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Phenotypic Effects of Mutations in the Jab1/MPN Domain of the Essential Pre-mRNA Splicing Factor Prp8 Amy Rines Northwestern University 2009 A POTENTIAL LINK BETWEEN SPLICING AND UBIQUITIN •Prp8 is an essential splicing factor of the spliceosome, which carries out RNA splicing to remove noncoding introns from mRNA. •Prp8 has few characteristic domains, but does contain a variant Jab1/MPN domain. •The Jab1/MPN domain is normally found within deubiquitinating enzymes that remove ubiquitin from proteins marked for destruction. •The non-canonical Jab1/MPN domain of Prp8 does not enable enzyme function, but is still essential for Prp8 expression and splicing efficiency. •The Jab1/MPN region has also has been shown to bind ubiquitin. •This criticality of Jab1/MPN and its ubiquitin-binding ability in Prp8 suggest that ubiquitin could regulate splicing through protein-protein interactions involving Prp8. •Characterizing this domain by localizing its critical amino acids is important for discovering the precise link between ubiquitin and splicing. RESULTS OF TEMPERATURE AND COPPER SENSITIVITY TESTS ON PRP8 MUTANT GROWTH Temperature-Sensitive Mutants at 37˚C WT PQ VL •Temperature-sensitive phenotypes revealed mutations with the strongest, most general growth defects. •PQ, between beta sheet 2 and 3, exhibited a near lethal temperature-sensitive phenotype at the restrictive temperature of 37˚C. •VL, in alpha helix 1, and FAD, in beta sheet 4, exhibited moderate temperaturesensitive phenotypes at the restrictive temperature. •None of the mutations had abnormal phenotypes at the permissive temperature of 30˚C or at the cold-sensitive temperature of 16˚C. FAD Copper-Sensitive Mutants at .5 mM Cu WT LLK F W SKL •Copper-sensitive phenotypes revealed mutations with more subtle growth defects that resulted from a specific strain on splicing. •LLK and F, both in alpha helix 1, W, in beta sheet 3, and SKL, in alpha helix 2, had copper-sensitive phenotypes at concentrations at or above .1 mM Cu. •The growth of each mutant decreased as the copper concentrations increased. MATERIALS AND METHODS •Missense mutations to alanine were introduced via site-directed mutagenesis. •The mutated Prp8 plasmids were introduced into S. cerevisiae. •Inoculated colonies were spotted onto plates at various dilutions and grown at 16˚C, 30˚C, and 37˚C (restrictive). •The mutated plasmids were also transformed into a parental yeast strain lacking the gene CUP1, which encodes copper resistance. •This strain was co-transformed with a plasmid containing an artificially introduced CUP1 gene fused to an actin promoter with an intervening intron. Precise splicing of the intron was required for the CUP1 metallothionein to be produced and for copper insensitivity to be conferred. Intron ACT Exon CUP1 Exon •Phenotypic growth defects were noted at copper concentrations from .05 mM to 1 mM Cu. Mutations in the Jab1/MPN Domain of Prp8 EEQNVYVLPKNLLKKFIEISDVKIQVAAFIYGMSAKDHPKVKEIKTVVLVPQLGHVGSVQISNIPDI CONCLUSIONS •The abnormal growth phenotypes are evidence that ubiquitin may regulate splicing through binding at the mutant residues. •These amino acid clusters are likely particular important to the domain’s function, and future research will test their protein expression and ubiquitin-binding to elucidate where ubiquitin interacts with Prp8. GDLPDTEGLELLGWIHTQTEELKFMAASEVATHSKLFADKKRDCIDISIFSTPGSVSLSAYNLTDE ACKNOWLEDGMENTS Red amino acids yielded temperature-sensitive phenotypes at 37˚C when mutated to alanine Green amino acids yielded copper-sensitive phenotypes above .1 mM Cu when mutated to alanine Blue amino acids yielded no phenotype when mutated to alanine Black amino acids have not been mutated Thank you to Priya Bellare and Erik Sontheimer of Northwestern University for their advice and guidance. REFERENCES 1. Maytal-Kivity, V., Reis, N., Hofmann, K., and Glickman, M.H. 2002. MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. BMC Biochem. 3: 28. 2. Bellare, P., Kutach, A.K., Rines, A.K., Guthrie, C., and Sontheimer, E.J. 2006. Ubiquitin binding by a variant Jab1/MPN in the essential pre-mRNA splicing factor Prp8p. RNA. 12:292-302. 3. Hicke, L., Schubert, H.L., and Hill, C.P. 2005. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6: 610–621.