An in Vitro System for Accurate Methylation of Internal Adenosine Residues in Messenger RNA

Abstract

Some internal adenosine residues in messenger RNA are methylated posttranscriptionally in the nucleus. Most of the methylated adenosine residues in prolactin mRNA are in the 3' untranslated region. The site of methylation in the 3' end of prolactin mRNA was determined. This methylation reaction is highly specific; of the three adenosine residues in consensus sequences located in the 3' end, only one is methylated. An in vitro methylation system was developed in which bovine prolactin mRNA, synthesized in vitro with T7 RNA polymerase, was accurately methylated in a HeLa cell nuclear extract. The adenosine residue that was methylated in vitro was the same as the one methylated in vivo. This cell-free system, which accurately methylates the N6-position of adenosine residues in mRNA, will allow further study of the mechanism of adenosine methylation.