) lengthening in ASD-risk genes (Extended Data Fig. 4ch). Therefore, we concluded that the decreased polyadenylation of ASD-risk mRNAs in ASD brains was unlikely a consequence of their reduced CPEB4 levels-on the contrary, decreased CPEB4 by itself had the opposite effect.
To explore the effects of the increase in Δ4 CPEB4 transcript isoforms, we generated mice with conditional neuronal-specific overexpression of CPEB4Δ4 transcript (TgCPEB4Δ4 mice, Fig. 4a), which did not display perinatal lethality-based on live births-and were indistinguishable from their WT and single-transgenic littermates from birth to weaning (Extended Data Fig. 5a). However, at weaning (3 weeks), up to 40% of TgCPEB4Δ4 mice begun to develop cranial dysmorphology suggestive of hydrocephalus. TgCPEB4Δ4 mice with cranial dysmorphology died prematurely, with a mortality peak at 7 weeks of age (Extended Data Fig. 5b). The TgCPEB4Δ4 mice with normal cranial morphology did not present any obvious abnormality nor die prematurely, but they were significantly smaller than their control littermates starting at 3 weeks of age (Extended Data Fig. 5c). In the remaining study, analysis was restricted to TgCPEB4Δ4 mice with normal cranial morphology. Transgene expression in TgCPEB4Δ4 mice takes place in neurons of forebrain structures, such as Cx and St (Extended Data Fig. 5d-e). Importantly, overexpression of CPEB4Δ4 transcript resulted in a Δ4/Ex4+ transcript ratio (Fig. 4b-c) similar to that observed in brains of idiopathic ASD individuals (Fig. 2h). Total CPEB4 protein levels in TgCPEB4Δ4 mice moderately increased in young adults (1.5 months), did not increase at 12 months and a tendency to decrease was observed in 2-year-old mice, despite increased transcript levels at all ages (Extended Data Fig. 5f-g). Likewise, as in human ASD brains, the other CPEBs were essentially unaltered in TgCPEB4Δ4 mice (Extended Data Fig. 5f-g). Strikingly, global poly(A)-tail length changes in TgCPEB4Δ4 mice significantly overlapped with those observed in idiopathic ASD individuals (Fig. 4d, Supplementary Table 4 and Extended Data Fig. 6a) and replicated the predominant deadenylation of ASD-risk genes, a pattern correlated with increasing ASD-risk gene confidence (Fig. 4e). The latter result was robust to different stratification analyses (Extended Data Fig. 6b-c and Supplementary Table  5). Thus, creating a CPEB4 transcript isoform imbalance in mice that mimics the increase in Δ4 isoforms observed in ASD individuals was sufficient to induce the ASD-associated poly(A) signature.
Similar to the observations in human ASD brains, shortened poly(A) tail-length of ASD-risk gene transcripts correlated with reduced protein levels in Cx and St of TgCPEB4Δ4 mice ( Fig. 4f and Extended Data Fig. 6d). Zbtb20, Tnrc6b, Chd2, Foxp1, Wac, Auts2 and Gpc6 are among the deadenylated SFARI cat.1-4 genes (Auts2 deadenylation also validated by Hire-PAT, Extended Data Fig. 6e) whose transcripts are bound by CPEB4, and they all showed significant decreases of their protein levels without decreased transcript levels (Fig.  4f-g and Supplementary Table 4). It is worth noting that RBFOX1 protein-one of the few splicing factors that enable microexon processing in neurons28 and that is known to regulate alternative splicing of CPEB429is also decreased in TgCPEB4Δ4 mice (Fig. 4h) and that Cpeb4 itself shows poly(A) tail shortening in TgCPEB4Δ4 mice (Supplementary Table 4) which may explain why protein levels do not match the increased transcript levels. Unaltered protein levels of non-deadenylated neuronal-and glial-specific genes rules out a non-specific decrease in protein translation efficiency in TgCPEB4Δ4 mice (Extended Data Fig. 6f).
We also generated TgCPEB4Δ4 mice in a CPEB4-KO heterozygous background (Extended Data Fig. 7a). Notably, CPEB4 transcript isoform imbalance persists in TgCPEB4Δ4:CPEB4-KO GT /+ mice without increased CPEB4 protein levels (Extended Data Fig. 7b-c). ASD genes that were diminished in the TgCPEB4Δ4 mice showed similar decreases in protein levels in the TgCPEB4Δ4:CPEB4-KO GT /+ mice, while control neuronal and glial genes were unaltered (Extended Data Fig. 7d-e). These data strongly suggest that the observed effects are due to the transcript isoform imbalance rather than to increased CPEB4 protein levels. Consistent with this, we did not observe decreased protein levels of ASD genes in CPEB4-KO GT /+ mice -which only have decreased CPEB4 protein (Extended Data Fig. 7f).

