Separate Pyrimidine-Nucleotide Pools for Messenger-RNA and Ribosomal-RNA Synthesis in HeLa S 3 Cells

Abstract

Kinetic analyses of mRNA and 28-S RNA labeling by [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP. [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleoside attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA.The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 pM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.Incorporation of labeled uridine into RNA has often been used as a measure of RNA synthesis. It also forms the basis of new half-life determinations of mRNA [I -41. In all these determinations it was assumed that one UTP pool served all the different RNA species. When we applied newly developed methods for the direct determination of specific radioactivities of individual RNA species [5, 61 and of UTP [7, 81 indications of rather different steady-state specific radioactivities for 28-S RNA zierms mRNA and (total) UTP were obtained [9, 101. These data pointed to the existence of different precursor pools for the various RNA species. This means that the turnover determinations mentioned above are inaccurate, as well as the conclusions drawn from labeling experiments with pyrimidine nucleotide precursors. Therefore, a study on steady-state specific radioDedicated to Feodor Lynen on the occasion of his 65th birthday. activities of uridine and cytidine in several RNA species under carefully controlled conditions of logarithmic growth and constant exogenous [3H]uridine concentrations over several generation times was started. The data obtained provide strong evidence that ribosomal and messenger RNA formation depend on separate pyrimidine nucleotide precursor pools.

The Synthesis and Degradation of Poly(A)‐containing mRNAs in Mouse Neuroblastoma Cells Treated with Dibutyryl cAMP or with Ro20‐1724 *

“…As the 18-h labeled mRNAs reflect the steady-state mRNA population, an increase in their relative labeling should have resulted in a corresponding increase in the steady-state, polyadenylated mRNA levels; however, we found no such change. This suggests that the increased labeling of the polyadenylated mRNAs with 3H-adenosine was a labeling artifact, perhaps a consequence of nonequilibration of nuclear and nucleolar precursor pools (Wiegers et al, 1976).…”

Pyrimidine Metabolism in Lemna minor

“…On the basis of steady-state specific radio uridine in individual RNA species of HeLa S5 (23) described a large, de novo generated, py pool (UTP and CTP for mRNA and dCTP I feedback inhibition by exogenous nucleosides turnover, pool (UTP and CTP for rRNA) wi feedback-sensitive by exogenous uridine. TI nucleic acid synthesis in the extranucleola smaller in the nucleolus.…”

Why are dietary nucleotides essential nutrients?

“…The large intracellular nucleotide pool was demonstrated to be under metabolic control in the sense that de novo synthesis could be suppressed by exogenous nucleotide supply (Goody et al 1975). More specifically, rRNA synthesis was shown to be supplied from a pool of pyrimidines, synthesized de novo (Wiegers et al 1976). These data did not suggest that purines and pyrimidmes were essential nutrients, indeed excess intake had negative health implications in relation to gout and inborn errors of purine metabolism.…”