Estrogen administration to male Xenopus causes the cytoplasmic destabilization of the hepatic serum protein coding mRNAs, most notably, albumin, yet has little effect on mRNAs encoding intracellular proteins such as ferritin. This report describes an estrogen-inducible ribonuclease activity found in liver polysomes that degrades albumin mRNA 4 times faster in vitro than it degrades ferritin mRNA. This differential rate of degradation was observed upon incubation of polysome extract with free liver RNA, isolated liver mRNPs, or transcripts from plasmid vectors. A cleavage fragment consisting of a doublet of approximately 194 nucleotides in length was consistently observed upon digestion of transcripts for the full length or 5' half of albumin mRNA. The generation of this cleavage fragment was used as an assay to study properties of the polysome nuclease activity. The 194 doublet is produced by the action of a Mg(2+)-independent endonuclease. This distinguishes the Xenopus liver enzyme from the enzymes that degrade histone or c-myc mRNA in vitro. It is inactivated by 400 mM NaCl or heating at 90 degrees C, but not by placental ribonuclease inhibitor or N-ethylmaleimide. Finally, the polysomal nuclease activity does not degrade double-stranded RNA. We believe the estrogen-induced nuclease activity contains an enzyme(s) that may mediate hormone-regulated changes in mRNA stability in this tissue.

The Xenopus laevis 68-kd and 74-kd albumin amino acid sequences are examined with respect to their relationship to the other known members of the albumin/alpha-fetoprotein/vitamin D-binding protein gene family. Each of the three members of this family presents a unique pattern of conserved regions indicating a differential selective pressure related to specific functional characteristics. Furthermore, an evolutionary tree of these genes was deduced from the divergence times calculated from direct nucleotide sequence comparisons of individual gene pairs. These calculations indicate that the vitamin D-binding protein/albumin separation occurred 560-600 million years (Myr) ago and the albumin/alpha-fetoprotein divergence 280 Myr ago. This observation leads to the hypothesis according to which the albumin/alpha-fetoprotein gene duplication occurred shortly after the amphibian/reptile separation. Consequently, and unlike mammals, amphibians and fishes should lack an alpha-fetoprotein in their serum at larval stages, which is consistent with a recent analysis of serum proteins in Xenopus laevis larvae. This hypothesis now will have to be tested further in additional lower vertebrates.

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.

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.

The three proteins principally involved in the regulation of iron metabolism are transferrin, the transferrin receptor and ferritin. Transferrin is the principal iron transport protein present in vertebrate plasma. It consists of a single polypeptide chain of approximately 80 kDa with two iron binding domains (1). We have isolated a cDNA encoding the complete sequence of transferrin mRNA from a library prepared from male Xenopus laevis liver. The sequence of Xenopus transferrin mRNA and its derived peptide sequence are presented below. Xenopus transferrin is similar in size to the corresponding molecules from human (1) and chicken (2) and shares the same repeated domain structure. Human and chicken transferrin have 2 and 3 sites of N-linked gylcosylation, respectively. The glycosylation sites are located in the C-terminal half of these proteins. There are no sites for N-linked gylcosylation in Xenopus transferrin. Xenopus transferrin has 47% and 46% amino acid identity with the chicken EMBL accession no. X54530 and human proteins, and 63% similarity to both. This degree of homology is significantly less than that found for Xenopus and human ferritin, which share 68% amino acid identity and 84.6% overall similarity (3). ACKNOWLEDGEMENTS

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.

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Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing 125I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.