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,
• MeSH
• DNA Repair Enzymes genetics   MeSH
• Gene Regulatory Networks MeSH
• Nuclear Proteins genetics   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Neurodevelopmental Disorders * genetics   MeSH
• RNA Splicing MeSH
• RNA Splicing Factors genetics   MeSH
• Spliceosomes * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Repair Enzymes MeSH
• Nuclear Proteins MeSH
• PRPF19 protein, human MeSH Browser
• RNA Splicing Factors MeSH

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3' splice sites (3'ss). Both proteins preferentially bind uridine-rich sequences upstream of 3'ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3'ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3'ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3'ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

• Keywords
• 3′ splice site, Functional genomics, PUF60, SF3B4, U2AF2, cancer, differential scanning fluorimetry, driver mutation, exon inclusion, gel shift assay, lariat branch point, leukemia, mRNA, pre-mRNA splicing,
• Publication type
• Journal Article MeSH

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

• MeSH
• Amino Acid Motifs MeSH
• Exons * MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Heterogeneous-Nuclear Ribonucleoproteins metabolism   MeSH
• Nuclear Proteins metabolism   MeSH
• Short Interspersed Nucleotide Elements MeSH
• Humans MeSH
• Ribonucleoprotein, U1 Small Nuclear metabolism   MeSH
• Mutation, Missense * MeSH
• RNA Splice Sites * MeSH
• RNA-Binding Proteins metabolism   MeSH
• Repressor Proteins chemistry   deficiency   metabolism   MeSH
• Sequence Analysis, RNA MeSH
• RNA Splicing Factors chemistry   deficiency   metabolism   MeSH
• Splicing Factor U2AF MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• HCC1 autoantigen MeSH Browser
• Heterogeneous-Nuclear Ribonucleoproteins MeSH
• Nuclear Proteins MeSH
• Ribonucleoprotein, U1 Small Nuclear MeSH
• RNA Splice Sites * MeSH
• poly-U binding splicing factor 60KDa MeSH Browser
• RNA-Binding Proteins MeSH
• Repressor Proteins MeSH
• RNA Splicing Factors MeSH
• Splicing Factor U2AF MeSH
• SNRNP70 protein, human MeSH Browser