Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay.

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.

Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells.

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.

Xenopus laevis serum albumin: sequence of the complementary deoxyribonucleic acids encoding the 68- and 74-kilodalton peptides and the regulation of albumin gene expression by thyroid hormone during development.

In adult Xenopus serum, albumin gene expression is regulated by estrogen through the selective destabilization of its mRNA during the vitellogenic response. The present study reports the cDNA sequence of both the 68K and 74K Xenopus albumin mRNAs, their derived amino acid sequence, and the regulation of albumin gene expression during embryogenesis. Albumin mRNA has a 39 nucleotide 5' untranslated region terminating in a consensus translation initiation site. The derived amino acid sequence yields a 24-amino acid hydrophobic leader sequence (terminating in Lys-Arg) that shares significant homology with the leader peptide of rat albumin. Overall there is 37% sequence identity between rat and frog albumin, with exact conservation of all but one Cys residue and the Pro residues responsible for the three domain structure of the mature protein. The 74K albumin (unlike the 68K albumin) is glycosylated; a point mutation converting Lys256 to Asn introduces an N-linked glycosylation site that is similar to one found in the sequence of mammalian alpha-fetoproteins. A larval albumin-like protein was not detectable by silver staining in serum of tadpoles before the beginning of metamorphosis at stage 48. Albumin mRNA is absent from early tadpoles (stages 22-47); however, it is rapidly induced at stage 48 as one of the earliest manifestations of metamorphosis. Exposure of embryos to 10(-8) M T3, which regulates amphibian metamorphosis, resulted in the premature induction of albumin mRNA, such that it is evident by stage 43.

Posttranscriptional regulation of albumin gene expression in Xenopus liver: evidence for an estrogen receptor-dependent mechanism.

We have previously shown that estrogen administration to male Xenopus laevis results in the posttranscriptional suppression of serum albumin mRNA concurrent with the transcriptional activation of the genes for the yolk protein precursor vitellogenin. To determine whether the posttranscriptional regulation of albumin gene expression is mediated through a mechanism involving the high affinity estrogen receptor protein or through a receptor-independent mechanism involving a middle affinity cytoplasmic estrogen-binding protein we examined the effects of the competitive estrogen receptor antagonist 4-hydroxytamoxifen. Administration of 4-hydroxytamoxifen 24 h before estradiol completely blocked both the suppression of albumin mRNA and the transcriptional activation of the vitellogenin genes. Albumin gene transcription remained constitutive under all treatment regimens. Competitive binding experiments demonstrated that 4-hydroxytamoxifen has an affinity for the estrogen receptor similar to that of estradiol. However, 4-hydroxytamoxifen displays little or no interaction with the middle affinity cytoplasmic estrogen-binding protein. These data indicate that the estrogen receptor occupies a key role in the posttranscriptional regulation of albumin mRNA.

Extranuclear estrogen-regulated destabilization of Xenopus laevis serum albumin mRNA.

The present study examined 1) whether the estrogen-regulated destabilization of albumin mRNA occurs in the nuclear or extranuclear fraction of the liver cell, and 2) whether the selective posttranscriptional regulation of albumin mRNA stability might result from covalent changes introduced in the processing or polyadenylation of the primary transcript. The disappearance of albumin mRNA after estrogen is restricted to the extranuclear fraction of the cell. Transient changes in steady state levels of the mature nuclear transcript were observed that mirrored the transient estrogen-induced changes previously reported for albumin gene transcription. When assayed 24 h after estrogen (when albumin RNA is virtually undetectable in the extranuclear fraction) the steady state levels of both the primary and mature albumin transcripts found in the nucleus were the same as observed in control animals. Estrogen had no effect on the splicing or selection of polyadenylation sites on the 3'-UTR as determined by high resolution gel analysis of the 3'-UTR and DNA sequencing of cDNA clones isolated from a liver library from an estrogen-treated male Xenopus. Most eukaryotic mRNAs have poly(A) tracts several hundred residues in length, and recent studies have demonstrated that a change in the stability of a number of mRNAs correlates directly with the degree of polyadenylation. Albumin contrasts sharply with this, first because it has an exceptionally short poly(A) tail of 17 residues, and second because the degree of polyadenylation is totally unrelated to its destabilization in response to estrogen. These findings indicate that a unique pathway is involved in the regulation of albumin RNA stability by estrogen in Xenopus.

Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis.

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.

A simple modification of the estrogen receptor exchange assay: validation in nuclei from the rat uterus and a mouse mammary tumor.

