Despite intensive study in numerous laboratories, our understanding of the molecular mechanisms which specify the accurate and efficient splicing of intron-containing mRNA nuclear transcripts has progressed only slightly beyond the phenomenological. The same is true for other steps in the maturation of messages, including the generation of 3' termini and polyadenylation. Attention has recently been drawn to a group of six small nuclear RNAs (snRNAs), whose extraordinary structural conservation, together with a variety of circumstantial evidence, suggest their participation in functionally conserved steps in eukaryotic gene expression. For example, U1 RNA is hypothesized to mediate intron removal via complementarity to conserved sequences at the splice junction(s). Because of the lack of tractable experimental systems among the higher eukaryotes, these hypotheses have yet to be critically tested. We propose to exploit the powerful genetic techniques uniquely available in yeast to achieve these ends. We are employing two complementary strategies. The first exploits our recent demonstration of the existence in Saccharomyces cerevisiae of snRNAs encoded by single copy genes; this allows us to perform definitive tests of these models by analyzing the consequences of null and conditional alleles in each of these genes. Moreover, the isolation of extragenic suppressors of these mutations will identify cellular components which interact with the snRNAs; these should include the snRNP proteins, as well as potential """"""""target"""""""" RNAs: the pre-mRNA substrates. Our second approach takes advantage of our recent identification of a mutant 5' splice junction in a yeast mRNA intron. In this case, the ability to isolate extragenic suppressors should allow a direct test of the """"""""Ul guide model"""""""" for RNA splicing

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National Institute of General Medical Sciences (NIGMS)
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Genetics Study Section (GEN)
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University of California San Francisco
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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
Mayerle, Megan; Raghavan, Madhura; Ledoux, Sarah et al. (2017) Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency. Proc Natl Acad Sci U S A 114:4739-4744
Mayerle, Megan; Guthrie, Christine (2017) Genetics and biochemistry remain essential in the structural era of the spliceosome. Methods 125:3-9
Mayerle, Megan; Guthrie, Christine (2016) A new communication hub in the RNA world. Nat Struct Mol Biol 23:189-90
Mayerle, Megan; Guthrie, Christine (2016) Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing. RNA 22:793-809
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
Soucek, Sharon; Zeng, Yi; Bellur, Deepti L et al. (2016) The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA. Mol Cell Biol :
Lipp, Jesse J; Marvin, Michael C; Shokat, Kevan M et al. (2015) SR protein kinases promote splicing of nonconsensus introns. Nat Struct Mol Biol 22:611-7
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

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