RNA catabolism in cultured cells : modified nucleoside excretion, effects of RNA synthesis inhibitors on RNA metabolism, and the origin of excreted modified nucleosides

The aims of this dissertation are: To determine what methylated nucleosides are excreted by cultured cells; to determine the excretion rates of these compounds; and to determine the origin of excreted modified nucleosides.

These studies are facilitated by a new rapid procedure for purifying nucleic acid components from biological fluids based on batch adsorption and elution from commercially prepacked silica cartridges. In this procedure nucleic acid components are separated into three groups (bases, deoxy and 2'-0-methyl nucleosides; nucleosides; and charged nucleosides). This fractionation is a necessary preparative step prior to identification of the nucleic acid components on an analytical scale.

I have shown that cultured fibroblasts, do indeed, excrete modified nucleosides into the culture media. A number of the excreted base and ribose methylated nucleosides were chemically identified. For the most part, the modified components of RNA are liberated as nucleosides during catabolism and not further metabolized. However, there are several exceptions. First, the methylated nucleoside 7-MeGuo, is not excreted as a nucleoside but rather as the base, 7-MeGua, indicating cleavage of the glycosidic bond. Second, 2'-0-MeAdo, a major ribose methylated nucleoside in tRNA, as well as rRNA, is not excreted. However, 2'-0-MeIno is excreted even though it is not present in RNAs in significant levels. One possible source of 2'-0-MeIno is deamination of 2'-'MeAdo. Third, the base methylated nucleoside, ribothymidine, can be salvaged by the fibroblast cells and converted to deoxythymidylate for DNA synthesis. All other methylated nucleosides are excreted without modification or intracellular accomulation. Ribothymidine is also excreted.

Finally, and most importantly, I have shown that excreted methylated nucleosides are derived from the turnover of rRNA and tRNA, not only tRNA as was previously thought. This opens the possibility that the increased excretion of modified nucleosides in cancer patients might not be from increased tRNA turnover, as previously suggested, but could result from changes in rRNA as well as tRNA metabolism.