In vitroprocessing of a plant pre-mRNA in a HeLa cell nuclear extract

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Abstract
In order to determine whether there is a general difference in the splicing mechanism of animal and plant pre-mRNAs, we cloned part of the gene for the small subunit of the ribulose 1,5-bisphosphate carboxylase containing both introns into the SP64 vector. RNA was synthesized with SP6 polymerase and used as substrate for in vitro processing in a HeLa cell nuclear splicing extract. Analyses of the processed RNA demonstrate that both introns of the plant pre-mRNA are efficiently removed in an ordered fashion yielding a faithfully ligated mRNA. Two branch points were identified for intron A and three for intron B. The branched nucleotides are adenosine residues in all cases and are located within a distance from the 3' splice site found to be crucial for lariat formation in animal pre-mRNAs. The implications of these results are discussed in light of our previous observation, that a functional pre-mRNA of the human growth hormone gene was not processed in plant tissue in vivo.