Methylations of adenosine residues (m6A) in pre-mRNA are important for formation of late simian virus 40 mRNAs.

Cycloleucine, a competitive inhibitor of methionine transferase was used to generate in vivo partially methylated mRNA in SV40-infected BSC-1 cells. Cycloleucine at 0.5 mg/ml causes more than a 30% decrease in internal m6As of late SV40 mRNA with only minor effect on the dimethyladenosine of the 5' caps m7GpppmAm. After treatment with 2 and 5 mg/ml of cycloleucine, internal m6As were reduced by 10- and 100-fold, respectively. The inhibition of BSC-1 mRNA methylations paralleled that observed for late SV40 mRNAs. In cells exposed to 2 mg/ml cycloleucine production of late SV40 mRNA was inhibited by 80% whereas the amount of SV40 nuclear RNA was only slightly reduced. Size fractionation of SV40 nuclear RNA from cycloleucine-treated cells revealed a loss of SV40 19 S RNA with a corresponding increase of fragmented RNA sedimenting between 11 to 5 S, so that the total amount of SV40 RNA in the nucleus was almost unchanged. Analysis of viral transcription complexes from cells treated with cycloleucine indicated that SV40 transcription was not affected by cycloleucine. SV40-transformed cells, in contrast to BSC-1 cells, were able to process and transport undermethylated RNA. When transformed cells were treated with 2 mg/ml cycloleucine no changes in quantities or size of cytoplasmic and nuclear RNA were detected. The data argues for a role of internal m6A moieties in modulating the processing-linked transport of mRNA from the nucleus to the cytoplasm of nontransformed cells. Transformed cells may escape these controls due to structural alterations in their perinuclear regions.