Browsing by Subject "Yeasts"
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Item Functional Prions in Mammalian Innate Immune Signaling(2014-07-07) Cai, Xin; Zinn, Andrew R.; Beutler, Bruce; Chen, Zhijian J.; Goldstein, Joseph L.Pathogens and cellular danger signals activate mammalian cytosolic sensors such as RIG-I and NLRP3 which signal through respective adaptor proteins MAVS and ASC to produce robust innate immune and inflammatory responses. MAVS and ASC harbor N-terminal CARD and PYRIN domains, respectively, essential for their signaling ability. Using the Sup35 based yeast prion assay, we show that CARD and PYRIN function as bona fide prions in yeast when fused to Sup35C. In response to respective upstream sensors RIG-I and NLRP3, both CARD and PYRIN form self-perpetuating, SDS-resistant polymers that are inherited cytoplasmically through multiple cell divisions. Similar to other cases of prion switch, CARD exhibits nucleation- and polymerization-dependent prion conversion in yeast. Likewise, a yeast prion domain (NM) can functionally replace CARD and PYRIN in mammalian innate immune and inflammasome signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, homologous domains from a conserved NOD- like receptor (NWD2) and classic prion (HET-s) in fungi can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion- like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.Item In Vivo Studies of Yeast Mitochondrial Intron Splicing : Ectopic Branching and a Screen for Nuclear Encoded Splicing Factors(2006-08-11) Nyberg, Tarah Michelle; Perlman, Philip S.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.