Crude nuclear preparations from the rat uterus and the MXT mouse mammary tumor contain endogenous nucleases whose activity results in the loss of estrogen-binding sites from these fractions due to DNA breakdown. Although this is not a significant problem for studies in the uterus, the majority of the receptors present in nuclei from the tumor were lost from the particulate fraction under the standard conditions for the [3H]estradiol exchange assay. The addition of 5 mM Ca++ to either of these nuclear preparations resulted in the aggregation of the released estrogen receptors with particulate components and, hence, a total inhibition of receptor release into the soluble (wash) fractions. Ca++ had no effect on the affinity of the receptors for [3H]estradiol, although in some cases it slowed the overall rate of binding. The addition of Ca++ to the nuclear estrogen exchange assay may therefore prove to be a simple yet powerful aide in studies on the potential hormonal responsiveness of human and experimental mammary tumors.

Differential induction of hepatic estrogen receptor and vitellogenin gene transcription in Xenopus laevis.

Low levels of estrogen receptor (200-500 per cell) are present in the liver of hormonally naive male Xenopus. However, administration of estradiol results in a rapid 2- to 5-fold increase in cellular estrogen receptor content concurrent with the de novo transcriptional activation of the genes for the yolk protein precursor vitellogenin. Studies on Xenopus embryogenesis suggest that estrogen receptor induction is required for the activation of vitellogenin transcription. The purpose of the present study was to examine the mechanism of estrogen receptor induction in male Xenopus liver. The experimental protocol used 4-hydroxytamoxifen, an antiestrogen with a high affinity for the estrogen receptor, to inhibit the effects of estradiol. Changes in estrogen receptor content were then determined through the use of an exchange assay. 4-Hydroxytamoxifen alone suppressed the level of estrogen receptor from 800 sites per cell in hormonally naive animals to 250 sites per cell. Administration of estradiol 24 h after the antiestrogen resulted in the induction of estrogen receptor to a level equivalent to that found in control animals (800 sites per cell). However, under the same conditions, estradiol was unable to overcome the antiestrogen inhibition of vitellogenin gene transcription. Although 4-hydroxytamoxifen displayed a high affinity for the hepatic estrogen receptor, it did not inhibit the binding of estradiol to a middle affinity cytoplasmic estrogen-binding protein. These results suggest that different mechanisms are involved in the induction of estrogen receptor and vitellogenin gene transcription.

Identification and characterization of a cDNA encoding ribosomal protein S12 from Xenopus laevis.

The complete nucleotide (nt) sequence of the cDNA clone XL-S12, encoding a Xenopus laevis (Xl) homologue of the mammalian ribosomal protein S12, has been determined. The sequence predicts a Xl S12 protein of 132 amino acids (aa) with a molecular mass of 14.7 kDa. Xl S12 shares 95 and 97% aa sequence identity with the human and murine S12 proteins, respectively. Analysis of nt substitution patterns and rates indicates that S12 is a very highly constrained protein, evolving at an estimated rate of only 0.03 x 10(-9) non-synonymous (protein-altering) substitutions per site per year.

Identification of a novel member of the pentraxin family in Xenopus laevis.

Pentraxins are a family of acute phase reactants. Two family members, C-reactive protein (CRP) and serum amyloid P component (SAP), are known in a range of mammalian species. CRP and SAP are both about 200 residues long, and arose from a gene duplication event, apparently before the divergence of the mammalian orders. To elucidate the origins of mammalian pentraxins, we have searched for pentraxin-coding genes in the amphibian Xenopus laevis. We have identified a gene determining a protein (XL-PXN1) which is about twice the size expected: the XL-PXN1 gene appears to be a fusion between regions encoding an amino-terminal peptide of unknown function and a carboxy-terminal pentraxin. The pentraxin domain is more divergent from CRP and SAP than they are from each other: it provides an outgroup for analysis of the evolution of mammalian pentraxins and confirms that putative CRP and SAP proteins partly characterized in non-vertebrate species cannot be true homologues of the mammalian proteins.

Impact of halogenated compounds on calcium homeostasis in hepatocytes.

Halocarbons (CCl4, 1,1-dichlorethylene) cause a wide spectrum of effects and injury in hepatocytes. One early effect of these compounds is the inhibition and destruction of the endoplasmic reticulum (ER) calcium pump. Subsequent to inhibition of this pump, the ER calcium pool is depleted and cytosolic levels of calcium are increased for a prolonged period of time. This effect of halocarbons has been characterized and is similar in vivo and in vitro. The importance of this redistribution of cell calcium in expression of halocarbon injury of hepatocytes has not been fully resolved. Several degradative enzymes (phospholipases, proteases) have been implicated as calcium-dependent mediators in toxicity. Our preliminary studies of the effect of calcium redistribution suggest that activation of a calcium-sensitive endonuclease in liver does not play a central role in initiating the lethal effect of halocarbons on hepatocytes.

Transcriptional and post-transcriptional inhibition of albumin gene expression by estrogen in Xenopus liver.

