Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element.

The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3' splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5' pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an approximately 62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.

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.

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.

Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3'-untranslated region by the mRNA endonuclease polysomal ribonuclease 1.

In Xenopus, estrogen induces the stabilization of vitellogenin mRNA and the destabilization of albumin mRNA. These processes correlate with increased polysomal activity of a sequence-selective mRNA endonuclease, PMR-1, and a hnRNP K homology-domain RNA-binding protein, vigilin. Vigilin binds to a region of the vitellogenin mRNA 3'-untranslated region (3'-UTR) implicated in estrogen-mediated stabilization. The vigilin-binding site in the vitellogenin B1 mRNA 3'-UTR contains two consensus PMR-1 cleavage sites. The availability of purified PMR-1 and recombinant vigilin made it possible to test the hypothesis that RNA-binding proteins interact with cis-acting elements to stabilize target mRNAs by blocking cleavage by site-specific mRNA endonucleases. Vigilin binds to the vitellogenin mRNA 3'-UTR site with at least 30-fold higher affinity than it exhibits for the albumin mRNA segment containing the mapped PMR-1 cleavage sites. This differential binding affinity correlates with differential in vitro susceptibility of the protein-RNA complexes to cleavage by PMR-1. Whereas recombinant vigilin has no detectable protective effect on PMR-1 cleavage of albumin mRNA, it retards in vitro cleavage of the vitellogenin mRNA 3'-UTR by purified PMR-1. The PMR-1 sites in the vitellogenin mRNA 3'-UTR are functional because they are readily cleaved in vitro by purified PMR-1. These results provide direct evidence for differential susceptibility to endonuclease-mediated mRNA decay resulting from the differential affinity of a RNA-binding protein for cis-acting stability determinants.

The poly(A)-limiting element is a conserved cis-acting sequence that regulates poly(A) tail length on nuclear pre-mRNAs.

Most vertebrate mRNAs exit the nucleus with a 200+-residue poly(A) tail and are deadenylated to yield heterogeneous polymers of 50-200 adenosine residues on any given mRNA. We previously reported that Xenopus albumin mRNA and pre-mRNA have an unusually short, discrete 17-residue poly(A) tail and showed that regulation of poly(A) length is controlled independently by two cis-acting poly(A)-limiting elements (PLE A and PLE B) located in the terminal exon. The present study sought to determine the generality of this regulatory mechanism. Transferrin mRNA also has a discrete <20-nt poly(A) tail, and deletion mapping experiments identified an element homologous to the albumin gene PLE B within the terminal exon of the transferrin gene that conferred poly(A) length regulation on a globin reporter mRNA. Based on this similarity the PLE B sequence was used in a database search to identify candidate mRNA targets for regulated polyadenylation. Of the several hundred sequences identified in this manner we focused on HIV-EP2/Schnurri-2, a member of a family of genes encoding related zinc finger transcription factors. A striking feature of the PLE-like element in these genes is its location 10-33 bp upstream of the translation stop codon. We demonstrate that HIV-EP2 mRNA has a <20-nt poly(A) tail, for which the identified PLE-like sequence is responsible. These results indicate that the presence of a PLE can predict mRNAs with <20-nt poly(A) tails, and that nuclear regulation of poly(A) tail length is a feature of many mRNAs.

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.

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family.

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.

Identification of two cis-acting elements that independently regulate the length of poly(A) on Xenopus albumin pre-mRNA.

Unlike most eukaryotic mRNAs studied to date, Xenopus serum albumin mRNA has a short (17-residue), discrete poly(A) tail. We recently reported that this short poly(A) tail results from regulation of the length of poly(A) on albumin pre-mRNA. The purpose of the present study was to locate the cis-acting element responsible for this, the poly(A)-limiting element or PLE. An albumin minigene consisting of albumin cDNA joined in exon 13 to the 3' end of the albumin gene produced mRNA with <20 nt poly(A) when transfected into mouse fibroblasts. This result indicates both that cis-acting sequences that regulate poly(A) length are within this construct, and that nuclear regulation of poly(A) length is conserved between vertebrates. Poly(A) length regulation was retained after replacing the terminal 53 bp and 3' flanking region of the albumin gene with a synthetic polyadenylation element (SPA). Conversely, fusing albumin gene sequence spanning the terminal 53 bp of the albumin gene and 3' flanking sequence onto the human beta-globin gene yielded globin mRNA with a 200-residue poly(A)tail. These data indicate that the PLE resides upstream of the sequence elements involved in albumin pre-mRNA 3' processing. Poly(A) length regulation was restored upon fusing a segment bearing albumin intron 14, exon 15, and 3' flanking sequence onto the beta-globin gene. We demonstrate that exon 15 contains two PLEs that can act independently to regulate the length of poly(A).

Cleavage properties of an estrogen-regulated polysomal ribonuclease involved in the destabilization of albumin mRNA.

Previous work from this laboratory [Dompenciel,R.E., Garnepudi,V.R. and Schoenberg,D.R. (1995)J. Biol. Chem.270, 6108-6118] described the purification and properties of an estrogen-regulated endonuclease isolated from Xenopus liver polysomes that is involved in the destabilization of albumin mRNA. The present study mapped cleavages made by this enzyme onto the secondary structure of the portion of albumin mRNA bearing the major cleavage sites. The predominant cleavages occur in the overlapping APyrUGA sequence AUUGACUGA present in a single-stranded loop region, and in AUUGA located within a bulged AU-rich stem. A structural mutation which converted the major loop cleavage site to a hairpin bearing one APyrUGA element eliminated cleavage at the intact site. This confirms that the polysomal RNase is specific for single-stranded RNA. Additional point mutations in the major loop characterized the nucleoside sequence requirements for cleavage. Finally, snake venom exonuclease was used to demonstrate the polysomal RNase generates products with a 3' hydroxyl. Binding of an estrogen-induced protein to a portion of the 3'UTR of vitellogenin mRNA may be involved in its stabilization by estrogen [Dodson,R.E. and Shapiro,D.J. (1994)Mol. Cell. Biol.14, 3130-3138]. The core binding site for this protein bears the sequence APyrUGA, suggesting that stabilization may be accomplished by occlusion of a cleavage site for the polysomal RNase.

