Partial purification and properties of a pre-mRNA splicing activity

Abstract

Precursor RNA substrates for splicing reaction were synthesized in vitro from a plasmid DNA in which the early region 2 gene of adenovirus 2 was fused to an efficient bacteriophage promoter (Salmonella phage 6). Pre-mRNA splicing activity from nuclear extracts of MOPC-315 mouse myeloma cells was partially purified 108-fold by three chromatographic steps. The in vitro splicing reaction catalyzed by the partially purified fractions was efficient (60-80% substrate conversion) and accurate at the nucleotide level. The reaction occurred with crude or purified fractions without any detectable lag and nucleotides (ATP or GTP) were absolutely required. Monoclonal anti-Sm antibodies that quantitatively immunoprecipitate U1 small nuclear ribonucleoprotein particles totally inhibited the splicing activity of the purified fractions, indicating that U1 small nuclear RNPs had co-purified with the activity and were absolutely required for the splicing reaction.