Efficient coupled transcription and mRNA splicing in vitro using plasmids derived from early region 3 of adenovirus 2 and a nondefective adenovirus-simian virus 40 hybrid.

Accurate and highly efficient (80%) splicing of a single mRNA precursor to processed products was achieved using HeLa cell extracts to synthesize and process RNA in vitro from recombinant plasmids containing specific DNA segments from adenovirus 2 (Ad2) and the nondefective adenovirus-simian virus 40 (Ad+2ND1) hybrid. One plasmid, pRID, contains a segment of Ad2 DNA spanning chromosome map coordinates 75.9-83.4. The other plasmid, pRW9, contains the analogous viral region from Ad+2ND1. RNA synthesis from pRID in vitro occurs for more than 60 min and is directed by RNA polymerase II. RNA products consistent in size with the expected precursor and the two processed mRNAs are made. RNA blot hybridization analyses showed that these products are complementary to the Ad2 insert in the plasmid and that the appropriate intervening sequence was absent from the smallest processed mRNA. Comparison of the splice patterns of RNA made in vitro to those of RNAs taken from infected cells using the nuclease S1 technique demonstrated the accuracy of intron removal.