Regulated nuclear polyadenylation of Xenopus albumin pre-mRNA.

Cytoplasmic regulation of the length of poly(A) on mRNA is a well-characterized process involved in translational control during development. In contrast, there is no direct in vivo evidence for regulation of the length of poly(A) added during nuclear pre-mRNA processing in somatic cells. We previously reported that Xenopus serum albumin [Schoenberg et al. (1989) Mol. Endocrinol. 3, 805-815] and transferrin [Pastori et al. (1992) J. Steroid Biochem. Mol. Biol. 42, 649-657], mRNA have exceptionally short poly(A) tails ranging from 12 to 17 residues, whereas vitellogenin mRNA has long poly(A). An RT-PCR protocol was adapted to determine the length of poly(A) added onto pre-mRNA, defined here as that species bearing the terminal intron. Using this assay we show that vitellogenin pre-mRNA has the same long poly(A) tail as mature vitellogenin mRNA. In contrast, albumin pre-mRNA has the same short poly(A) as found on fully-processed albumin mRNA. These results indicate that the short poly(A) tail on albumin mRNA results from regulation of poly(A) addition during nuclear 3' processing.

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.

Purification and characterization of an estrogen-regulated Xenopus liver polysomal nuclease involved in the selective destabilization of albumin mRNA.

A previous report from this laboratory described an estrogen-regulated endoribonuclease activity on Xenopus liver polysomes which had properties one might expect for a messenger ribonuclease involved in the regulated destabilization of albumin mRNA (Pastori, R. L., Moskaitis, J. E., and Schoenberg, D. R. (1991) Biochemistry 30, 10490-10498). This report describes the purification and properties of this ribonuclease. The purified nuclease fraction contained a doublet of 62 and 64 kDa and a small amount of a 40-kDa peptide. In situ analysis on both denaturing and nondenaturing gels using an albumin transcript as substrate showed all three proteins possess nuclease activity. Peptide mapping and Western blot with a polyclonal antiserum showed the 62- and 64-kDa peptides to be isoforms, and the 40-kDa peptide to be a degradation product of the larger species. Two-dimensional gel electrophoresis further separated the 62- and 64-kDa species into three pairs of proteins, with isoelectric points of 9.6, 9.8, and 9.8. The purified ribonuclease rapidly degraded a full-length albumin transcript, yet had no effect on either a full-length albumin antisense transcript or full-length ferritin transcript. A number of properties of the purified nuclease were characterized, including the effects of salt, divalent cations, EDTA, sulfhydryl reagents, and temperature. Treatment of the polysomal nuclease with micrococcal nuclease had no effect, indicating that this enzyme does not require an RNA cofactor for activity. Finally, primer extension mapped the major cleavage site to an overlapping repeated sequence APyrUGA, with cleavage between and adjacent to the two pyrimidine residues generating fragments with 5'-hydroxyls.

S-adenosyl-L-homocysteine hydrolase from Xenopus laevis--identification, developmental expression and evolution.

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is an important enzyme in the trans-sulphuration pathway, mediating the conversion of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. We have identified a cDNA clone from Xenopus laevis, encoding a protein of 433 aa, which is highly conserved with S-Adenosyl-L-homocysteine hydrolases (Adohcyases) from other species. Expression of Adohcyase mRNA in X.laevis tadpoles is detectable from developmental Stage 27 onwards. Phylogenetic analysis of available Adohcyase sequences indicates that species cluster essentially as predicted from morphological data. Furthermore, we estimate that S-adenosyl-L-homocysteine hydrolase is evolving very slowly, almost 10 times slower than the average rate.

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.

Human immunodeficiency virus type 1 Rev is required in vivo for binding of poly(A)-binding protein to Rev-dependent RNAs.

In the absence of Rev or the Rev-responsive element, the Rev-dependent human immunodeficiency virus type 1 (HIV-1) RNAs do not behave as mRNAs; rather, they exhibit nuclear defects in splicing and/or nuclear export and cytoplasmic defects in stability and translation. A translational initiation factor, eIF-5A, has recently been shown to bind specifically to the Rev activation domain. As the binding of poly(A)-binding protein 1 (PAB1) to the poly(A) tail of mRNAs is involved in both the stability and translation of cytoplasmic mRNAs, we investigated whether Rev might influence the association of PAB1 with cytoplasmic HIV-1 RNAs. Antibodies were generated against PAB1. We used these antibodies in an immunoprecipitation assay to detect specific binding of PAB1 to cytoplasmic mRNAs. We found that in the presence of Rev, PAB1 was associated with Rev-dependent and Rev-independent RNAs in the cytoplasm of transfected cells. However, in the absence of functional Rev, we found little or no PAB1 associated with Rev-dependent RNAs. These RNAs were capable of binding PAB1 in vitro. These results demonstrate that HIV-1 RNAs are defective in PAB1 association in the absence of Rev.

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 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.

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.

Estrogen-induced ribonuclease activity in Xenopus liver.

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.