Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation.
The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532, USA.
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D000595:Amino Acid Sequence; D003080:Cold Temperature; D017124:Conserved Sequence; D039561:Eukaryotic Initiation Factor-4E; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020365:RNA Helicases; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism*; D013489:Suppression, Genetic; D013696:Temperature; D020798:Two-Hybrid System Techniques
D039561:Eukaryotic Initiation Factor-4E; D005656:Fungal Proteins; D009841:Oligonucleotides; C077018:PRP8 protein, S cerevisiae; D010448:Peptide Initiation Factors; C401305:Prp24 protein, S cerevisiae; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C104158:BRR2 protein, S cerevisiae; D020365:RNA Helicases