Inhibition of Ribosomal Ribonucleic Acid Maturation in Rat Liver by 5-Fluoroorotic Acid Resulting in the Selective Labeling of Cytoplasmic Messenger Ribonucleic Acid

Abstract

Both orotate and 5-fluoroorotate are precursors of hepatic RNA. Whereas [6-14C]orotate was extensively incorporated into ribosomal RNA after a 3-hour period, 5-fluoro[2-14C]-orotate was selectively incorporated into nonribosomal ribonucleoprotein particles. These analogue-labeled RNA-protein complexes were released from polyribosomes by EDTA treatment and were distinctly different from ribosomal subunit particles, based on their polydisperse sedimentation in sucrose gradients, relatively lower buoyant density in CsCl gradients, and insensitivity to actinomycin D. Orotic acid and 5-fluoroorotic acid were incorporated equally well into nuclear mRNA-containing ribonucleoprotein particles. These results suggested that the analogue was selectively incorporated into an RNA which exists in the cell as a relatively low density RNA-protein complex. Analysis of microsomal RNA showed little or no incorporation of 5-fluoroorotic acid into either 18 S or 28 S rRNA for at least 3 hours. A small level of incorporation into rRNA was observed 24 hours after the administration of labeled analogue. Electrophoretic analysis of nuclear RNA demonstrated that the analogue was rapidly incorporated into 45 S ribosomal precursor RNA but that its subsequent incorporation into mature 18 S and 28 S rRNA was significantly inhibited. The kinetics of labeling of the various nuclear and cytoplasmic rRNA species was identical whether one labeled with 5-fluoroorotate alone, or with labeled orotate simultaneously with an equimolar dose of unlabeled analogue. Based on these results, and its relative nontoxicity at the dose levels employed, it is proposed that 5-fluoroorotic acid may in mammalian systems be useful for the selective inhibition of rRNA synthesis, resulting in the selective incorporation of a labeled precursor into a cytoplasmic RNA having several properties of mRNA.