ASD-related phenotypes of TgCPEB4Δ4 mice
We then tested whether TgCPEB4Δ4 mice showed potential ASD-related anatomical, electrophysiological and/or behavioural abnormalities. We observed reduced brain weight for TgCPEB4Δ4 mice (Extended Data Fig. 8a) as well as for TgCPEB4Δ4:CPEB4-KO GT /+ mice, but not for CPEB4-KO GT /+ mice, with volume reductions in Cx, St and hippocampus together with lateral ventricle enlargement (Fig. 5a) and a two-fold increase in caspase-3 positive cells, without decreased neuronal density (Extended Data Fig. 8b-c). Dendritic spine dysgenesis is frequent in ASD and mouse models30. We found a 9.2% decrease in total spine density in TgCPEB4Δ4 mice ( Fig. 5b) but not in CPEB4 KO GT /+ mice (Extended Fig.  8d). Whole-cell recordings of miniature excitatory postsynaptic currents (mEPSCs) in layer V pyramidal neurons of somatosensory Cx revealed no differences in the mean amplitude but a 32% reduction in the mean frequency in TgCPEB4Δ4 (Fig. 5c), resembling neurexindysfunction mice31. No such electrophysiological alterations were observed in CPEB4-KO GT /+ mice (Extended Data Fig. 8e). Together with the deficit in spine density, these findings are compatible with a presynaptic shortfall in neurotransmitter release and/or a reduction in numbers of excitatory synapses.
We then assayed behaviour. In the open field (OF) test, TgCPEB4Δ4 mice showed stereotypical running at the cage periphery (Fig. 5d), which was not due to anxiety-related behaviour since, in the elevated plus-maze test, TgCPEB4Δ4 mice actually showed reduced levels of anxiety (Fig. 5e). In the ultrasonic vocalization (UsV) test, TgCPEB4Δ4 mice emitted significantly less UsVs (Fig. 5f). In the social approach (SA) test, TgCPEB4Δ4 mice did not show a preference to interact with the cage containing a mouse over the empty one, indicating a dysfunction in sociability (Fig. 5g). TgCPEB4Δ4:CPEB4-KO GT /+ mice, but not CPEB4-KO GT /+ mice, mimicked these ASD-like behaviours (Extended Data Fig. 8f-h), demonstrating that they are due to the altered transcript isoform ratio and not to altered total CPEB4 protein levels. Interestingly, TgCPEB4Δ4 mice treated with doxycycline to express the transgene only during embryonic and early postnatal life (ON/OFF-TgCPEB4Δ4 mice) displayed the stereotypic running in OF and the deficit in SA, while TgCPEB4Δ4 mice treated to express the transgene only after the age of 3 weeks (OFF/ON-TgCPEB4Δ4 mice) did not (Extended Data Fig. 9), indicating that these ASD-like phenotypes in TgCPEB4Δ4 mice originate during development. Together, the stereotypic running, the UsVcommunication deficit, and the diminished social interaction indicate that TgCPEB4Δ4 mice display components of autistic-like behaviour that have been seen in multiple mouse models harbouring single ASD-risk mutations.
Hundreds of minimally penetrant mutations are implicated in ASD6 of which only a fraction will coincide in a given individual. Accordingly, environment-triggered misexpression of multiple ASD-related genes may contribute4, and CPEB4 is well positioned to act as one such neurodevelopment perturbation-driven regulator. It is conceivable that developmentmodifying factors that have been proposed for non-genetic ASD causality1,32 could modulate CPEB4 developmental functions through inducing its mis-splicing. In fact, prenatal cytomegalovirus infection-which has been related to ASD33-has been shown to remodel RNA splicing and polyadenylation in a CPEB1-dependent manner and to increase CPEB4 expression34.
The new molecular signature of transcript-deadenylation in idiopathic ASD brains, combined with proteomics data and gene modules dysregulated in ASD brain, may unravel additional risk genes or pathophysiological pathways. Finally, preclinical studies have mainly relied on monogenic syndromic-ASD mouse models. Since CPEB4 mis-splicing orchestrates expression of a plethora of ASD-risk genes, preclinical testing in TgCPEB4Δ4 mice might be relevant for a wide range of ASDs.

Human brain tissue samples
Brain specimens used in immunoblot, RNA sequencing, absolute qRT-PCR, digital-droplet PCR and poly(U) chromatography in this study from frontal and temporal cortex of Autism Spectrum Disorder (ASD) patients and controls (CTRL) were provided by University of Maryland Brain and Tissue Bank, NIH NeuroBioBank (NBB) (Baltimore,MD) and the Autism Tissue Program (ATP) brain bank at The Harvard Brain and Tissue Bank (Belmont, MA). Written informed consent for brain removal after death for diagnostic and research purposes was obtained from brain donors and/or next of kin. Brain sample and donor metadata is available in Supplementary Table 6.