The purpose of this study was to investigate the suppression of albumin mRNA by estrogen in Xenopus liver. A single dose of estradiol rapidly suppressed albumin mRNA to 30% of the control level. Albumin mRNA remained at this new steady-state level for 9 days, after which it returned to the control level. Transcription 'run-on' experiments in isolated liver nuclei demonstrated a transient decrease of 60-90% in albumin transcription after 2-6 h and approached constitutive transcription by 12 h. Albumin gene transcription then remained constant for the following 12 days. Prolonged and enhanced suppression of albumin mRNA was observed in animals treated repeatedly with estrogen for 12 days. In these animals, albumin gene transcription was decreased 80-90% from the constitutive control level. These data indicate that albumin mRNA is suppressed by both transcriptional and post-transcriptional mechanisms.

Differential regulation and polyadenylation of transferrin mRNA in Xenopus liver and oviduct.

Estrogen destabilizes transferrin mRNA in male Xenopus liver in the same manner as observed for albumin and gamma-fibrinogen. The present study examined estrogen regulation of transferrin gene expression in female Xenopus liver and oviduct. In female Xenopus liver estrogen causes the same enhanced degradation of transferrin mRNA from the cytoplasm as seen in males. In contrast, transferrin is induced 3- to 4-fold in both oviduct nuclear and cytoplasmic RNA. The similar increase in transferrin RNA in both preparations suggests a transcriptional mechanism is responsible for this stimulation. Therefore, transferrin expression is differentially regulated in these tissues by the same hormone. Previous experiments showed that Xenopus serum albumin mRNA has a very short (17 residue) poly(A) tail that may play a role in its hormone-regulated instability. Transferrin mRNA has a similarly short poly(A) tail in liver of both male and female Xenopus. Estrogen has no effect on transferrin polyadenylation in liver. Similarly short poly(A) is found on transferrin mRNA from estrogen-deprived oviducts in explant culture. However, addition of estradiol to the medium results in the appearance of a 50-200 nucleotide poly(A) concurrent with induction. Therefore, transferrin mRNA is differentially polyadenylated in Xenopus liver and oviduct. In the latter tissue polyadenylation is under hormonal control.

The Xenopus laevis homologue of the 64-kDa subunit of cleavage stimulation factor.

Cleavage stimulation factor (CstF) is composed of three subunits of 50, 64 and 77 kDa, respectively. We report here the identification of a cDNA clone from Xenopus laevis encoding a homologue of the 64-kDa subunit of human CstF. Comparative sequence analysis reveals that these two proteins are highly conserved with the exception of a unique repeat structure found in the human, but not in the X. laevis, protein. Analysis of expression of this mRNA during X. laevis tadpole development indicates a requirement for this protein throughout all stages of development.

Albumin is encoded by 2 messenger RNAs in Xenopus laevis.

A cDNA clone library was prepared from liver poly(A) RNA pf non-estrogenized Xenopus laevis. Albumin coding sequences were screened by hybridization to a cDNA prepared from poly(A) RNA enriched by sucrose density gradient centrifugation, and by a sensitive solid-phase radioimmunoassay to detect clones that contain templates for albumin antigenic determinants. Nine clones were obtained by this approach, and all but one have the cDNA inserted in phase with the beta-lactamase gene of pBR322. Mapping of these clones with restriction endonucleases yielded 2 distinct patterns, suggestive of heterogeneity in the coding sequences. This was confirmed by heteroduplex analyses of hybrids formed between clones representative of each of the 2 classes. Both classes of albumin cDNA clones were used to select mRNAs of the same size (2.3kb) that code for peptides that are indistinguishable by SDS gel electrophoresis. Examination of the organization of the albumin genes by blot hybridization of the cDNA clones to restriction fragments of Xenopus DNA failed to detect any differences at the genomic level. The considerable diversity of the albumin cDNAs is suggestive of a multiplicity of albumin genes, rather than differential processing of a common precursor RNA.

The nuclease that selectively degrades albumin mRNA in vitro associates with Xenopus liver polysomes through the 80S ribosome complex.

A ribonuclease activity that has characteristics expected for an enzyme that catalyzes the regulated destabilization of serum protein-coding mRNAs following estrogen administration was previously identified on Xenopus liver polysomes. This enzyme activity is estrogen inducible and selectively degrades mRNAs (e.g., albumin, gamma-fibrinogen) that are unstable following estrogen administration to male frogs. This paper reports on the relationship between this enzyme activity and the association of 40S and 60S ribosomal subunits. Ribonuclease activity (as defined by the generation of a specific cleavage fragment from albumin RNA) is found in polysome fractions that contain the majority of the liver mRNA. This activity sediments on sucrose gradients with the large polysome complexes observed in liver of vitellogenic animals. EDTA treatment generates 40S and 60S ribosome subunits and a significant amount of 80S ribosome monomers. Under these conditions, polysomal ribonuclease activity is found both free in solution and with the 80S material. Puromycin treatment generates predominantly 40S and 60S ribosomal subunits. Polysomal ribonuclease activity is found only in solution following puromycin treatment. These data indicate that the Xenopus liver polysomal nuclease requires the association of both ribosomal subunits for complex formation with polysomes. The polysomal nuclease behaves as a basic protein on Mono Q chromatography, with the fractionated material retaining the same differential activity toward albumin versus ferritin mRNA.

