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 T1 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 branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.