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1.
PLoS One ; 15(7): e0233582, 2020.
Article in English | MEDLINE | ID: mdl-32735620

ABSTRACT

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.


Subject(s)
Alternative Splicing , Choanal Atresia/pathology , Deafness/congenital , Gene Expression Regulation, Developmental , Heart Defects, Congenital/pathology , Induced Pluripotent Stem Cells/cytology , Models, Biological , Ribonucleoprotein, U5 Small Nuclear/deficiency , Spliceosomes/physiology , Apoptosis , Cell Differentiation , Cellular Reprogramming Techniques , Choanal Atresia/genetics , Clone Cells , Deafness/genetics , Deafness/pathology , Epithelial-Mesenchymal Transition , Exons/genetics , Face/embryology , Facies , Female , Head/embryology , Heart Defects, Congenital/genetics , Humans , Neural Crest/cytology , Promoter Regions, Genetic/genetics , RNA Splice Sites , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoprotein, U5 Small Nuclear/genetics , Sequence Deletion , Transcription Factor 7-Like 2 Protein/genetics , Wnt Signaling Pathway
2.
Hum Genomics ; 13(1): 63, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31806011

ABSTRACT

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.


Subject(s)
Cell Differentiation , Chondrocytes/pathology , Osteoblasts/pathology , Peptide Elongation Factors/deficiency , Peptide Elongation Factors/genetics , Ribonucleoprotein, U5 Small Nuclear/deficiency , Ribonucleoprotein, U5 Small Nuclear/genetics , Signal Transduction , Tumor Suppressor Protein p53/metabolism , Animals , Base Sequence , Bone and Bones/embryology , Bone and Bones/pathology , Cartilage/embryology , Cartilage/pathology , Cell Line , Child, Preschool , Chondrocytes/metabolism , Female , Gene Knockdown Techniques , Humans , Male , Mutation/genetics , Osteoblasts/metabolism , Pedigree , Peptide Elongation Factors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoprotein, U5 Small Nuclear/metabolism , Time Factors , Zebrafish
3.
PLoS One ; 8(4): e62125, 2013.
Article in English | MEDLINE | ID: mdl-23637979

ABSTRACT

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.


Subject(s)
Cell Cycle , RNA Helicases/chemistry , RNA Helicases/metabolism , Ribonucleoprotein, U5 Small Nuclear/chemistry , Ribonucleoprotein, U5 Small Nuclear/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Down-Regulation/drug effects , Ecdysone/pharmacology , HEK293 Cells , Humans , Molecular Weight , Mutation , RNA Helicases/deficiency , RNA Helicases/genetics , RNA Interference , RNA Splicing/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Ribonucleoprotein, U5 Small Nuclear/deficiency , Ribonucleoprotein, U5 Small Nuclear/genetics
4.
Nature ; 474(7350): 173-8, 2011 May 25.
Article in English | MEDLINE | ID: mdl-21614000

ABSTRACT

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.


Subject(s)
Alternative Splicing , Gene Expression Regulation, Fungal , Ligases/metabolism , RNA Splice Sites/genetics , RNA, Fungal/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Gene Deletion , Humans , Ligases/deficiency , Ligases/genetics , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/genetics , Protein Binding , Protein Conformation , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/deficiency , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoprotein, U5 Small Nuclear/deficiency , Ribonucleoprotein, U5 Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/deficiency , Ribonucleoproteins, Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Schizosaccharomyces/chemistry , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Spliceosomes/chemistry , Spliceosomes/metabolism , Ubiquitin-Protein Ligase Complexes/deficiency , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitins
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