Mechanism of 5-azacytidine-induced transfer RNA cytosine-5-methyltransferase deficiency

The administration of 5-azacytidine to mice leads to a specific, rapid, time-dependent, and dose-dependent decrease of transfer RNA (tRNA) cytosine-5-methyltransferase activity of mouse liver and the synthesis of tRNA specifically lacking 5-methylcytidine. The mechanism of this enzyme deficiency was investigated. The pretreatment of mice with RNA synthesis inhibitors such as actinomycin D and D-galactosamine prevented the enzyme deficiency induced by 5-azacytidine administration. These results suggested that RNA synthesis was a prerequisite for the induction by 5-azacytidine of the enzyme inhibition in vivo. Indeed, a slowly sedimenting RNA (4 to 7S) from the livers of mice treated with 5-azacytidine, when present in an in vitro tRNA methyltransferase assay, decreased specifically the activity of tRNA cytosine-5-methyltransferase. The pretreatment of mice with actinomycin D or D-glactosamine prior to the administration of 5-azacytidine effectively prevented the formation of such inhibitory RNA in vivo as determined by an in vitro tRNA methyltransferase assay. These results indicate that the administration of 5-azacytidine to mice leads to the rapid synthesis of a low-molecular-weight RNA fraction which is capable of specifically inactivating tRNA cytosine-5-methyltransferase activity in vivo and in vitro.