Animals
As HD mouse model, we used R6/1 mice transgenic for the human exon-1-Htt gene35 because our unpublished results show that these mice have altered levels of CPEBs (specifically CPEB1 and CPEB4) which correlate with changes in poly(A)-tail length of numerous mRNAs. R6/1 mice were in B6CBAF1/J background. Heterozygous CPEB4KO GT /+ mice27 harbor a gene trap between exons 1 and 2 which prevents formation of the full length CPEB4 protein while allowing expression of the N-terminal low complexity domain, LCD)36, thus resulting in results in partial reduction of CPEB4 protein. CPEB4 KO mice20 harbor homozygous deletion of constitutive exon 2 resulting in a premature stop codon and full suppression of CPEB4 protein. CamkII-tTA (tTA)37, CPEB4KO GT /+, and CPEB4 KO mice were in C57BL/6J background. Conditional mice expressing human CPEB4 lacking exon 4 (TgCPEB4∆4) were generated (for details, see "Generation of TgCPEB4∆4 mice" below) for this study and used in C57BL/6J background. All mice were housed in CBMSO animal facility. Mice were housed four per cage with food and water available ad libitum and maintained in a temperature-controlled environment on a 12/12 h light-dark cycle with light onset at 08:00. Animal housing and maintenance protocols followed the local authority guidelines. Animal experiments were performed under protocols (P15/P16/P18/P22) approved by the Centro de Biología Molecular Severo Ochoa Institutional Animal Care and Utilization Committee (Comité de Ética de Experimentación Animal del CBM, CEEA-CBM), and Comunidad de Madrid PROEX 293/15.

RNA immunoprecipitation (RIP)
Four male WT and R6/1 mice (HD mice) were sacrificed by cervical dislocation at the age of 7-8 month-old. Total striatum (St) was quickly dissected on an ice-cold plate and cut into pieces. The pool of the four WT or R6/1 striata was washed twice with phosphate buffer solution (PBS), crosslinked with 0.5% formaldehyde in PBS for 7 min at room temperature and treated with glycine 1M for 5 min. After two washes with cold PBS, pool samples were homogenized in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxicolate, 0.1% SDS, 1 mM EDTA, protease inhibitor (Complete, Roche, 11697498001), RNAse inhibitor (Ribolock, Life Technologies, EO0381)) and centrifuged at 13000g for 10 min at 4ºC. Dynabeads protein A (Life Technologies, 10001D) were washed twice with PBS and incubated with anti-CPEB4 antibody (10 μg, Abcam, ab83009), anti-CPEB1 antibody (5 μg, Proteintech, 13274-1-AP) or rabbit IgG (5 μg, Sigma, I5006) for 2h. Next, dynabeads were washed once with PBS and twice with triethanolamine 0.2 M pH 8.2, incubated with dimethyl pimelimidate 20 mM for 30 min, treated with Tris 50mM pH 8.0 and washed twice with lysis buffer. Lysates were precleared with unconjugated dynabeads for 20 min at 4ºC in a wheel, an aliquot was stored at -80ºC ("Input") and the rest of the extract was immunoprecipitated with the antibody-conjugated dynabeads overnight at 4ºC in a wheel. Immunoprecipitates were washed six times in cold lysis buffer.
For protein extraction, samples were incubated with Laemmli buffer (10% SDS, 0.325 M Tris HCl pH7.5, Glycerol 25%) for 20 min at 60ºC. Dynabeads were removed with the help of a magnet and samples were boiled after adding DTT 0.1 M and bromophenol blue 0.1%.
For RNA extraction, immunoprecipitates were resuspended in 100 µl of proteinase K buffer (200 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% SDS) containing 0.7 µg/µl of proteinase K (Roche, 03115852001) and incubated 1h at 42ºC and 1h at 65ºC. RNA was extracted using the TRIzol reagent (Invitrogen, 15596018). Briefly, samples were resuspended in 300 µl of TRIzol. Then, 20 µg of glycogen was added. Samples were vortexed, incubated for 5 min at room temperature and centrifuged at 14000g for 15 min at 4ºC. The aqueous phase was recovered, mixed with 1 volume of isopropanol, incubated for 5 min at room temperature and precipitated at 14000g 30 min at 4ºC. The pellet was washed with 300 μl of ice-cold 75% ethanol and centrifuged at 14000g 10 min at 4ºC. The pellets were resuspended in 100 μl of nuclease-free water. To ensure the complete removal of the phenol, the RNA was precipitated again. To this aim, 10 μl of sodium acetate 3 M pH 5.6 and 250 μl of 75% ethanol were added. Samples were vortexed and stored at -20ºC for 1h. Then, they were precipitated at 14000g for 30 min at 4ºC. The pellet was washed with 75% ethanol and centrifuged again at 14000g for 10 min at 4ºC. Pellets were air-dried for 5 min and resuspended in nuclease-free water.

