Spliceosome malfunction causes neurodevelopmental disorders with overlapping features

Abstract

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Keywords: Development; Genetic diseases; Genetics; Neurodevelopment; iPS cells.

Figure 1. Clinical photographs of affected individuals with U2AF2 variants demonstrating a shared facial gestalt and molecular analyses demonstrating U2AF2 variants alter pre-mRNA splicing.

(A) Individual identifiers correlated with those in Supplemental Table 1. Shared craniofacial features include a prominent/broad forehead, a high anterior hairline, deep-set eyes, short palpebral fissures, downslanting palpebral fissures, a broad nasal root, a narrow nasal bridge with upturned nose, a thin upper lip, micrognathia, a wide mouth, wide-spaced teeth, and a short neck. (B) Two average faces generated from controls (top) and all 19 available photos of U2AF2 individuals (bottom) summarizing an identifiable facial gestalt. (C) An intolerance landscape plot generated by MetaDome for U2AF2 variant (NM_001012478.1) analysis (top panel) and a lollipop plot (middle panel) with a schematic outline of the U2AF2 protein domains (lower panel) showing 7 recurrent variants [p.(Arg149Trp), p.(Arg149Gln), p.(Arg150Cys), p.(Val153Met), p.(Arg150His), p.(Val186Met) and p.(Gly265Asp)] and other conserved variants identified in individuals 1–46 and in the DDD study [p.(Pro157Leu) and p.(Thr252Ile)]. (D) Eight U2AF2 variants reduced expression of the longer isoform in the minigene splicing assay, indicative of the pathogenicity of these variants. Normalized ratios are illustrated by the box-and-whisker plot at the bottom (minimum to maximum, showing all the points). n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, by ordinary 1-way ANOVA with Dunnett’s multiple comparisons test.

Figure 2. DrosophilaU2af50–knockdown, hPSCs, and rescue studies with 2 hyper-recurrent variants.

(A and B) Flies expressing CD8GFP in the setting of U2af50 knockdown with the elav-Gal4 driver died before or soon after eclosion. Expression of U2AF2^WT drastically increased the survival rate on the first day after eclosion (DAE) (A), and almost all the flies were still healthy on the seventh DAE (B), whereas variants only slightly increased the survival rate at the first DAE. n = 3 groups, with 17–51 flies per group for males and 21–77 flies per group for females. Data indicate the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA followed by Tukey’s test. (C–E) Pan-neuronal U2af50 knockdown led to a decrease in the MB area in larvae, whereas U2AF2^WT and variants partially (vertical lobe) or fully (horizontal lobe) rescued the phenotype. n = 14–17 brains. (C) Representative images of MBs. Scale bar: 50 μm. (D) Quantification of the vertical lobe area. (E) Quantification of the horizontal lobe area. Data indicate the mean ± SEM. *P < 0.05 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s test. (F) U2af50 knockdown by the D42-Gal4 driver resulted in increased heat-induced paralysis. Expressing U2AF2^WT and variants in U2af50 knockdown partially attenuated the phenotype. n = 6–13 groups, with 7–14 flies per group. Data indicate the mean ± SEM. *P < 0.05 and ***P < 0.001, by 2-way ANOVA followed by Tukey’s test. (G) Representative immunofluorescence images of induced neurons on day 2. Scale bars: 100 μm. (H) Quantification of the average neurite length per cell normalized by the Hoechst-labeled cell number. Graphs show the mean ± SD of 4 biological replicates. **P < 0.01 and ***P < 0.001, by ordinary 1-way ANOVA with Dunnett’s multiple-comparison test.

Figure 3. Modeling PRPF19 in in vitro cellular and in vivo fly models.

