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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM021119-24
Application #
2331945
Study Section
Special Emphasis Panel (NSS)
Project Start
1977-02-01
Project End
2000-01-31
Budget Start
1997-02-01
Budget End
1998-01-31
Support Year
24
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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
Abelson, John; Blanco, Mario; Ditzler, Mark A et al. (2010) Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nat Struct Mol Biol 17:504-12

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