RNA methyltransferase activity from healthy and cowpea mosaic virus-infected etiolated cowpea hypocotyls

Abstract

RNA methyltransferase (MTase) activity in extracts from etiolated cowpea hypocotyls was measured by determining the radioactivity in the acid-insoluble precipitate formed from an assay using ( 3H-methyl)-S-adenosyl- l-methionine as a methyl donor and either Escherichia coli (strain D 10 met-RC rel− RNase I −) methyl-poor tRNA or plant viral RNAs as methyl acceptors. The RNAs of tobacco mosaic virus (TMV), brome mosaic virus (BMV), and E. coli methyl-poor tRNA were methylated four to five times more efficiently than were the RNAs of cowpea mosaic virus (CPMV) and belladonna mottle virus. The MTase activities in extracts from healthy and CPMV-infected etiolated cowpea hypocotyls were compared seeking changes following virus infection. No significant difference in total MTase activity was observed between extracts of healthy and CPMV-infected etiolated hypocotyls, using E. coli methyl-poor tRNA or TMV RNA as methyl acceptors. Methyl nucleosides identified in TMV RNA methylated in vitro with extracts from both healthy and infected hypocotyls were 7-methylguanosine, 1-methyladenosine, 5-methylcytosine, 2′- O-methylguanosine, 5-methyluridine and 6-methyladenosine. Sequential methylation experiments suggested that MTase extracts from healthy and CPMV-infected hypocotyls differed in specificity when RNAs from BMV were used as the methyl acceptor. Methylation of TMV and BMV RNAs did not affect their specific infectivity, but the efficiency of translation in an in vitro wheat germ system was reduced up to 98%.