Purification of inosine monophosphate by thin layer chromatography and detection of inosine monophosphate within cellular messenger RNA

This dissertation describes the development and use of novel thin layer chromatography (TLC) purification methods that allowed for the specific and sensitive detection of inosine monophosphate (IMP) within cellular mRNA. Radioactive 5' nucleoside monophosphate (5' NMP) mixtures were prepared from mRNA, and then spiked with nonradioactive 5' IMP. This nonradioactive 5' IMP served as a marker for the migration of radioactive 5' IMP and allowed for the conclusive identification of radioactive 5' IMP derived from mRNA. Three consecutive rounds of TLC purified 5' IMP away from the more abundant ribonucleotides found within RNA (adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, and uridine monophosphate), as well as other minor modified nucleotides found within RNA. These purification methods allowed for the detection of 1 to 5 parts IMP per 1 million parts NMPs (1-5 ppm). Radioactive 5' NMPs were prepared from polyadenylated RNA isolated from a number of different rat tissues. TLC analyses of these 5' NMPs showed that 5' IMP was most abundant in brain mRNA, and the tissue specific abundance of 5' IMP directly correlated with the expression of genes encoding adenosine deaminases that act on RNA (ADARs). Quantitation of this endogenous IMP provided the first estimate of the amount of inosine in cellular mRNA. It was also demonstrated that in polyoma virus infected cells, IMP levels in cellular RNA increased with viral titer. Polyoma early (sense) and late (antisense) RNAs are thought to form double-stranded RNAs (dsRNAs) within the cell nucleus and undergo significant modification by ADARs. Cells were infected with polyoma virus and nuclear and cytoplasmic RNA was subsequently isolated at various hours postinfection. TLC analyses showed that 5' IMP was present in nucleotides derived from nuclear RNA isolated at the late stage of polyoma infection but 5' IMP was not present in nucleotides derived from nuclear RNA isolated at the early stage of polyoma infection. These results suggest that stable dsRNA duplexes exist within the nucleus during the late stage of polyoma infection and that these duplexes are modified by ADARs.