(A) An intolerance landscape plot generated by MetaDome for PRPF19 variant (NM_014502.5) analysis (top panel) and a schematic outline of the PRPF19 protein domains (lower panel). (B) Western blot and co-immunoprecipitation analysis of overexpressed PPRF19^WT, PRPF19^Gly404Ser, PPRF19^Leu499Phe, and PPRF19^{His273Thrfs*37}with FLAG tag. Graph shows the mean ± SEM. n = 3 independent experiments. ***P < 0.001, by 2-tailed, paired t test. (C–E) Pan-neural Prp19 knockdown led to reduced MB area. Expression of human PRPF19^WT, PRPF19^Gly404Ser, or PPRF19^Leu499Phe fully or partially rescued the area, while PPRF19^{His273Thrfs*37} failed to attenuate the structural defects. n = 6–11 brains. (C) Representative images. Scale bar: 50 μm. (D) Quantification of the vertical lobe area. (E) Quantification of the horizontal lobe area. Data indicate the mean ± SEM. *P < 0.05 and ***P < 0.001, by 1-way ANOVA (D and E). (F–H) In the social behavior assay, flies expressing the 2 PRPF19 missense variants in the setting of Prp19 knockdown displayed disrupted social behavior as revealed by their aberrant distribution in the device and reduced interdistance. In comparison, expression of PRPF19^WT in the setting of Prp19 knockdown partially restored the impaired social behavior. n = 3 groups, with 26–42 flies per group. (F) Representative images showing fly distribution in the social behavior assay. (G) Quantification of the distance between any of the 2 flies in 1 trial. *P < 0.05 and ***P < 0.001, by 2-tailed, unpaired t test. (H) Cumulative frequency of fly interdistance in the device.*P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA followed by Šidák’s test. .

Figure 4. RT-qPCR confirms potential substrates or downstream effectors that robustly rescue MB defects in U2af50-knockdown brains.

(A and B) RT-qPCR data confirmed 4 exon candidates from fly brain RNA-Seq and show that Iswi, Rbfox1, and RpS19a might be the shared downstream splicing targets of U2af50 and Prp19. n = 3. (A) Compared with WT, increased exon inclusions in Iswi, RpS19a, and Rbfox1 were significantly upregulated in U2af50-knockdown brains. *P < 0.05 and ***P < 0.001, by 2-tailed, unpaired t test. (B) The expression level of the same differentially used exons in Iswi, RpS19a, and Rbfox1 was also upregulated in Prp19-knockdown brains. Data indicate the mean ± SEM. *P < 0.05 and ***P < 0.001, by 2-tailed, unpaired t test. (C–E) The expression of Rbfox1, Iswi, or RpS19a fully or partially rescues the decreased MB area in U2af50-knockdown brains. n = 8–16. (C) Representative images. Scale bar: 50 μm. (D) Quantification of the vertical lobe area. ***P < 0.001, by 1-way ANOVA followed by Dunnett’s multiple-comparison test. (E) Quantification of the horizontal lobe area. Data indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Dunnett’s multiple-comparison test.

Figure 5. The identified RBFOX1 missense variants alter the gene-splicing pattern.

(A) Intolerance landscape plot generated by MetaDome for RBFOX1 (NM_018723.4) variant analysis (top panel) and a lollipop plot (middle panel) with a schematic outline of the RBFOX1 protein domains (lower panel) showing 1 recurrent variant, p.(Arg118Gln), and other conserved variants identified in individuals 5 and 6. (B) Schematic representation of the murine TrkB gene (top) and the TrkB-BAC minigene (bottom). The exons in black are commonly expressed in both the full-length (TrkB.FL) and the truncated T1 (TrkB.T1) isoforms. The TrkB.T1 isoform was generated by including the specific T1 exon (green), whereas the TrkB.FL isoform was generated by including exons in orange. The 164 kb TrkB-BAC minigene includes upstream, the transmembrane coding exon and, downstream, in addition to the TrkB.T1 coding exon, the 2 exons encoding the juxtamembrane region preceding the tyrosine kinase region. The cDNAs coding for the missing extracellular domain and the tyrosine kinase region of TrkB were fused inframe to an upstream exon (98 bp) and a downstream exon (131 bp) of the BAC region (dashed lines). A neomycin resistance cassette (NEO) is present in the minigene for selection, while a synthetic CAG promoter drives the minigene expression. (C) Representative immunoblot analysis of the clonal cell line expressing the TrkB-BAC minigene transiently transfected with plasmid vectors expressing the mouse Rbfox1 and the human RBFOX1 (as positive controls), the F158A mutant (as a negative control), and the de novo RBFOX1 variants. Ntrk2 (TrkB) protein levels were tested 48 hours after transfection with an antibody against the TrkB intracellular domain to specifically detect the full-length protein (TrkB.FL). (D) Immunoblot quantification analysis of TrkB.FL protein levels from 3 independent experiments, as in C. n = 3. Data indicate the mean ± SEM. ***P < 0.001, by ordinary 1-way ANOVA with Dunnett’s multiple-comparison test.