Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.     

Subcellular localization of histone messenger RNAs on cytoskeleton-associated free polysomes in HeLa S3 cells

We have examined the subcellular distribution of histone mRNA-containing polysomes in HeLa S3 cells to assess the possible relationship between localization of histone mRNAs and the regulation of cellular histone mRNA levels. The distribution of histone mRNAs on free and membrane bound polysomes was examined as well as the association of histone mRNA-containing polysomes with the cytoskeleton. The subcellular localization of histone mRNAs was compared with that of HLA-B7 mRNAs which encode a cell surface antigen. Histone mRNAs were localized predominantly on the free polysomes, whereas the HLA-B7 mRNA was found almost exclusively on membrane bound polysomes. However, both species of mRNA were found associated with the cytoskeleton. Interruption of DNA synthesis by hydroxyurea treatment resulted in a rapid and selective destabilization of histone mRNAs in each subcellular fraction; in contrast, the stability of HLA-B7 mRNA appeared unaffected. The results presented confirm that histone mRNAs are predominantly located on non-membrane bound polysomes and suggest that these polysomes are associated with the cytoskeletal framework.     

Purification of histone messenger ribonucleoprotein particles from HeLa cell S-phase polysomes. Characterization of associated proteins

We have purified HeLa histone mRNA from polysomes of S-phase cells which had been synchronized by hydroxyurea treatment. This mRNA was shown to direct the in vitro synthesis of all five histones which amount to at least 90-95% of its total translational activity. Polysomal histone mRNP was also purified and identified by cell-free translation and hybridization to a clone of histone DNA from E. esculentus. The protein moiety of this mRNP contained three prominent species of molecular weight 86,000, 73,000 and 53,000 daltons. The presence of the 73,000 species previously assessed to be bound to poly(A) is discussed in view of the fact that histone mRNA does not contain a pail. As globin mRNA, histone mRNA as well as histone mRNP were translated with equal efficiency in cell-free extracts from either S-phase or hydroxyurea blocked HeLa cells.     

The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain

1. The turnover of cerebral histones and DNA after injection of [4,5-(3)H]leucine or [methyl-3-(3)H]thymidine, respectively, was studied in the developing chick. 2. Chromatin was prepared from chick nuclei that had been purified by centrifugation through 1.9m-sucrose. 3. Nuclear proteins were fractionated into three major histone classes, F1 (lysine-rich), F2(b) (slightly lysine-rich) and [F3+F2(a)] (arginine-rich), and a non-histone protein residue. 4. The proportions of the histone classes remained constant throughout the period of development studied. 5. All histone fractions decayed at a similar rate, initially with a half-life of around 5 days, later with a half-life of 19 days. 6. Non-histone proteins from chromatin decayed in a heterogeneous manner with a wide range of half-lives. 7. Short-term labelling studies showed that all histone fractions were synthesized at the same rate. 8. Some non-histone proteins were very rapidly synthesized relative to histones. 9. DNA had a longer half-life than any histone fraction studied. A biphasic exponential decay curve with half-lives of 23 and 50 days was found. 10. It was concluded that the turnover of histones can occur independently of that of DNA and that different histone classes have similar rates of synthesis and decay.     

Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.

Most host mRNAs are degraded soon after infection of cells with herpes simplex virus type 1 (HSV-1). This early shutoff or early destabilization response is induced by a virion component, the virion host shutoff (vhs) protein. HSV-1 mutants, vhs1 and vhs-delta Sma, which produce defective or inactive vhs protein, fail to induce early shutoff. We have used an in vitro mRNA decay system to analyze the destabilization process. Polysomes from uninfected human erythroleukemia cells, used as a source of target mRNAs, were mixed with polysomes or with post-polysomal supernatant (S130) from HSV-1- or mock-infected murine erythroleukemia cells. Normally stable gamma-globin mRNA was destabilized by approximately 15-fold with S130 from wild-type virus-infected cells but was not destabilized with S130 from mock-infected cells or from cells infected with either of the two HSV mutants. The virus-induced destabilizing activity had no significant effect on the in vitro half-lives of two normally unstable mRNAs, histone and c-myc. No destabilizing activity was detected in polysomes from infected cells. We conclude that a virus-induced destabilizer activity can function in vitro, is located in the S130 of infected cells, and accelerates the decay rates of some, but not all, polysome-associated host mRNAs.     

