Our experiments are designed to elucidate the role of post-transcriptional methylation of mRNA molecules in the processing of nuclear mRNA precursors into mature cytoplasmic mRNAs. We have mainly focused on the expression of growth hormone and prolactin genes in normal and transformed pituitary cells. Recent progress on this project has occurred in two major areas. First, we have developed a new approach for defining the location of naturally occurring N?6?-methyladenosine (m?6?A) residues which are located at internal positions within individual mRNA sequences. This highly sensitive technique should be broadly applicable because it does not rely on prior in vivo labeling of mRNA with radioactive methyl groups. Using this approach we have determined the location of m?6?A in bovine prolactin mRNA and found that these modified nucleotides are contained within a short, 3' terminal fragment (129 nucleotides) of this 915 nucleotide-long mRNA. The non-random location of m?6?A residues within prolactin mRNA suggests a specific function which can now be assessed by a combination of techniques involving DNA-mediated gene transfer and site-directed mutation of the bovine prolactin gene. Similar studies have defined the selective distribution of M?6?A residues in influenza viral RNAs. Secondly, by using an inhibitor which blocks mRNA methylation, we were able to follow the fate of undermethylated mRNA in HeLa cells. Our results indicate that once the nuclear processing and/or transport of mRNA into the cytoplasm is significantly affected by undermethylation. These nuclear effects can now be studied using individual mRNA sequences which accumulate to high steady-state levels when transcribed from chimeric genes introduced into cells by DNA-mediated gene transfer. (G)
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