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.
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