Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells.

The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, branch point to 3' splice site (BPS-3'SS) distance and splice site strengths, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.

EFTUD2 gene deficiency disrupts osteoblast maturation and inhibits chondrocyte differentiation via activation of the p53 signaling pathway.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM) is characteristic of multiple skeletal anomalies comprising craniofacial anomalies/dysplasia, microcephaly, dysplastic ears, choanal atresia, and short stature. Heterozygous loss of function variants of EFTUD2 was previously reported in MFDM; however, the mechanism underlying EFTUD2-associated skeletal dysplasia remains unclear. RESULTS: We identified a novel frameshift variant of EFTUD2 (c.1030_1031delTG, p.Trp344fs*2) in an MFDM Chinese patient with craniofacial dysmorphism including ear canal structures and microcephaly, mild intellectual disability, and developmental delay. We generated a zebrafish model of eftud2 deficiency, and a consistent phenotype consisting of mandibular bone dysplasia and otolith loss was observed. We also showed that EFTUD2 deficiency significantly inhibited proliferation, differentiation, and maturation in human calvarial osteoblast (HCO) and human articular chondrocyte (HC-a) cells. RNA-Seq analysis uncovered activated TP53 signaling with increased phosphorylation of the TP53 protein and upregulation of five TP53 downstream target genes (FAS, STEAP3, CASP3, P21, and SESN1) both in HCO and in eftud2-/- zebrafish. Additionally, inhibition of p53 by morpholino significantly reduced the mortality of eftud2-/- larvae. CONCLUSIONS: Our results confirm a novel de novo variant of the EFTUD2 gene and suggest that EFTUD2 may participate in the maturation and differentiation of osteoblasts and chondrocytes, possibly via activation of the TP53 signaling pathway. Thus, mutations in this gene may lead to skeletal anomalies in vertebrates.

Cell cycle abnormalities associated with differential perturbations of the human U5 snRNP associated U5-200kD RNA helicase.

Splicing of pre-messenger RNAs into functional messages requires a concerted assembly of proteins and small RNAs that identify the splice junctions and facilitate cleavage of exon-intron boundaries and ligation of exons. One of the key steps in the splicing reaction is the recruitment of a tri-snRNP harboring the U5/U4/U6 snRNPs. The U5 snRNP is also required for both steps of splicing and exon-exon joining. One of the key components of the tri-snRNP is the U5 200kd helicase. The human U5-200kD gene isolated from Hela cells encodes a 200 kDa protein with putative RNA helicase function. Surprisingly, little is known about the functional role of this protein in humans. Therefore, we have investigated the role of the U5-200kD RNA helicase in mammalian cell culture. We created and expressed a dominant negative domain I mutant of the RNA helicase in HEK293 cells and used RNAi to downregulate expression of the endogenous protein. Transient and stable expression of the domain I mutant U5-200kD protein using an ecdysone-inducible system and transient expression of an anti-U5-200kD short hairpin RNA (shRNA) resulted in differential splicing and growth defects in the 293/EcR cells. Cell cycle analysis of the dominant negative clones revealed delayed exit from the G2/M phase of the cell cycle due to a mild splicing defect. In contrast to the domain I dominant negative mutant expressing cells, transient expression of an anti-U5-200kD shRNA resulted in a pronounced S phase arrest and a minute splicing defect. Collectively, this work demonstrates for the first time establishment of differential human cell culture splicing and cell cycle defect models due to perturbed levels of an essential core splicing factor.

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing.

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.