Abstract
This chapter reviews that the use of enzymes for selective hydrolysis, coupled with high-resolution liquid chromatography for assay of products, provides an efficient means of determining the specific patterns of methylation in eukaryotic mRNA molecules. Continuous labeling with levels of L-[methyl-3H] methionine permits normal growth of Novikoff cells to examine the methylation of specific sites of cytoplasmic mRNA as a function of time. The main site of cytoplasmic mRNA labeling is at the second position (N'') of the 5'-terminal sequence. Data obtained by comparing methylnucleoside composition of these sequences and the ratio of doubly to singly O-methylated termini as a function of labeling time is consistent with a model in which m7G, N'm and the m6A located in the mRNA molecule are all products of nuclear methylation events. Subsequently there is a cytoplasmic methylation of some singly O-methylated structures at the second (N'') position yielding the doubly O-methylated structure. The kinetics of methyl labeling and the changing composition within the caps show a distinct pattern, possibly reflecting a selection or enrichment of a stable class of mRNA molecules, many of which contain the doubly labeled structure at their 5'-terminus and are of smaller size.