The fates of undermethylated mRNA cap structures of vesicular stomatitis virus (New Jersey) during in vitro transcription

Abstract

A dual label pulse-chase analysis employing S-adenosyl-L-[methyl-3H]methionine and [beta-32P]GTP has been devised to identify precursors to the cap structure 7mG(5')ppp(5')Am present on the 5'-termini of vesicular stomatitis virus mRNAs. Both monomethylated cap structures, 7mG(5')ppp(5')A and G(5')ppp(5')Am, have been detected in vitro in New Jersey serotype reactions containing suboptimal concentrations of S-adenosyl-L-methionine. The simultaneous chasing of both radiolabeled substrates allowed the determination of the transcriptive fate of each pulse-labeled cap structure in the total RNA population. Ten percent of the 7mG(5')ppp(5')A cap structure and 27% of the G(5')ppp(5')Am cap structure generated during the pulse were chased into 7mG(5')ppp(5')Am. These results suggested that while there may have been a preferred order of 5'-cap methylation, the order was not compulsory. The dual label analysis also revealed that only 34% of the pulse-labeled G(5')ppp(5')A cap structure could be chased into methylated cap structures. A nascent RNA chain length-dependent "methylation window" is proposed.