Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei

Abstract

To study the mechanisms of RNA splicing we have synthesized beta-globin mRNA precursors by in vitro transcription using a plasmid in which a human beta-globin gene is fused to an efficient bacteriophage promoter. The structural requirements for accurate splicing of the in vitro synthesized pre-mRNAs were investigated by injection into Xenopus oocyte nuclei. We detect splicing only if the pre-mRNA is capped in vitro prior to injection; uncapped RNA is rapidly degraded. In addition, we find that in vitro synthesized pre-mRNAs that are not polyadenylated or lack a normal 3' end are spliced following injection into oocytes. The observation that a purified pre-mRNA can be spliced in oocytes indicates that transcription and splicing are not obligatorily coupled. When an in vitro synthesized beta-globin pre-mRNA containing a splice junction mutation is microinjected, the affected junction is not spliced, indicating that sequences necessary for accurate splicing in human cells are also necessary for splicing in oocytes.