Translation of globin RNA spliced in vitro

Pre-mRNA (precursor mRNA) enriched for globin-gene transcription products was prepared from murine erythroleukaemia cells induced to differentiate with dimethyl sulphoxide. The pre-mRNA was prepared from nuclear RNA by oligo(dT)-cellulose chromatography followed by sucrose-gradient centrifugation. The pre-mRNA thus obtained was utilized as the substrate for a splicing assay based on sequential splicing and translation reactions. Small nuclear ribonucleoprotein particles, which were shown to inhibit translation, were removed by an intermediate high-speed centrifugation step. The assay developed is based on a three-step procedure: (1) incubation of pre-mRNA with HeLa-cell nuclear extract in the presence of ATP; (2) incorporation of 3H-labelled amino acid into proteins by wheat-germ extract by using RNA from step 1; and (3) immunoprecipitation of globin chains with rabbit anti-(mouse globin) antibody. No incorporation of label into immune complexes was observed in the absence of HeLa-cell extract or ATP. Preincubation with these components was unnecessary for the incorporation of label into immune complexes when mature cytoplasmic RNA was used as a template for protein synthesis. The assay was further validated by fluorography after polyacrylamide-gel electrophoresis, when the only band detectable corresponded to globin chains. These results demonstrate, for the first time, translational activity of transcripts spliced in vitro. The current assay can be utilized with a few micrograms of pre-mRNA and may have potential for further characterization of the splicing system.