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 vitro RNA splicing assay was developed to analyze how an exonic splicing enhancer stimulates splicing of bovine papillomavirus type 1 (BPV-1) late pre-mRNAs. The optimal concentration of HeLa nuclear extract (HNE) in a standard RNA 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 efficient splicing reaction. Surprisingly, EDTA-untreated HNE in the absence of exogenous Mg2+ catalyzed very efficiently splicing of the RNA. Addition of Mg2+ from 0.1 to 0.5 mM, only enhanced slightly the splicing in EDTA-untreated HNE and excessive Mg2+ concentration (above 1.5 mM) in the reaction resulted in production of aberrant splicing products or intermediates. In contrast, addition of Mn2+ to EDTA-untreated HNE severely suppressed splicing. In addition, it was observed that the RNA transcribed from vector sequences downstream 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, when supplied in trans, also suppressed splicing. These results suggest that a DNA template used to make RNA transcripts should avoid these sequences as much as possible.
Zheng
Z M
ZM
Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute/National Institutes of Health, Bethesda, MD 20892, USA. zhengt@dce41.nci.nih.gov
Baker
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D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002457:Cell Extracts / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics; D004330:Drosophila; D029721:Drosophila Proteins; D004492:Edetic Acid / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D019476:Insect Proteins / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; D019476:Insect Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D003035:Cobalt; D008345:Manganese; D004492:Edetic Acid; D008274:Magnesium