In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.

Early region 2 of the adenovirus 2 genome (map position 61-75) specifies two poly(A)+ nuclear RNAs (28S and 23S) that appear to be precursors of the 20S cytoplasmic mRNA [Goldenberg, C. J. & Raskas, H. J. (1979) Cell 16, 131-138]. Splicing of these nuclear RNAs in vitro has been obtained with a whole cell extract prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The splicing reaction was demonstrated by two procedures: (i) hybridization of pulse-labeled RNA fractionated by size and (ii) annealing of RNAs with radioactive DNA probes followed by nuclease digestion. The first procedure provided evidence that sequences from the large 2300-nucleotide intron (74.6-68.8) were excised and 5' transcripts were spliced to the mRNA body. Utilizing both S1 and Exo VII nucleases, the second procedure demonstrated excision of sequences from the smaller 720-nucleotide intron (68.5-66.3), the splicing of sequences from the second leader (68.8) to the mRNA body, and the formation of an mRNA body of 1700 nucleotides, the size found in vivo. These findings provide evidence that an in vitro system that splices viral RNAs to yield products comparable to those found in vivo is now available.