In vitro splicing of SV4O late mRNA in isolated nuclei from CV-1 cells

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Abstract
An in vitro splicing system utilizing isolated nuclei of SV4O infected cells has been developed. Nuclei were isolated from CV-1 cells at a late stage of SV4O infection after a pulse-labeling with 3 H-uridine. In nuclei prepared under mild isotonic conditions, 19S viral coded RNA synthesized in vivo was converted in vitro into 16S mRNA. In contrast, the nuclei prepared with RSB, a hypotonic medium, showed a very low splicing activity only. Addition of a “nuclear extract” to these nuclei restored the activity almost to the original level. These results indicate that 1) l9S RNA is indeed a precursor to 16S mRNA 2) the splicing of 19S RNA into 16S RNA takes place in the nucleus, and 3) at least a part of the enzyme system required for splicing could be extracted from the nucleus. This in vitro system may be useful for the assay of the splicing enzyme(s).