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Nucleic Acids Research, 1995, Vol. 23, No. 3 320-326
© 1995

RNA

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing

Stefan Teigelkamp+, Erica Whittaker§ and Jean D. Beggs*

Institute of Cell and Molecular Biology, University of Edinburgh King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK

*To whom correspondence should be addressed

Received December 5, 1994. Accepted December 20, 1994.

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.

+Present address: Institute für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany

§Present address: PPL Therapeutics, Roslin, Edinburgh EH25 9PP. UK


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