Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.
Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.
Teigelkamp
S
S
Institute of Cell and Molecular Biology, University of Edinburgh, Cambridge, UK.
Newman
A J
AJ
Beggs
J D
JD
D001483:Base Sequence; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D005656:Fungal Proteins / Q000378:metabolism* / Q000528:radiation effects; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000378:metabolism
D003432:Cross-Linking Reagents; D017931:DNA Primers; D005656:Fungal Proteins; C077018:PRP8 protein, S cerevisiae; D012322:RNA Precursors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins