Accurate in vitro splicing of human beta-globin RNA

Abstract

Human beta-globin RNA transcribed from an exogenous DNA template is spliced in vitro by concentrated whole cell extracts from HeLa cells. Using the primer extension technique, we have shown that the small intervening sequence is spliced accurately and that the sequence of the product across the splice junction is identical to that of beta-globin mRNA prepared from human reticulocytes. The efficiency of the splicing reaction is low. The RNA transcript containing both introns and terminated upstream from the polyadenylation site is spliced most efficiently. The transcript which is terminated downstream from the polyadenylation site is not spliced at all. Thalassemic beta-globin RNA which carries an extra splice site in the small intron is also spliced, albeit with a low yield.