In Vivo Studies of Yeast Mitochondrial Intron Splicing : Ectopic Branching and a Screen for Nuclear Encoded Splicing Factors

The splicing mechanism of group II introns is analogous to that of nuclear introns and it is generally thought that both share a common ancestor. This work contains two studies of group II intron splicing in yeast mitochondria. Previous studies done in collaboration with Dr. Anna Pyle at Yale identified several important determinants for in vitro branch-site selection of intron aI5gamma : the presence of a bulged A(A880), the 5' flanking GU base pair and the branch location within domain VI. I confirmed the in vitro findings in vivo and show that displacing the branch adenosine by one nucleotide in either direction can support branching at the shifted bulged A in vivo. Returning the base-pairs flanking the shifted branch-points to GU pairs increased both the efficiency and fidelity of branching at the ectopic branch A. However, for the shifted down ectopic branch A, it is not the presence of the GU pair flanking the branch that restores branching but the presence of a GC pair located two base-pairs above the branch. This finding is consistent with our observations that for the wild-type branch location, the branch environment above and below the branch are distinct. It appears that the short stem below the branch is important for the second splicing step. The goal of the second project was to identify novel nuclear genes that are involved in mitochondrial intron splicing. Based on the yeast genome project and several recent proteomic studies of yeast mitochondria, we identified 808 nuclear genes coding for potential mitochondrial proteins that can be deleted without lethality. Of these, 476 deletion strains retain a complete copy of the mtDNA (13 introns) and have a respiratory growth defect. Those strains were screened by northern blot analysis for intron splicing defects. I observed the expected splicing defects in strains deleted for MSS18, CBP2 and PET54. I observed a novel splicing pattern in strains deleted for IMP1, CBS2, PET111, MNE1, AAT1, ATP10 and PIF1.