Parameters that affect in vitro splicing of bovine papillomavirus type 1 late pre-mRNAs

During the course of study on regulated viral pre-mRNA splicing, an in vitr o RNA splicing assay was developed to analyze how an exonic splicing enhanc er stimulates splicing of bovine papillomavirus type 1 (BPV-1) late pre-mRN As. The optimal concentration of HeLa nuclear extract (HNE) in a standard R NA splicing reaction depends on the individual substrate pre-mRNA. Splicing of a BPV-1 late pre-mRNA required 40% HNE and 2 h incubation at 30 degrees C. Higher HNE was detrimental to splicing and longer incubation times lead to RNA degradation. In the reaction containing 40% EDTA-treated HNE, 1.5-3 .0 mM Mg2+ or 3 mM Mn2+, but not Co2+, were required to catalyze an efficie nt splicing reaction. Surprisingly, EDTA-untreated HNE in the absence of ex ogenous Mg2+ catalyzed very efficiently splicing of the RNA. Addition of Mg 2+ from 0.1 to 0.5 mM, only enhanced slightly the splicing in EDTA-untreate d HNE and excessive Mg2+ concentration (above 1.5 mM) in the reaction resul ted in production of aberrant splicing products or intermediates. In contra st, addition of Mn2+ to EDTA-untreated HNE severely suppressed splicing. In addition, it was observed that the RNA transcribed from vector sequences d ownstream of the polylinker region of pSP72 vector, when connected to the 3 ' terminus of chimeric Drosophila doublesex-BPV-1 SE1 pre-mRNAs, suppressed dramatically splicing. RNA transcribed from the pSP72 polylinker region, w hen supplied in trans, also suppressed splicing. These results suggest that a DNA template used to make RNA transcripts should avoid these sequences a s much as possible. Published by Elsevier Science B.V.