Characterization of the newly-formed internucleotide bond of in vitro spliced mRNAs

Precursor RNAs were synthesized in vitro from a plasmid in which the early region 2 (E2) of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and utilized as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. We have shown previously (Goldenberg, C.J., PNAS, August, in press, 1984) that in vitro splicing in those extracts was accurate at the nucleotide level. We now show that: i) the new internucleotide bond at the splice junction generated in vitro is a 3',5'-phosphodiester bond; and ii) the phosphate that forms the splice between the exons is derived from the pre-mRNA.