The overall goal of this study is to understand the biosynthesis and function(s) of methylphosphate cap structures found in human U6 small nuclear RNA and many other eukaryotic small RNAs. We purified the methylphosphate capping enzyme from HeLa cells and found that the same activity caps other known methylphosphate-capped small RNAs. All known methyltransferases methylate usually basic amino acid residues in proteins or sugar/base moieties in nucleic acids. This is the first methyltransferase that methylates a phosphate residue in nucleic acids and thus, the mechanism might be distinct from that of other known methyltransferases. We plan to further characterize the capping enzyme to understand the mechanism of methylphosphate cap formation. Towards this goal, the following specific aims will be pursued: 1. Obtain and express a cDNA clone encoding the 130 kDa methylphosphate capping enzyme. 2. Use the purified enzyme and study the biochemistry of its interactions with the RNA substrates. 3. Obtain polyclonal antibodies against the 130 kDa capping enzyme to study its subcellular localization and the mechanism of capping. 4. Study the phosphorylation state of the capping enzyme in relation to the metabolic state of the cell to investigate the possible role of phosphorylation/dephosphorylation in regulation of the capping enzyme activity. 5. In addition to the above studies on the methylphosphate capping enzyme, another enzymatic activity responsible for m6A formation in human U6 snRNA will be purified. We developed an efficient in vitro system for the formation of m6A in human U6 snRNA and partially purified this enzymatic activity. Our goal is to understand the relationship between this U6 snRNA m6A methyltransferase and mRNA m6A methyltransferase.
