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Amanda Solem Graduate
Student amanda.solem@yale.edu 
Group II intron protein-facilitated splicing: Mss116 and ai5? Group II introns are large catalytic RNAs that self-splice independently in vitro at artificially high metal ion concentrations; however, in vivo most group II introns require protein cofactors (1). These protein cofactors may either be intron-encoded maturases, which usually function specifically with certain introns, or host-encoded proteins (2). Mss116 is a yeast nuclear-encoded DEAD-box protein targeted to the mitochondria which affects in vivo splicing of multiple introns, especially ai5? (3; Perlman, personal communication). ai5 is a mitochondrial group II intron from S. cerevisiae which has been studied extensively in vitro in the absence of protein. Neither interactions between Mss116 and group II introns nor the mechanism by which Mss116 facilitates efficient splicing of these RNAs are well understood. I am interested in determining whether Mss116 facilitates ai5? splicing and folding in vitro, investigating RNA-protein interactions, and determining whether Mss116 is a functional DEAD-box helicase. I will use kinetic splicing assays and footprinting techniques to determine whether Mss116 facilitates ai5? splicing and folding in vitro. To investigate Mss116/ ai5? interactions, I will determine binding parameters such as the Kd through nitrocellulose filter binding or gel shift experiments. Finally, I will perform ATPase assays and characterize substrate specificity and direction of helicase activity through helicase activity assays to determine whether Mss116 is a functional DEAD-box helicase. These experiments represent a step towards understanding the mechanism by which Mss116 facilitates RNA splicing. 1. Michel F. and Ferat J. (1995) Annu. Rev. Biochem. 94, 435-461. 2. Lambowitz A.M., Caprara M.G., Zimmerly S. and Perlman P.S. (1999) The RNA World (second edition) Chapter 18, 451-485, Cold Spring Harbor Laboratory Press. 3. Seraphin B. (1989) Nature 337, 84-87. |
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