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.     

Quantitation of the accumulation of histone messenger RNA during oogenesis in Xenopus leavis

The accumulation of messenger RNA coding for histone H3 in oogenesis of Xenopus laevis was studied by quantitative hybridization techniques, using a cloned genomic DNA fragment as a probe. This probe was isolated from cloned Xenopus histone DNA and contains most of the H3 coding sequences. Histone H3 mRNA accumulation was found to be completed before the maximum lampbrush stage. Hybridization of RNA blots with DNA probes containing genes for histones H2A, H2B, and H4 suggests the same accumulation pattern for the mRNAs coding for these histones as for histone H3 mRNA. The amount of H3 mRNA in the mature oocyte was established to be 130 ± 68 pg, i.e., about 5 × 10⁸ copies.     

Degradation of sperm histones in vitro by cytoplasm of the sea urchin egg

Sperm-specific histone variants in the sea urchin Paracentrotus lividus are replaced early after fertilization with a specific embryonic set of histone variants. A possible in vitro model for the involvement of a degradation mechanism in the replacement of sperm-specific histones is presented. Soluble sperm histones are shown to be degraded quickly by egg cytoplasm. The proteolytic activity is maximal at pH 3.0; H1 and H2A histones are the most sensitive while H3 and H4 are the most resistant. H2B histones have an intermediate sensitivity. Histone degradation by egg cytoplasm or by purified fractions of it can be inhibited by chymostatin and leupeptin and, to a lesser degree, by pepstatin.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component.

The turnover rates of some mRNAs vary by an order of magnitude or more when cells change their growth pattern or differentiate. To identify regulatory factors that might be responsible for this variability, we investigated how cytosolic fractions affect mRNA decay in an in vitro system. A 130,000 X g supernatant (S130) from the cytosol of exponentially growing erythroleukemia cells contains a destabilizer that accelerates the decay of polysome-bound c-myc mRNA by eightfold or more compared with reactions lacking S130. The destabilizer is deficient in or absent from the S130 of cycloheximide-treated cells, indicating that it is labile or is repressed when translation is blocked. It is not a generic RNase, because it does not affect the turnover of delta-globin, gamma-globin, or histone mRNA and does not destabilize a major portion of polysomal polyadenylated mRNA. The destabilizer accelerates the turnover of the c-myc mRNA 3' region, as well as subsequent 3'-to-5' degradation of the mRNA body. It is inactivated in vitro by mild heating and by micrococcal nuclease, suggesting that it contains a nucleic acid component. c-myb mRNA is also destabilized in S130-supplemented in vitro reactions. These results imply that the stability of some mRNAs is regulated by cytosolic factors that are not associated with polysomes.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

A method for the purification of histone fraction F3 by affinity chromatography

Calf thymus histone fraction F3 has been purified in high yield by affinity chromatography. The protein is attached reversibly to an organomercurial—Sepharose—by the thiol groups of its cysteinyl residues, while all other histones lacking -SH groups pass through the column. Histone F3 is subsequently eluted with -SH reagents of low molecular weight, such as cysteine or β-mercaptoethanol. The purified F3 shows electrophoretic heterogeneity due to different degrees of acetylation of the parent polypeptide chain. Under oxidizing conditions it forms a complex mixture of dimers and oligomers, all of which have some available -SH groups.     

A protein with antimicrobial activity in the skin of Schlegel's green tree frog Rhacophorus schlegelii (Rhacophoridae) identified as histone H2B

An extract of the skin of Schlegel's green tree frog, Rhacophorus schlegelii (Anura: Rhacophoridae), contained a protein that inhibited the growth of the Gram-negative bacterium Escherichia coli but was inactive against the Gram-positive bacterium Staphylococcus aureus. The protein was purified to near homogeneity by reverse-phase HPLC and amino acid sequence analysis of the products of an endoproteinase Glu-C digest identified the protein as histone H2B. The complete primary structure of the 125 amino acid residue Rhacophorus histone H2B was determined by nucleotide sequence analysis of a cloned cDNA encoding the protein. Mass spectrometry demonstrated that the protein isolated from the skin was not post-translationally modified. Histone fragments with antimicrobial activity were not identified in the Rhacophorus skin extract nor were cationic, alpha-helical antimicrobial peptides of the kind isolated from the skins of several other frog families. The data provide further evidence that histones play a role in the defense against microorganisms.