PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase Ti treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branchpoint-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattem of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.

BiochemistryAffinity purification of spliceosomes reveals that the precursor RNA processing protein PRP8, a protein in the U5 small nuclear ribonucleoprotein particle, is a component of yeast spliceosomes ABSTRACTNuclear pre-mRNA splicing in Saccharomyces cerevisiae, as in higher eukaryotes, occurs in large RNAprotein complexes called spliceosomes. The small nuclear RNA components, U1, U2, U4, U5, and U6, have been extensively studied; however, very little is known about the protein components of yeast spliceosomes. Here we use antibodies against the precursor RNA processing protein PRP8, a protein component of the U5 small nuclear ribonucleoprotein particle, to detect its association with spliceosomes throughout the splicing reaction and in a post-splicing complex containing the excised intron. In addition, an indirect immunological approach has been developed that conflrms the presence of precursor RNA processing protein PRP8 in isolated spliceosomes. This method has possible general application for the analysis of ribonucleoprotein particle complexes.

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