RNA isolation, Whole Transcriptome Amplification (WTA) and microarrays processing
Input and immunoprecipitated RNA were purified using Agencourt RNAClean XP bead suspension (Beckman Coulter, A66514). Library preparation and amplification were performed following the distributor's (Sigma-Aldrich) recommendations for WTA2 from purified immunoprecipitated or diluted input RNA. SYBR Green (Sigma-Aldrich, 163795-75-3) was added to the amplification reaction, which was performed in a CFX Realtime instrument (Bio-Rad) to monitor amplification yield. When the SYBR Green signal reached a plateau after 27 cycles, the reaction was stopped. Amplified cDNA was purified and quantified on a Nanodrop ND-1000 spectrophotometer (Thermo-Fischer). 8 µg of cDNA were subsequently fragmented by DNAseI and biotinylated by terminal transferase obtained from GeneChip Mapping 250K Nsp Assay Kit (Affymetrix, 900753). After hybridization for 16h at 45ºC, washing and staining was performed in the Affymetrix GeneAtlas Fluidics Station. The arrays were scanned in the GeneAtlas Imaging Station. All processing was performed according to manufacturer's recommendations. CEL files were generated from DAT files using Affymetrix Command Console software. To generate the log2 expression estimates, overall array intensity was normalized between arrays and the probe intensity of all probes in a probe set summarized to a single value using RMA (Robust Multichip Average) algorithm38.
In order to compare samples from different conditions, fold changes were computed after MA mean and variance normalization using a Generalized Additive Model (GAM). An empirical Bayes partial density model was then used to compute the posterior probability of differential expression. Differentially expressed genes were defined as those with a maximum False Discovery Rate (FDR) of 5% and a log2 fold change threshold of 1.75. We calculated the Input versus inmunoprecipitated fold change (FC) for CPEB1 and CPEB4 binders considering a transcript positive when at least one probe showed a FC above 1.75 in WT or HD mice.

Enrichment and co-expression network analysis of CPEB4 and CPEB1 binders
Enrichment analysis studies use one-sided Fisher's exact test to evaluate whether a gene set, in this case CPEB4 or CPEB1 binders determined by RIP, is enriched over background, providing a P-value and enrichment value. We used curated ASD candidate gene list from Simons Foundation Autism Research Initiative (SFARI) AutDB database, referred as ASD SFARI list and a more restrictive, smaller ASD only gene list, where genes linked to intellectual disability were removed. The gene set (CPEB4 and CPEB1 binders) was also used to study enrichment in functional co-expression modules that represent shared pathology in ASD brain. These gene modules derived from previous unbiased weighted gene co-expression network analysis (WGCNA) obtained by gene array15 and by RNA-seq16 studies from ASD postmortem samples.

Analysis of canonical and functional CPE sequences
3'UTR (untranslated region) sequences from selected gene sets were extracted from Ensembl (http://www.ensembl.org/)39 and incidence of canonical and functional CPE (cytoplasmic polyadenylation element) sequences was detected using the algorithm described in40 (http://genome.crg.es/CPE/). The list of brain genes was obtained from the human protein atlas (http://proteinatlas.org/humanproteome/brain), neuronal, astrocytic and oligodendrocyte-enriched genes from41, synaptic-enriched genes from42 and the ASD gene lists was obtained in July 2017 from SFARI database (categories 1 to 4, https:// gene.sfari.org/autdb/GS_Home.do). ASD39 gene list consists of the 39 genes harboring rare de novo protein disrupting mutations identified in the two most largest whole exome sequencing studies in simplex ASD17,43. Takata gene list consists of the 61 genes enriched for damaging de novo mutatios in ASD identificated by Takata et al19. All lists of ASD causing genes are shown in Supplementary Table 2.

Simulations
To compare feature enrichment in our subset of high confidence AS genes (SFARI cat. 1-2), we first selected several control gene groups: total genome, brain-enriched (from the human protein atlas), neuronal-enriched41 and synaptic-enriched42 and removed those genes previously linked to ASD (i.e. any SFARI category). Then, for each simulation and control group, each ASD gene was matched randomly with a gene from the control group based on its 5' UTR (+/-75 nt), 3' UTR (+/-150 nt) or CDS length (+/-200 nt), genomic size (+/-2,000 bp) or ratio of Neuronal vs. Glial expression (+/-0.1). For the latter, RNA-seq data for isolated populations of neurons, astrocytes, microglia, new oligodendrocytes, and oligodendrocyte precursors was obtained from44 and expression values calculated using vast-tools24. An average value for all glial cell types was calculated for each gene and the ratio between the expression in neurons vs. the glial (NvsG) average used for stratification. Next, the percentage of CPEs, CPEB4 binders and genes with poly(A)-tail shortened and lengthened in ASD human, TgCPEB4Δ4, CPEB4 KO GT /+ and CPEB4 KO mice were calculated in each control and stratified test subset. This process was repeated 10,000 times and P-values were calculated as the number of times the stratified control show the same or higher percentage (or lower, if testing for depletion) than the test set, divided by 10,000. All results are shown in Supplementary Table 5.

Quantification of CPEBs gene expression in human postmortem ASD cases
CPEBs expression levels in idiopathic ASD (n=43 samples from 26 individuals) and CTRL (n=63 samples from 33 individuals) postmortem prefrontal (Ba9) and temporal (Ba41-42-22) cortex samples were evaluated from RNA-seq data16. Briefly, the paired-end raw reads were mapped to the human reference genome assembly GRCh37.73 using Tophat245, and the counts were quantified using HTSeq46. Gene length, G+C content and library size were normalized (referred to as "Normalized FPKM") using the cqn package in R47. Linear mixed effects (LME) were used, modeling to account for effects from biological covariates (condition, age, sex, brain region), technical variables related to sample processing (RIN, brain bank, sequencing batch), technical variables related to sequencing quality metrics and individual ID was set as a random effect accounting for the fact that multiple samples came from the same individual.

