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Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae
[Thesis]. Manchester, UK: The University of Manchester; 2018.
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Abstract
Removal, or splicing, of intervening sequences from the pre-messenger RiboNucleic Acid (pre-mRNA) to form mature mRNA in eukaryotes is carried out by the spliceosome. During the stepwise assembly of the spliceosome, the catalytic core of the spliceosome is created as the B complex is activated. Proteins of U5 small nuclear RiboNucleo Protein particles (snRNPs), Prp8, Brr2, Snu114 and Dib1, are core splicing factors which play crucial roles in activation of the B complex. As Prp8 releases its inhibition on the helicase Brr2, the U4/U6 duplex unwinds and completes the formation of the catalytic core of the spliceosome. GTP has to bind Snu114, a GTPase, to support U4/U6 duplex unwinding while Dib1 leaves B complex during the activation of B complex. Compound heterozygosity of core splicing factor DIB1 (TXLN4A) and haploinsufficiency of core splicing factor SNU114 (EFTUD2) cause the craniofacial disorders Burn McKeown Syndrome (BMKS) and Mandibulofacial Dysostosis, Guion-Almeida type (MFDGA) respectively. Patients with BMKS and MFDGA share similar disease phenotypes. As BMKS and MFDGA have overlapping phenotypes and Snu114, in its GTP state, activates the B complex while Dib1 has to leave to allow B complex activation, we hypothesize that Snu114 and Dib1 are functionally linked. A synthetic genetic screen was performed to identify if genetic interactions exist between Snu114 and Dib1. For the synthetic genetic screen, a haploid yeast strain, having SNU114-DIB1 double knock out and the complementing wild type genes on a URA3 plasmid, was successfully constructed. The constructed strain was then transformed with different combinations of snu114 and dib1 mutants. Two snu114 mutations, V266P and K146I, were in the GTP domain while P860K was found in the IV domain of Snu114. Mutants of dib1 were dib1 (K34A+D36R) mutant 1, dib1 (E72A+E75A) mutant 3 and dib1 (E66A+D69A) mutant 4. Using a plasmid shuffle assay, where the URA3 plasmid with the complementing wild type genes was counter-selected with 5-Fluoroorotic acid, a positive genetic interaction between the dib1 (K34A+D36R) mutant 1 and snu114 mutant V266P was observed. A putative positive genetic interaction suggests that Snu114, in its GTP state, acts antagonistically to Dib1 in the activation of complex B and suggests that Snu114 is responsible for the removal of Dib1 from the B complex to form the activated B complex. Although further investigation is needed to explain how Snu114, in its GTP state, could remove Dib1, the positive interaction between Snu114 and Dib1 provides evidence on why BMKS and MFDGA share similar phenotypes.