Fate of Histone Messenger RNA in Synchronized HeLa Cells in the Absence of Initiation of Protein Synthesis

Abstract

The fate of cytoplasmic histone mRNA was studied under conditions in which initiation of protein synthesis in synchronized HeLa cells in S phase was blocked by increasing the osmolarity of the growth medium with NaCI. In contrast to the interruption of DNA replication with hydroxyurea, which results in an exponential degradation of translatable histone mRNA with a half-life of about 10-13 min, blocking the initiation of protein synthesis leads to only a marginal loss of biologically active histone mRNA in the cytoplasm. When the initiation of protein synthesis was interrupted by treating cells with 150 mM NaC1, 40-50% of the total cytoplasmic histone mRNA previously translated in polyribosomes appears in the cytoplasm integrated into mRNA . protein particle(s) sedimenting between 15 S and 30 S. On the other hand, in untreated S-phase cells or in cells blocked with hydroxyurea only 3 -6 of the total translatable histone mRNA is found in the cytoplasm not bound to ribosomes or their subunits. In addition, the degradation of histone mRNA in hydroxyurea-blocked S-phase cells is prevented when the initiation of protein synthesis is inhibited with NaCl.These studies clearly indicate that the inhibition of initiation of protein synthesis per se is not the cause for the rapid degradation of cytoplasmic histone mRNA observed when DNA replication is turned off and that the inactivation of these mRNAs is a process dependent on continuous protein synthesis.The coordinate syntheses of histones and DNA in cultured mammalian cells appear to involve highly specific regulatory mechanisms for histone mRNA production, translation and decay. During the cell cycle, histone mRNAs enter the cytoplasm at the onset of the DNA replication period [1,2] and seem to be stable for the period of one S phase [3]. They are then destroyed at the end of S phase following zeroorder kinetics [3]. When DNA synthesis is blocked with hydroxyurea or cytosine arabinoside, histone mRNA is no longer transported to the cytoplasm [l -61 and translatable histone mRNA disappears rapidly and selectively from the cytoplasm following first-order kinetics with a half-life of about 10-13 min [7]. We have recently shown that cycloheximide prevents the hydroxyurea-induced inactivation of cytoplasmic histone mRNA and have suggested that a step in the initiation of histone mRNA translation might be inhibited under conditions where DNA replicationAbbreviations. CM-cellulose, carboxymethyl-cellulose; mRNA Enzymes. Pancreatic RNase or polyribonucleotide 2-oligonucleotide-transferase (cyclizing) (EC 3.1 -4.22); creatine phosphokinase or ATP:creatine phosphotransferase (EC 2.7.3.2).protein, messenger ribonucleoprotein.is blocked leading to the rapid breakdown of these mRNAs [7].To further explore the possibility that inhibition of reinitiation of histone mRNA translation is the cause of its rapid inactivation we have performed experiments in which the initiation of total protein synthesis in HeLa cells was inhibited by increasing the osmolarity of the growth medium with Na...

Nucleus‐associated polyribosomes are engaged in the replication‐ and transcription‐dependent histone synthesis in early fish embryos

“…Such a spatial organization may provide a structural basis for the functional organization of the system involved in coordinated biogenesis of chromatin, where coupling of histone and DNA synthesis can be effected through short-range, as opposed to the diffusiondependent, interactions at the level of the nuclear envelope. This proposal, detailed in [ 141, is compatible with other models proposed to explain the coupling [5,6,12]. It differs in that it envisages a minimal contribution of diffusion in the wellknown, and still unexplained, extremely rapid transport of newly made histone mRNAs and histones [5,6,9,10] and dependence of histone template translation on antecedent DNA synthesis [5&l.…”

“…The mechanism of this coupling, although repeatedly discussed [5,6,12], remains elusive. On the one hand, the mechanism seemed to involve a remote control since histones are believed to be synthesized in free polyribosomes [6-81. On the other hand, the search for diffusible factors to mediate this control (i.e., to govern translation or survival of histone mRNAs depending on the state of the replication system) has been unsuccessful [5,12].…”

Rapid reversible changes in the rate of histone gene transcription and histone mRNA levels in mouse myeloma cells.

“…This reduction is due to a decrease in the half-life of the histone mRNA coupled with a decrease in the rate of transcription (14,24). Inhibitors of protein synthesis block the reduction in histone mRNA levels (5,26) primarily by stabilizing the mRNA but also by altering the rate of transcription (24).…”

Construction of chimeric plasmids containing histone H5 cDNA from hen erythrocyte. DNA sequence of a fragment derived from the 5′ region of H5 mRNA

“…Thus, while synthesis of the main histone classes is coupled to DNA replication (3)(4)(5), H5 is synthesized after cessation of DNA synthesis (6,7) resulting in the partial replacement of H5 for Hi in the chromatin (8,9). Synthesis of H5 is, however, also observed concurrently with that of the other histones in erythroblasts (8,9) and even in proerythroblasts transformed by avian erythroblastosis virus (AEV) (10,11) (Fig.1 ).…”

“…AEV (RAV-2) transformed chicken erythroblast precursor cells (5) were kindly provided by Dr. T.Graf. Cells were labeled with 10-30 wCi of [3Hllysine (75 Ci/mol, Amersham) as de-cribed previously (16).…”