Our long-term objectives are to exploit the powerful techniques available in the yeast Saccharymocyes cerevisiae for an understanding of messenger RNA splicing at the molecular level. Our future work relies on our recent demonstration of a 1:1 correspondence between five small nuclear RNAs (snRNAs) in this yeast and mammalian structural organization. One focus of your program -- motivated by the discovery of group II self-splicing RNAs - - will be to seek catalytic roles for these snRNAs. We will identify those residues that are the best candidates for such functions using a combined genetic, phylogenetic, and biochemical approach. First, structural domains inferred from phylogenetic comparisons will be deleted and assayed by complementation of deletion strains. When an essential domain is identified, its function will be further assessed by inter-species """"""""swaps""""""""; this provides a rapid and rational method of bulk mutagenesis. Finally, evolutionarily invariant nucleotides in these chimeras will by subjected to site-specific mutagenesis, to identify change which lead to lethal or, ideally, conditionally lethal phenotypes. To determine the specific functional lesion, we will assay the pattern of splicing intermediates in vivo, and the distribution of spliceosomal complexes within the ordered assembly pathway in vitro. The complementary focus of this project is to understand what roles the spliceosomal proteins play, many of which are known to be essential for viability. We will explore the hypothesis that at least certain of these proteins participate in proofreading functions, consistent with the large number of steps in the spliceosome assembly pathway that require ATP. In particular, we have cloned and sequenced a nucleotide; rna16-1 has a consensus ATP binding site and other features of a recently reported superfamily, members of which include EIF4alpha and a protein required for mitochondrial mRNA splicing. We propose genetic and biochemical tests of the model that RNA16 functions in branchpoint recognition to effect an ATP- dependent conformational switch; by this view, rna16-1 is a """"""""clock mutant"""""""" that acts as a suppressor by decreasing the time allowed for incorrect splicing substrates to dissociate. This model has important consequences for elucidating the molecular mechanisms used to maintain biologically tolerable error rates in complex macromolecular precesses.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Molecular Biology Study Section (MBY)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Francisco
Schools of Medicine
San Francisco
United States
Zip Code
de Bruyn Kops, Anne; Guthrie, Christine (2018) Identification of the Novel Nup188-brr7 Allele in a Screen for Cold-Sensitive mRNA Export Mutants in Saccharomyces cerevisiae. G3 (Bethesda) 8:2991-3003
Nissen, Kelly E; Homer, Christina M; Ryan, Colm J et al. (2017) The histone variant H2A.Z promotes splicing of weak introns. Genes Dev 31:688-701
Ledoux, Sarah; Guthrie, Christine (2016) Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity. J Biol Chem 291:11954-65
Patrick, Kristin L; Ryan, Colm J; Xu, Jiewei et al. (2015) Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast. PLoS Genet 11:e1005074
Blanco, Mario R; Martin, Joshua S; Kahlscheuer, Matthew L et al. (2015) Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics. Nat Methods 12:1077-84
Hadjivassiliou, Haralambos; Rosenberg, Oren S; Guthrie, Christine (2014) The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing. RNA 20:656-69
Ryan, Colm J; Roguev, Assen; Patrick, Kristin et al. (2012) Hierarchical modularity and the evolution of genetic interactomes across species. Mol Cell 46:691-704
Plocik, Alex M; Guthrie, Christine (2012) Diverse forms of RPS9 splicing are part of an evolving autoregulatory circuit. PLoS Genet 8:e1002620
Bergkessel, Megan; Whitworth, Gregg B; Guthrie, Christine (2011) Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast. RNA 17:1461-78
Mitrovich, Quinn M; Tuch, Brian B; De La Vega, Francisco M et al. (2010) Evolution of yeast noncoding RNAs reveals an alternative mechanism for widespread intron loss. Science 330:838-41

Showing the most recent 10 out of 85 publications