Identification of in vivo mRNA decay intermediates corresponding to sites of in vitro cleavage by polysomal ribonuclease 1.

Previous work from this laboratory identified a polysome-associated endonuclease whose activation by estrogen correlates with the coordinate destabilization of serum protein mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a novel member of the peroxidase gene family. A characteristic feature of PMR-1 is its ability to generate in vitro degradation intermediates by cleaving within overlapping APyrUGA elements in the 5'-coding region of albumin mRNA. The current study sought to determine whether the in vivo destabilization of albumin mRNA following estrogen administration involves the generation of decay intermediates that could be identified as products of PMR-1 cleavage. A sensitive ligation-mediated polymerase chain reaction technique was developed to identify labile decay intermediates, and its validity in identifying PMR-1-generated decay intermediates of albumin mRNA was confirmed by primer extension experiments performed with liver RNA that was isolated from estrogen-treated frogs or digested in vitro with the purified endonuclease. Ligation-mediated polymerase chain reaction was also used to identify decay intermediates from the 3'-end of albumin mRNA, and as a final proof of principle it was employed to identify in vivo decay intermediates of the c-myc coding region instability determinant corresponding to sites of in vitro cleavage by a polysome-associated endonuclease.

Characterization of mRNA endonucleases.

Endonucleases are key effectors of mRNA degradation, particularly for mRNAs whose turnover rates are regulated by extracellular stimuli. The rapid clearance of mRNA degradation products in vivo and the need to selectively identify mRNA endonucleases in the presence of many other cellular ribonucleases make the study of these enzymes particularly challenging. We have successfully purified and cloned one such enzyme, termed polysomal RNase 1, or PMR-1. Presented here are protocols either developed in our laboratory or adapted from the work of others that we have used successfully in characterizing PMR-1. We first describe methods to determine whether a particular mRNA is degraded in vivo through an endonuclease-initiated mechanism, and then present approaches for developing an in vitro mRNA degradation system. Next we describe experiments one should perform to optimize reaction conditions, determine cofactor requirements for an endonuclease, map in vitro cleavage sites, and characterize endonucleolytic cleavage products. Finally we describe kinetic parameters one should evaluate in characterizing the enzymology of mRNA endonucleases, with particular concern focused on the relative selectivity of these enzymes for cleavage at preferred sites within target mRNAs.

Nuclear association states of rat uterine oestrogen receptors as probed by nuclease digestion.

The solubilization of oestrogen receptors from uterine nuclei by micrococcal nuclease and deoxyribonuclease I was examined after the injection of oestradiol or Nafoxidine into castrated female rats. At 1h after an injection of oestradiol, 30% (0.18pmol/mg of DNA) of the nuclear oestrogen receptors was solubilized by 5 min of mild digestion with either nuclease. No further receptor release occurred, although DNA hydrolysis continued throughout a 20min interval. The limitation in receptor solubilization was not due to an artifact of digestion conditions or insufficient nuclease concentrations. Similar patterns of receptor solubilization and DNA hydrolysis were obtained with both nucleases whether the animals had been injected with oestradiol 1h before death or if the uteri from uninjected animals were incubated with [(3)H]oestradiol for 1h in vitro. When uterine nuclei were digested with these enzymes 12h after the animal was injected with oestradiol there was little change in the quantity of nuclease-sensitive sites (0.11pmol/mg of DNA); however, the quantity of nuclease-resistant sites decreased 10-fold. These values correspond quantitatively to the changes in salt-resistant and salt-extractable sites observed over a 12h interval after oestradiol treatment. Nuclease digestion of uterine nuclei obtained 16h after Nafoxidine treatment gave a pattern qualitatively and quantitatively similar to that observed 1h after oestradiol treatment, a result consistent with the agonist/antagonist action of this compound. An analysis by sucrose-density-gradient centrifugation of the time course of nuclease-dependent receptor solubilization indicated that the solubilized receptors were not associated with discrete nucleosomal fragments. We believe that these data indicate that only a portion of the receptors translocated to the nucleus become associated with chromatin, and this association may occur on regions of chromatin that are preferentially susceptible to nucleolytic cleavage.