Sapra AK, et al. (2008) The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis. Biochem J 416(3):365-74
Abstract: Saccharomyces cerevisiae PRP17 null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first splicing reaction and are arrested for the second reaction at temperatures greater than 34C. Here we show these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16-helicase. These data suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs show Prp17 interacts with U2, U5 and U6 snRNPs but it is not a core component of any one snRNP. Prp17 association with in vitro assembled spliceosome complexes, on actin pre-mRNAs, was also investigated. While the U5 snRNP proteins- Prp8 and Snu114, are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step. However, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus, Prp17 joins the spliceosome prior to both catalytic reactions. Our data indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first-step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex (NTC), functional elements in U2 and U5 snRNAs and other second step splicing factors.
|Status: Published||Type: Journal Article||PubMed ID: 18691155|
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