Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snPNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.
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Univ Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USAUniv Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USA
Price, Argenta M.
Goernemann, Janina
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Univ Wisconsin, Sch Med & Publ Hlth, Dept Biomol Chem, Madison, WI 53706 USA
Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, GermanyUniv Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USA
Goernemann, Janina
Guthrie, Christine
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Univ Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USAUniv Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USA
Guthrie, Christine
Brow, David A.
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Univ Wisconsin, Sch Med & Publ Hlth, Dept Biomol Chem, Madison, WI 53706 USAUniv Calif San Francisco, Dept Biochem & Biophys, San Francisco, CA 94143 USA
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Univ Alberta, Dept Biochem, Edmonton, AB T6G 2H7, CanadaUniv Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
Garside, E. L.
Whelan, T. A.
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Univ British Columbia, Biodivers Res Ctr, Vancouver, BC V6T 1Z4, Canada
Univ British Columbia, Dept Bot, Vancouver, BC V6T 1Z4, CanadaUniv Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
Whelan, T. A.
Stark, M. R.
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Univ Northern British Columbia, Dept Chem, Prince George, BC V2N 4Z9, CanadaUniv Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
Stark, M. R.
Rader, S. D.
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Univ Northern British Columbia, Dept Chem, Prince George, BC V2N 4Z9, CanadaUniv Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
Rader, S. D.
Fast, N. M.
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Univ British Columbia, Biodivers Res Ctr, Vancouver, BC V6T 1Z4, Canada
Univ British Columbia, Dept Bot, Vancouver, BC V6T 1Z4, CanadaUniv Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
Fast, N. M.
MacMillan, A. M.
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Univ Alberta, Dept Biochem, Edmonton, AB T6G 2H7, CanadaUniv Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
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State Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, BrazilState Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, Brazil
Garcia Garces, Hans
Hamae Yamauchi, Danielle
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State Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, BrazilState Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, Brazil
Hamae Yamauchi, Danielle
Theodoro, Raquel Cordeiro
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Univ Fed Rio Grande do Norte, Trop Med Inst Rio Grande Norte, Natal, RN, BrazilState Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, Brazil
Theodoro, Raquel Cordeiro
Bagagli, Eduardo
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State Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, BrazilState Univ Sao Paulo, Biosciences Inst Botucatu,Immunol Dept,MicroBiol, UNESP, Botucatu, SP, Brazil