In vitro - synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts ( Salmonella phage 6 - early region 2 pre - RNA / mRNA splicing / MOPC - 315 nudear extract / RNA sequencing )

Precursor mRNAs were synthesized in vitro from a plasmid in which the early region 2 gene of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and used as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro splicing was accurate at the nucleotide level. The reaction occurs rapidly and without any detectable lag. The concentration of the pre-mRNA precursor during incubation appears to be an important factor for high efficiency (60%-80%) of in vitro RNA splicing. Fractionation of the splicing components as well as modifications of the DNA template to study the nucleotide-sequence requirement for in vitro splicing can now be accomplished with this system. The mRNA precursors of many eukaryotic genes contain introns, which are removed by splicing. Introns have also been found in genes encoding transfer RNAs and ribosomal RNAs. The splicing machinery is thought to be different for each class of RNA (1-4). Although several models for mRNA splicing have been proposed, the biochemical mechanisms of intron removal and the regulation of this process are not known (5, 6). The invariable consensus 5' and 3' splice junction sequences identified as the result of the nucleotide sequence analysis ofnumerous eukaryotic genes are so far the only recognized RNA signals for mRNA splicing (4, 7). Several reports indicate that large portions of the introns can be removed without affecting the accuracy of splicing (8-12). Mutations in the nucleotide sequence of the splice junction consensus sequences decreased the splicing efficiency or generated alternative splice sites (13-15). The mechanisms as well as the enzymology of mRNA splicing should be addressed in in vitro systems, which would allow the characterization and purification of the individual components. It was reported previously that splicing of purified adenovirus precursor mRNAs occurs accurately and efficiently in nuclear extracts prepared from MOPC-315 mouse myeloma cells (16, 17). These experiments, however, were limited by the inability to purify precursor RNAs from infected cells in large quantities. In this report, I have used in vitro-synthesized pre-mRNAs as substrates for splicing. Truncated RNAs encoded by adenovirus 2 (Ad2) early region 2 (E2) were synthesized in vitro by using a plasmid in which a highly efficient bacteriophage promoter, Salmonella phage 6 (SP6), is fused to the E2 gene. In vitro transcription of this plasmid with SP6 RNA polymerase generated large quantities of single RNA species that could be labeled to high specific activity. Here, I describe experiments that demonstrate accurate and efficient (60%80%) in vitro splicing of the E2 in vitro-synthesized RNAs. The nucleotide sequence across the splice junctions in the processed RNAs unambiguously showed that in vitro splicing has the same precision as in vivo. Several kinetic parameters as well as optimum conditions and requirements for the splicing reaction were also defined. MATERIALS AND METHODS Cloning of the Ad2 E2 DNA fragments in the plasmid DNA containing the SP6 bacteriophage promoter was as indicated in the figure legends. In vitro transcriptions for 1 hr at 370C containing 1 ,Ag of DNA template and 16 units of SP6 RNA polymerase (New England Nuclear) in the presence of 10 ,uCi of [a-32P]GTP (600 Ci/mmol; 1 Ci = 37 GBq) were as described (19). Usually, 3 pg of RNA was obtained from 1 ,gg of the linearized,DNA template. The purified RNAs (36 pmol of the complete 3.3-kilobase (kb) pre-RNA equivalent to 0.3 ,g in a 15-ilI reaction mixture (volume) were incubated in the presence of a nuclear extract prepared from MOPC315 mouse myeloma cells as described (17). After the reaction was complete, the RNAs were extracted as described (17).