Accurate and efficient in vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome

Abstract

Polyadenylated and deproteinized nuclear RNA precursors encoded by early region 2 of the adenovirus 2 genome are spliced in vitro by nuclear extracts 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 nucleotide sequence across the splice junctions in the E2 RNAs processed in vitro was investigated by performing primer extensions in the presence of dideoxynucleotides and direct sequencing on polyacrylamide gels. We conclude that the in vitro splicing reaction is accurate and has the same precision as that of in vivo E2 cytoplasmic mRNA prepared from Ad2 infected cells. The efficiency of in vitro splicing by the nuclear extracts is very high. Approximately 80% of E2 RNA precursor, on a molar basis, are spliced in vitro to a mature RNA. These findings provide evidence that a nuclear extract prepared from MOPC-315 mouse myeloma cells is capable of accurate and efficient splicing of E2 RNA precursors.