Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 °C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Δ 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 (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) 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. Although 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 results 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 prp17Δ and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.
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Research Article|
November 26 2008
The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis
Aparna K. Sapra;
Aparna K. Sapra
1
1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
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Piyush Khandelia;
Piyush Khandelia
1
1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
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Usha Vijayraghavan
Usha Vijayraghavan
2
1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India
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Publisher: Portland Press Ltd
Received:
June 11 2008
Revision Received:
July 28 2008
Accepted:
August 11 2008
Accepted Manuscript online:
August 11 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 416 (3): 365–374.
Article history
Received:
June 11 2008
Revision Received:
July 28 2008
Accepted:
August 11 2008
Accepted Manuscript online:
August 11 2008
Citation
Aparna K. Sapra, Piyush Khandelia, Usha Vijayraghavan; The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis. Biochem J 15 December 2008; 416 (3): 365–374. doi: https://doi.org/10.1042/BJ20081195
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