Quantification of CPEBs transcript splicing and differential splicing analysis
We used n= 81 CTRL and n = 82 ASD cortical prefrontal and temporal samples from n= 47 ASD and n = 44 CTRL individuals16. We computed percent spliced in (PSI) values using: -Vast-tools (https://github.com/vastgroup/vast-tools). This software consists of multiple utilities to align and process raw RNA-seq reads to derive PSIs for all types of alternative Splicing24. Where two sequencing surrogate variables (seqSV1 and seqSV2) were used as covariates.

RNA extraction and cDNA synthesis
Total tissue RNA was extracted from prefrontal cortex -BA8/9 of CTRL (n = 15) and idiopathic ASD patients (n = 16) and striatum, cortex or forebrain from Control, CPEB4 KO GT /+, TgCPEB4∆4, CPEB4 KO/+ and TgCPEB4∆4:CPEB4 KO GT /+, mice using the Maxwell® 16 LEV simplyRNA Tissue Kit (Promega, AS1280). Quantification and quality of RNA was done on a Nanodrop ND-1000 spectrophotometer and Nanodrop 1000 v.3.7.1 (Thermo Scientific). Retrotranscription (RT) reactions were performed using the iScript cDNA Synthesis kit (Bio-Rad, PN170-8891) following manufacturer´s instructions. Briefly, 1000 ng of total RNA from each samples were combined with 10 μl of master mix (includes all necessary reagents among which a mixture of random primers and oligo-dT for priming). The reaction volume was completed up to 40 μl with DNAse/RNAse free distilled water (Gibco, PN 10977). Thermal conditions consisted of the following steps: 5 min at 25ºC; 20 min at 46ºC and 1 min at 95ºC.

Digital-Droplet PCR
mRNAs of each CPEB4 splicing isoforms were measured by digital-droplet PCR (ddPCR) in a BioRad QX200™ Digital Droplet™ PCR system (Bio-Rad, 1864100). All PCR reactions were made in three replicates and assayed in 96 well plates. The PCR reaction volume was 22 µL using the QX200™ ddPCR™ Evagreen Supermix (Bio-Rad, 1864034). Each reaction included 11 µL of ddPCR™ Evagreen Supermix, forward and reverse primers at 0.9 μM each and 4 ng of template cDNA. The PCR reaction mixture was loaded into an eightwell DG8™ Cartridge for QX200™ (Bio-Rad, 1864008) and droplets with Oil for EvaGreen (Bio-Rad, 1864005) were formed with the Bio-Rad QX200™ Droplet Generator (Bio-Rad, 1864101), following the manufacturer's instructions. During emulsion, the QX200 droplet generator partitions the samples into 20,000 nanolitre-sized droplets. The droplets were then transferred to a 96-well plate and sealed with a Bio-Rad PX1™ PCR Plate Sealer (Bio-Rad, 1814000). Optimal ddPCR annealing temperatures for the CPEB4 isoforms assays were determined by incorporating a temperature gradient from 55.6ºC to 66.6ºC. FL-CPEB4 and CPEB4∆3 were amplified using the following cycling conditions: 95ºC for 5 min, 40x (95ºC for 30 s and an annealing-extension step at 60ºC for 1 min) and 90ºC for 5 min. CPEB4∆4 and CPEB4∆3∆4 were amplified using: 95ºC for 5 min, 40x (95ºC for 30 s + 62ºC for 1 min) and 90ºC for 5 min. Finally, droplets were read on the QX200™ Droplet Reader (Bio-Rad, 1864003) and data were analyzed with Quantasoft Version 1.6.6.0320 (Bio-Rad). The primers used to amplify each CPEB4 isoform are detailed in Supplementary Table 7.

Real-time quantitative reverse transcriptase-PCR
Quantification was performed by real-time PCR using a CFX 384 Real Time System C1000 Thermal Cycler (Bio-Rad) in combination with SsoFast Eva Green (Bio-Rad, CN 172-5204) and 0.25 μM of primer pair was used. Data were analyzed by GenEx 5.3.7 software (Multid AnaLyses AB). The mRNA levels were normalized first relative to total RNA and then relative to the 18S ribosome subunit, β-ACTIN, GAPDH and β-TUBULIN gene expression in each sample. Absolute quantitative PCR was performed to determine the percentage of each CPEB4 splicing isoform in both human and mouse species using specific primers (Supplementary Table 7). For every primer couple, specificity was tested, PCR assay conditions were adjusted to obtain a single amplicon analyzed by both melting curve analysis and agarose gel electrophoresis. Amplicons of each CPEB4 isoform were serially diluted to generate a calibration curve. A duplication of this curve was made to give robustness. Next, total tissue RNA was extracted, and quantitative real-time RT-PCR was performed. Finally, the percentage of each CPEB4 isoform with respect to total CPEB4 copies was calculated.

