The 5' termini of many viral and cellular mRNAs contain sequences of the type m7G(5")pppNm. An
RNA (guanine-7-)-methyltransferase that specifically methylates the 5'-terminal
guanosine residue of RNAs ending in the dinucleoside
triphosphate G(5')pppN- has been purified from the cytoplasm of HeLa cells. Approximately two-thirds of the
methyltransferase activity detected in an assay employing umnethylated vaccinia virus
mRNA as acceptor was located in the cytoplasm when cells were disrupted by Dounce homogenization; 30% of the cytoplasmic activity was associated with ribosomes but was removed by washing with 0.5 M KCl. The
enzyme was purified 165-fold from the cytoplasm by removing
nucleic acid by phase partition followed by
ammonium sulfate precipitation and column chromatography on
DEAE-cellulose, denatured
DNA-
agarose, and CM-
Sephadex. The partially purified
enzyme preparation methylated heterologous tRNAs as well as
vaccinia mRNA, but the
tRNA methyltransferases could be separated from the
mRNA activity by
sucrose gradient sedimentation and gel filtration on
Sephadex G-200. The product of the partially purified
enzyme using
vaccinia mRNA as substrate was exclusively
7-methylguanosine located in the terminal dinucleoside
triphosphate. In addition to RNAs and synthetic
polyribonucleotides terminating in a dinucleoside
triphosphate, free G(5')pppG could be methylated but
GTP,
GDP, and G(5')pppG could not. The
enzyme also methylated the dinucleoside
diphosphate G(5')pppG but much less efficiently than G(5')pppG. An S20, W of 3.8, a Stokes radius of 3.6 nm, and a molecular weight of 56,000 were obtained from
sucrose gradient sedimentation and
Sephadex G-200 column chromatography.