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Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome

Tandiono, Moses

[Thesis]. Manchester, UK: The University of Manchester; 2016.

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Abstract

Proper formation of the cranial and facial features in vertebrates is a crucial event that requires the tight regulation of differentiation and proliferation of cranial neural crest cells (CNCC). Similar to Treacher Collins syndrome, both mandibulofacial dysostoses Guion-Almeida (MFDGA) and Burn McKeown Syndrome (BMKS) patients share some malformed craniofacial features. Genetic characterisation of MFDGA and BMKS has identified spontaneous heterozygous mutations and deletions in EFTUD2 (hSnu114) and in the promoter regions of TXNL4A (hDib1) combined with one null allele, respectively. Both genes encode for highly conserved U5 small nuclear ribonucleoproteins (snRNP), and the mutations results in reduced expression of hSnu114 and hDib1, both of which are essential components of the spliceosome required for pre-mRNA splicing. In this work, yeast models of MFDGA and BMKS were used in growth assays to identify growth sensitivities to ER stress factors, providing evidence that ER stress may affect CNCC survival, proliferation and differentiation during craniofacial development. The profiling of GAL1-regulated SNU114 and DIB1 strains displayed different proliferation rates when SNU114 and DIB1 expression was switched on or off, indicating that expression of both SNU114 and DIB1 needs to be tightly regulated to prevent mis-splicing of pre-mRNAs. The inhibition of SNU114 and DIB1 expression induced a markedly reduced and a slightly reduced tri-snRNP assembly in primer extension assay, respectively, revealing their functional link in tri-snRNP assembly and in pre-mRNA splicing. Additionally, an accumulation of larger U2 snRNA complexes were observed in both SNU114 and DIB1 depleted extracts. This study suggest a link in reduced tri-snRNP assembly to the mis-splicing of a subset of pre-mRNAs that are crucial not only for proliferation and differentiation, but for restoring ER homeostasis during NCC development. Finally, the elimination of antibiotic activity of a fusidic acid derivative through targeted chemical modifications indicates the generation of a splicing inhibitor that potentially targets Snu114p. It is suggested that the reduced expression of EFTUD2 and TXNL4A results in neural crest cell apoptosis due to aberrant mis-splicing of crucial genes involved in differentiation and proliferation during embryogenesis, leading to MFDGA and BMKS.