Western blot
Samples from human brain were stored at -80ºC and were ground with a mortar in a frozen environment with liquid nitrogen to prevent thawing of the samples, resulting in tissue powder. For mouse, brains were quickly dissected on an ice-cold plate and the different structures stored at -80ºC. Human and mouse extracts were prepared by homogenizing the brain areas in ice-cold extraction buffer (

Poly(U) chromatography
Human samples: brain specimens from prefrontal cortex -BA8/9 of ASD patients (n = 6) and CTRL (n = 5) males with age between 5-23 years old. To verify RNA integrity and poly(A)-tail quality, we performed Hire-PAT of a typical normalizer gene (ACTB) and found any alterations in CTRL or ASD brains.
Mouse samples: WT, CPEB4 KO GT /+ and CPEB4 KO (n=2) and Control vs. TgCPEB4∆4 mice (n=3) were sacrificed by cervical dislocation at the age of 6 weeks. The cortex and striatum together were quickly dissected on an ice-cold plate.
Human and mouse samples were homogenized and total RNA was extracted using the Maxwell® 16 LEV simplyRNA Tissue Kit (Promega, AS1280), and stored at -80ºC until use. The poly(A) RNA fraction was purified by poly(U) chromatography49. Poly(U)agarose (Sigma, p8563) was suspended in swelling buffer (0.05 M Tris-HCl, pH 7.5, 1 M NaCl) 35 ml/g, incubated overnight at room temperature and loaded into the chromatography column. An aliquot of total RNA was stored at -80ºC ("Input") and the rest was incubated with sample buffer (0.01 M Tris-HCl, pH 7. Europe PMC Funders Author Manuscripts min and centrifuged 15 min at 14000g at 4ºC. The supernatant was removed and the pellet was washed with 750 µL of ethanol and centrifuged at 14000g and 4ºC for 5 min. The supernatant was removed and the pellet was air-dried for 5 min. The RNAs were resuspended in 300 µL of nuclease-free water and then 300 µL of acid Phenol:Chloroform (5:1) were added to them. Samples were vortexed and centrifuged for 10 min at 14000g and 4ºC. The aqueous phase was recovered, mixed with 1 volume of chloroform, vortexed and centrifuged again. The aqueous phase was recovered and precipitated again using the isopropanol precipitation. When setting up the method, we perform digestion of the nonpoly(A) mRNA regions followed by end-labelling of the poly(A) for each eluted fraction and Urea-PAGE to confirm the average length in each fragment. We also compared poly(A)tail by HIRE-PAT assay of control genes in Input, Washed and Eluted fractions to verify that it worked properly.

Human PrimeView and GeneAtlas MG-430 PM microarrays analysis
cDNA library preparation and amplification were performed according to the manufacturer's instructions (Sigma-Aldrich) for the WTA2 kit from 25 ng starting material. The cDNA was amplified for 17 cycles and purified using PureLink Quick PCR Purification Kit (Invitrogen, K310001). Quantification of amplified cDNA was done on a Nanodrop ND-1000 spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, USA). 8.5 ug of the cDNA from each sample were fragmented and labeling with GeneChip Mapping 250K Nsp assay kit (Affymetrix, 900753) following the instructions of manufacturer.
Human: samples ready to hybridize were denatured at 99ºC for 2 min prior to incubation into the GeneChip Human PrimeView arrays (Affymetrix, 901838). Hybridization was performed for 16h at 45ºC / 60 rpm in the GeneChip Hybridization Oven 645 (Affymetrix, 00-0331). Washing and stain steps after hybridization were performed in the GeneChip Fluidics Station 450 (Affymetrix, 00-0079), following the specific script for PrimeView arrays. Finally, the arrays were scanned with GeneChip Scanner GCS3000 (Affymetrix) using default parameters, and the generation of CEL files for bioinformatics analysis was done with Command Console software (Affymetrix).
Mouse: hybridization was performed using the GeneAtlas Hyb, Wash and Stain Kit for 3' IVT arrays. Samples ready to hybridize were denatured at 96ºC for 10 min prior to incubation into Mouse MG-430 PM Array Strip (Affymetrix, 901570), the hybridization was performed for 16 h at 45ºC in the GeneAtlas Hybridization Oven (Affymetrix, 00-0331).
Washing and stain steps after hybridization were performed in the GeneAtlas Fluidics Station (Affymetrix, 00-0079), following the specific script for Mouse MG-430 PM Arrays. Finally, the arrays were scanned with GeneAtlas Scanner (Affymetrix) using default parameters, and the generation of CEL files for bioinformatics analysis was done with GeneAtlas software (Affymetrix).
Processing of microarray samples was performed using R50 and Bioconductor51. Raw CEL files were normalized using RMA background correction and summarization52. Standard quality controls were performed in order to identify abnormal samples53 regarding: a) spatial artifacts in the hybridization process (scan images and pseudo-images from probe level models); b) intensity dependences of differences between chips (MvA plots); c) RNA

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts quality (RNA digest plot); and d) global intensity levels (boxplot of perfect match logintensity distributions before and after normalization and RLE plots). Probeset annotation was performed using the information available in Affymetrix web page (https:// www.affymetrix.com/analysis/index.affx) using version na35.
Expression values were adjusted for technical biases as described in54 using a linear model and implemented with the R package "limma"55. For each biological replicate the log2 fold change was computed between "WASH" and "ELUTED" samples and used to find significant differences between WT vs. CPEB4 KO GT /+ and CPEB4 KO mice (n = 2), control vs. TgCPEB4∆4 mice (n = 3) and human CTRL (n = 5) vs ASD patients (n = 6). Differential expression was performed using a linear model with fluidics and amplification batch as covariates. P-values were adjusted with the Benjamini and Hochberg correction. We considered one transcript is shortened when P-value < 0.05 and FC is negative and lengthened when P-value < 0.05 and FC is positive, in at least one probe. If the same transcript showed opposite results for different probes, it was considered as not changed.

