Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center.
A U5 snRNP protein, hPrp8, interacts closely with the GU dinucleotide at the 5' splice site (5'SS), forming a specific UV-inducible cross-link. To test if this physical contact between the 5'SS and the carboxy-terminal region of Prp8 reflects a functional recognition of the 5'SS during spliceosome assembly, we mutagenized the corresponding region of yeast Prp8 and screened the resulting mutants for suppression of 5'SS mutations in vivo. All of the isolated prp8 alleles not only suppress 5'SS but also 3'SS mutations, affecting the second catalytic step. Suppression of the 5'SS mutations by prp8 alleles was also tested in the presence of U1-7U snRNA, a predicted suppressor of the U+2A mutation. As expected, U1-7U efficiently suppresses prespliceosome formation, and the first, but not the second, step of U+2A pre-mRNA splicing. Independently, Prp8 functionally interacts with both splice sites at the later stage of splicing, affecting the efficiency of the second catalytic step. The striking proximity of two of the prp8 suppressor mutations to the site of the 5'SS:hPrp8 cross-link suggests that some protein:5'SS contacts made before the first step may be subsequently extended to accommodate the 3'SS for the second catalytic step. Together, these results strongly implicate Prp8 in specific interactions at the catalytic center of the spliceosome.
Siatecka
M
M
The Rockefeller University, New York, New York 10021, USA.
Reyes
J L
JL
Konarska
M M
MM
D000483:Alleles; D000595:Amino Acid Sequence; D002352:Carrier Proteins; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D016153:Genes, Suppressor / Q000235:genetics; D015698:Genomic Library; D007438:Introns / Q000235:genetics; D008668:Metallothionein / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; C077018:PRP8 protein, S cerevisiae; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C007121:copper thionein; D008668:Metallothionein