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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA031810-11
Application #
3169937
Study Section
Biochemistry Study Section (BIO)
Project Start
1981-07-01
Project End
1995-01-31
Budget Start
1991-02-01
Budget End
1992-01-31
Support Year
11
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Ransohoff, R M; Narayan, P; Ayers, D F et al. (1987) Priming of influenza mRNA transcription is inhibited in CHO cells treated with the methylation inhibitor, neplanocin A. Antiviral Res 7:317-27
Narayan, P; Ayers, D F; Rottman, F M et al. (1987) Unequal distribution of N6-methyladenosine in influenza virus mRNAs. Mol Cell Biol 7:1572-5
Hampson, R K; Rottman, F M (1987) Alternative processing of bovine growth hormone mRNA: nonsplicing of the final intron predicts a high molecular weight variant of bovine growth hormone. Proc Natl Acad Sci U S A 84:2673-7

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