High-Resolution poly(A) tail (HIRE-PAT) assay
USB® Poly(A) Tail-Length Assay Kit (Affymetrix, 76455) based on HIRE-PAT method, was used. Frontal cortex -BA8/9 of ASD patients (n = 3) and CTRL (n = 3) and total striatal RNA from 1.5 month-old control and TgCPEB4∆4 mice (n = 3) was extracted using the Maxwell® 16 LEV simplyRNA Tissue Kit (Promega, AS1280) and stored at -80ºC until use. G/I tailing (1 μg of total RNA) and reverse transcription were performed according to the manufacturer's instructions. Poly(A) size was determined by subtracting the PCR amplicon size obtained with the Universal primer and forward specific primers. To verify that the measured poly(A) tail corresponds to specific gene three different forward specific primers were tested (Supplementary Table 7). PCR products were resolved on 2.5% agarose/ gelgreen (Biotium, 41004) gels run at 120V for 1.5h.

Gene Ontology analysis
CPEB4 binders with poly(A)-tail changes in prefrontal cortex of ASD individuals were analyzed with DAVID Bioinformatics Resources 6.7, KEGG pathway annotation56.

Human-mouse altered poly(A) tail length geneset comparison
In order to compare transcripts with altered poly(A) tail in ASD patients and CPEB4 modified mice, we first converted the gene set from mouse into their human orthologous Biomart (http://www.ensembl.org/biomart/)39. Then we calculated the statistical significance of the overlap between genes with poly(A) changes in human ASD and CPEB4modified mice by hypergeometric distribution test. We considered overlapping when the representation factor is > 1 and P-value < 0.05, and dissimilar when the representation factor is < 1 and P-value < 0.05.

Generation of TgCPEB4∆4 mice
Human CPEB4 cDNA lacking exon 4 (CPEB4Δ4) was cloned into a plasmid containing a bidirectional TetO sequence to also express LacZ reporter with a nuclear localization signal (pBI-G, Clontech, 631004). The construct was microinjected into single-cell CBAxC57BL/6 embryos and resulting TetO βGAL/CPEB4∆4 founder mice were backcrossed with WT C57BL/6J mice (TetO βGAL/CPEB4∆4). TetO βGAL/CPEB4∆4 mice were crossed with CamkII-tTA (tTA) mice37 to obtain the conditional double transgenic mice with forebrain neuron expression of CPEB4∆4 (TgCPEB4∆4 mice). There are different CamKII-tTA transgenic mouse lines37 and, for this study, we chose one with expression starting at late embryonic age57. Upon observation of premature death of the subset of TgCPEB4∆4 mice showing cranial dysmorphology, these were systematically culled when found.
To generate TgCPEB4Δ4 mice with transgene expression starting after weaning (OFF/ON-TgCPEB4Δ4 mice) pregnant females were isolated and kept on doxycycline (Sigma, D9891, 0.5g/L) until weaning of the litter. After weaning, the progeny was switched to plain water to allow expression of the transgene.
To generate TgCPEB4Δ4 mice that expressed the transgene only during embryonic and early postnatal development (ON/OFF-TgCPEB4Δ4 mice) pregnant females were isolated and kept on plain water until birth of the litter, when water was replaced by doxycycline solution (Sigma, D9891, 2g/L) so doxycycline intake starts in the pups through the milk. The progeny is kept on doxycycline throughout the rest of the experiment.

Immunohistochemistry
Mice were euthanized with CO 2 . Brains were immediately removed and dissected on an icecold plate and left hemispheres, processed for histology, were placed in 4% paraformaldehyde in Sorensen's phosphate buffer overnight and then immersed in 30% sucrose in PBS for 72h. Once cryoprotected, the samples were included in optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek Europe, 4583), frozen and stored at -80ºC until use. 30 µm sagittal sections were cut on a cryostat (Thermo Scientific), collected and stored free floating in glycol containing buffer (30% glycerol, 30% ethylene glycol in 0.02 M phosphate buffer) at -20ºC. Before staining, sections were washed with PBS to eliminate the cryoprotective buffer and immersed in 0.3% H 2 O 2 in PBS for 30 min to quench endogenous peroxidase activity. Sections were immersed for 1h in blocking solution (PBS containing 0.5% Fetal Bovine Serum, 0.3% Triton X-100 and 1% BSA) and incubated overnight at 4ºC with the corresponding primary antibody diluted in blocking solution. After washing, brain sections were incubated first with biotinylated anti-rabbit or anti-mouse secondary antibody and then with avidin-biotin complex using the Elite Vectastain kit (Vector Laboratories, PK-6101-2). Chromogen reactions were performed with diaminobenzidine (SIGMAFASTTM DAB, Sigma, D4293) for 10 min. Sections were mounted on glass slides and coverslipped with Mowiol (Calbiochem, 475904). Images were captured using an Olympus BX41 microscope with an Olympus camera DP-70 and Olympus cellSens Entry v.1.7 (Olympus Denmark A/S)

