The major objective of the proposed research is to learn the functional role(s) of modified nucleosides, predominantly N6-methyladenosine (m6A), which are located at internal sites in most mRNAs of higher eukaryotes. Previous studies, which led to the precise localization of m6As at seven sites in RSV virion RNA, will be extended to RSV mRNAs. If differences in methylation patterns are observed between the genomic RNA and specific viral mRNAs, the significance of m6A in regulation of either splicing or packaging of viral RNA will be investigated. Oligonucleotide-directed mutagenesis will be used to prevent methylation at specific sites in RSV RNA, and the effects of altering the methylation pattern will be investigated both in vivo and in vitro. Possible effects on RNA splicing, transport from the nucleus, packaging into virions, reverse transcription, translation, RNA secondary and tertiary structure, and stability will be examined. Purified m6A methylase will also be used in in vitro studies of the biological function of m6A, as well as in studies of the specificity of m6A modification. A consensus sequence for m6A sites will be derived for chicken cellular mRNAs and compared with that previously observed for mammalian mRNAs (RAC) and for RSV RNA (RGACU), to determine whether there are species-specific differences in m6A sites. The hypothesis that m6A at potential branch points affects splicing at nearby splice acceptor sites will be tested. Intron branches, which form lariat structures during mRNA splicing, usually occur at adenines, preceded by a purine and followed by a pyrimidine. This sequence is similar to the m6A consensus sequence. Therefore, it is possible that m6A is present at branch points and promotes 2'-P-5' lariat bond formation or alternatively that m6A at a potential branch point may block lariat formation. Branch structures will be isolated from total nuclear poly (A)+ RNA, and the relative amount of m6A will be measured directly. The presence of m6A elsewhere in intron sequences will also be examined to determine whether m6A is totally conserved during mRNA processing. These studies may give insight into regulation of retroviral RNA metabolism, as well as into that of cellular mRNAs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA033199-06
Application #
3171143
Study Section
Virology Study Section (VR)
Project Start
1982-07-01
Project End
1991-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Dalrymple, S A; Beemon, K L (1990) BK virus T antigens induce kidney carcinomas and thymoproliferative disorders in transgenic mice. J Virol 64:1182-91
Csepany, T; Lin, A; Baldick Jr, C J et al. (1990) Sequence specificity of mRNA N6-adenosine methyltransferase. J Biol Chem 265:20117-22
Ryden, T A; Beemon, K (1989) Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein. Mol Cell Biol 9:1155-64
Carlberg, K; Beemon, K (1988) Proposed gag-encoded transcriptional activator is not necessary for Rous sarcoma virus replication or transformation. J Virol 62:4003-8
Arrigo, S; Beemon, K (1988) Regulation of Rous sarcoma virus RNA splicing and stability. Mol Cell Biol 8:4858-67
Carlberg, K; Ryden, T A; Beemon, K (1988) Localization and footprinting of an enhancer within the avian sarcoma virus gag gene. J Virol 62:1617-24
Arrigo, S; Yun, M; Beemon, K (1987) cis-acting regulatory elements within gag genes of avian retroviruses. Mol Cell Biol 7:388-97
Loeb, D M; Woolford, J; Beemon, K (1987) pp60c-src has less affinity for the detergent-insoluble cellular matrix than do pp60v-src and other viral protein-tyrosine kinases. J Virol 61:2420-7
Kane, S E; Beemon, K (1987) Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations. J Biol Chem 262:3422-7
Beemon, K; Mattingly, B (1986) Avian sarcoma virus gag-fps and gag-yes transforming proteins are not myristylated or palmitylated. Virology 155:716-20

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