Inhibition of in vitro splicing of a mouse insulin pre-mRNA by covalent cross-links in the intron region.
Recent studies have indicated that in vitro splicing of a mouse insulin pre-mRNA by a HeLa cell nuclear extract is accompanied by the unwinding of substrate RNA. The present experiments were performed to determine whether this melting of the secondary structure of the precursor RNA is essential for the splicing reaction. 32P-labelled mouse insulin pre-mRNA synthesized in vitro in a SP6 transcription system was cross-linked with aminomethyltrimethyl psoralen and fractionated by polyacrylamide gel electrophoresis. RNA species containing different intramolecular cross-links were eluted from the gel and the sites of cross-links were mapped by primer extension analysis using synthetic oligonucleotide primers. Under conditions that allow accurate in vitro splicing of intact pre-mRNA, precursor molecules with psoralen cross-links within their intron region were not spliced by a HeLa cell nuclear extract. This observation strongly supports the assumption that unwinding of precursor RNA molecules is necessary for the splicing reaction.
Szeberényi
J
J
Department of Biology, University Medical School of Pécs, Hungary.
Wollenzien
P L
PL
Goldenberg
C J
CJ
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D007328:Insulin / Q000235:genetics*; D007438:Introns / Q000187:drug effects; D051379:Mice; D009693:Nucleic Acid Hybridization; D012322:RNA Precursors / Q000502:physiology*; D012326:RNA Splicing / Q000187:drug effects; D014158:Transcription, Genetic; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology
D003432:Cross-Linking Reagents; D007328:Insulin; D012322:RNA Precursors; C018822:aminomethyltrioxsalen; D014307:Trioxsalen