Abstract
Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing. No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts. Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP. Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs. Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions. Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly. These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs. Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs. Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Blotting, Northern
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Centrifugation, Density Gradient
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DNA Probes / chemistry
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Electrophoresis, Polyacrylamide Gel
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Fungal Proteins / genetics
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Fungal Proteins / metabolism*
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Mutation
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Nucleic Acid Conformation
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Precipitin Tests
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RNA Precursors / genetics
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RNA Precursors / metabolism
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RNA Splicing*
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
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Ribonucleoprotein, U4-U6 Small Nuclear / genetics
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Ribonucleoprotein, U4-U6 Small Nuclear / metabolism*
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Ribonucleoproteins, Small Nuclear / chemistry
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Ribonucleoproteins, Small Nuclear / genetics
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Ribonucleoproteins, Small Nuclear / metabolism*
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Saccharomyces cerevisiae / chemistry*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae Proteins*
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Sodium Chloride / pharmacology
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Spliceosomes / metabolism
Substances
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(U4-U6.U5)tri-snRNP-specific protein, S cerevisiae
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DNA Probes
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Fungal Proteins
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Nuclear Proteins
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PRP3 protein, S cerevisiae
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RNA Precursors
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RNA, Messenger
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Ribonucleoprotein, U4-U6 Small Nuclear
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Ribonucleoproteins, Small Nuclear
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Saccharomyces cerevisiae Proteins
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Sodium Chloride