The objective of this research is to understand the function of a ubiquitous post- transcriptional base modification, N6-methlyadenosine (m6A), in eukaryotic MRNA. The methyltransferase that mediates the formation of m6A is a complex multicomponent enzyme, and a cDNA encoding the S- adenosylmethionine-binding [subunit] of this enzyme is now available. This provides an essential tool for gene disruption experiments and for the further characterization of the multi-component enzyme.
The specific aims to be addressed are: First, the biological function of m6A residues in MRNA will be examined at the cellular level using antisense and ribozyme mediated disruption of m6A methyltransferase activity and the effect on pre-MRNA splicing MRNA transport will be determined. The role of m6A in mRNA biogenesis and function will then be examined in the intact organism by studying the effects of gene knockout mutants in mice. SEcond, the molecular composition of the other essential components of this large enzyme will be determined, with emphasis on identification of the factor(s) that are involved in the sequence- specific recognition of mRNA methylation sites. The functional relationship of this post-transcriptional modification to other pre-mRNA processing events, as suggested by the localization of m6A methyltransferase in nuclear interchromatin granule clusters, will be examined. N6-adenosine methylation in mRNA is an unexplored post- transcriptional event whose potential role in the regulation of gene expression will be studied in both normal and transformed cells. Alterations in methyl-group metabolism have been shown to play a role in carcinogenesis in a number of systems, although the role of RNA methylation in cancer has not been explored. These studies focus on a defined set of goals that will elucidate the physical characteristics of the mRNA m6A methyltransferase complex, and will establish the general biological function of m6A in mRNA. The completion of these objectives will provide a firm basis for future work extending this research into the areas of regulation of oncogene expression and cellular transformation.
|Clancy, Mary J; Shambaugh, Mary Eileen; Timpte, Candace S et al. (2002) Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res 30:4509-18|