Brain weight and volumetric analysis
1.5 month-old mice were completely anesthetized with an intraperitoneal pentobarbital injection (60 mg/kg Dolethal®, Vetoquinol). The whole brain was extracted and weighted in a precision scale (Metter Toledo, AB265-S). Left hemispheres were fixed in 4% paraformaldehyde, immersed in 30% sucrose, included in OCT compound (Tissue-Tek, Sakura Finetek Europe, 4583), frozen and stored at -80ºC. Sagittal sections (30 µm thick) were cut on a cryostat and every sixth section was counterstained with toluidine blue pH 4.0 (1g/l Toluidine Blue (Sigma, 198161), 0.8 M glacial acetic acid). Digital images were captured at a 2.5x magnification (Canon EOS 450D digital camera) and the hippocampal, striatal and motor and somatosensory cortical area from 20-22 sections for each animal was calculated by means of the ImageJ software59. Considering a separation of 180 μm between each section, total structure volume in each mouse was calculated.

Electrophysiology
For preparation of acute brain slices, we based on the N-methyl-D-glucamine (NMDG) protective recovery method according to60-62. Briefly, 5-6 weeks-old control vs. TgCPEB4∆4 (n = 5) mice and WT vs. CPEB4 KO GT /+ (n = 5) mice of both sexes were anesthetized with 2% tribromoethanol (0.15 ml/10 mg) and rapidly decapitated. The brains were dissected out and transferred to NMDG ice-cold artificial cerebrospinal fluid (ACSF) composed of (in mM): 93 NMDG, 2. Currents were low-pass filtered at 3 kHz, digitized at 20 kHz, and acquired using PatchMaster software (HEKA). All miniature postsynaptic currents were analyzed with the program Stimfit63. Recordings were first digitally filtered at 1 kHz. For each cell, all events were inspected to avoid false-positive events, and then an average of all events detected was made.

Cleaved caspase-3 quantification
1.5 month-old control and TgCPEB4Δ4 mice (n = 6) were analyzed. The total number of immunopositive cells with apoptotic shape was quantified in the cortex of three sections per animal using an Olympus BX41 microscope with an Olympus camera DP-70 (Olympus Denmark A/S). Means of the three sections were calculated. a, Comparison of genes with poly(A) changes in the same or the opposite direction between human ASD cases and TgCPEB4Δ4 mice, representation factor (RF). b, Fold change enrichment of brain, oligodendrocytic, astrocytic, neuronal, synaptic and ASD specific (SFARI cat.1-2) genes with shortened poly(A)-tail respect to total genome in TgCPEB4Δ4 mice. c, Fold change enrichment of ASD (SFARI cat. 1-2, n = 62) genes shortened in TgCPEB4Δ4 mice and lengthened in CPEB4 KO GT /+ and CPEB4 KO mice vs. total genome stratified by 5'UTR, 3'UTR, CDS, gDNA length and ratio neuronal/glial expression. d, Protein levels in St 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 neuronal and astrocytic specific genes in Cx of control and TgCPEB4Δ4 mice (n = 7). For gel source data, see Supplementary Figure 1. a, Hypergeometric test. b, One-sided Fisher's exact test, P-values of genes with shortened poly(A) in each group respect to ASD genes. c, Statistical details in simulations in method section. d-f, Two-sided unpaired t-test.

Extended Data
a-d, TgCPEB4Δ4 mice with transgene expression starting at the age of 3 weeks (OFF/ON-TgCPEB4Δ4 mice) do not display ASD-like behavioral phenotypes. a, β-GAL nuclear staining in forebrain neurons and CPEB4 immunohistochemistry in 3 month-old control and TgCPEB4Δ4 mice (n = 3). Cx, cortex; St, striatum; Hipp, hippocampus. b, Evolution of body weight (grams) of males (n = 29 controls, n = 11 OFF/ON-TgCPEB4Δ4) and females (n = 29 control, n = 10 OFF/ON-TgCPEB4Δ4). No premature death nor 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 center in OF test. d, Time interacting with either an empty cage, an unfamiliar mouse or without any interaction during 10 min. Control (n = 12) and OFF/ON-TgCPEB4Δ4 mice (n = 5). e-h, Silencing transgene expression in TgCPEB4Δ4 mice which have expressed the transgene during embryonic development does not revert ASD-like behaviors (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. f, Evolution of body weight (grams): males (n = 19 controls and n = 10 ON/OFF-TgCPEB4Δ4), females (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 center in OF test. h, Time interacting with either an empty cage, an unfamiliar mouse or without any interaction during 10 min. 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. 95% CIs. n.s nonsignificative, *P < 0.05, **P < 0.01, ***P < 0.001.