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)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA031810-09
Application #
3169936
Study Section
Biochemistry Study Section (BIO)
Project Start
1981-07-01
Project End
1990-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
9
Fiscal Year
1989
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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Carroll, S M; Narayan, P; Rottman, F M (1990) N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA. Mol Cell Biol 10:4456-65
Narayan, P; Rottman, F M (1988) An in vitro system for accurate methylation of internal adenosine residues in messenger RNA. Science 242:1159-62
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
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

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