Post-transcriptional modifications of mRNA. Purification and characterization of cap I and cap II RNA (nucleoside-2'-)-methyltransferases from HeLa cells

Abstract

The existence in HeLa cell extracts of two separate RNA (nucleoside-2')-methyltransferases involved in the modification of mRNA was established using assays that specifically measure the conversion of cap O [m7G(5')pppNpN-] to cap I [m7G(5')pppNmpN-] and cap I to cap II [m7G(5')pppNmpNm-]. Cap II methyltransferase activity was found almost exclusively in cytoplasmic fractions while cap I methyltransferase activity was also found in the nucleus, its apparent biological site of action. The two enzymes were purified by DEAE-cellulose and phosphocellulose chromatography and their optimal reaction conditions were determined. The substrate specificity of cap I methyltransferase was examined with particular regard to information that would help elucidate the natural order of capping and methylation was drawn from data presented here. Both purine and pyrimidine nucleosides in the N position of M7G(5')pppN- were methylated by purified cap I methyltransferase.