The long term goal of this research is to determine the mechanism underlying the highly selective post-transcriptional formation of N6-methyladenosine residues in eukaryotic mRNA and to determine the function of this modification in mRNA metabolism. The experimental approach will utilize a recently developed in vitro system which is capable of methylating the same adenosine residues as found in vivo in both exon and intron sequences of bovine prolactin mRNA. These studies are defined in three specific aims. 1. The features of the mRNA substrate that are recognized by this post-transcriptional modification system will be defined. The consensus nucleotide sequence for N6-adenosine methylation will be characterized and the influence of mRNA secondary structure determined. 2. The N6-adenosine methyltransferase which catalyzes this modification of mRNA will be purified from HeLa cell nuclear extracts. Amino acid sequence information derived from the purified methyltransferase will be used to develop probes for the isolation of cDNA clones encoding this enzyme. 3. The function of N6-adenosine methylation in mRNA metabolism will be approached though specific site-directed mutations of the predominant consensus methylation sequence in bovine prolactin mRNA, the use of methyltransferase inactivating antibodies in cell-free systems and the expression of antisense RNA constructs in transfected cells. The development of a cell-free N6-adenosine methyltransferase system and the antibody and antisense RNA reagents corresponding to this enzyme provide the tools to gain a better understanding of the mechanisms underlying the degree of specificity of N6-adenosine methylation and the capability of probing the biological function of this modification of eukaryotic mRNA in normal and transformed cells.
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