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Endocrinology Vol. 139, No. 3 894-904
Copyright © 1998 by The Endocrine Society

ARTICLES

Identification of a Novel cis-Element in the 3'-Untranslated Region of Mammalian Peptidylglycine {alpha}-Amidating Monooxygenase Messenger Ribonucleic Acid

Sandrine Fraboulet, Françoise Boudouresque, Christine Delfino and L’Houcine Ouafik

INSERM U-297, Faculté de Médecine Nord, IFR Jean Roche, 13916 Marseille, France

Address all correspondence and requests for reprints to: Dr. L’Houcine Ouafik, INSERM U-297, Faculté de Médecine Nord, IFR Jean Roche, boulevard Pierre Dramard, 13916 Marseille Cedex 20, France. E-mail: ouafik.h{at}jean-roche.univ-mrs.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptidylglycine {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal {alpha}-amidation of peptidylglycine substrates, yielding amidated products. Growing evidence suggests that the metabolism of PAM messenger RNAs (mRNAs) can be regulated within the cytoplasm. To understand the mechanisms controlling the metabolism of PAM mRNAs, we sought to identify cis elements of the 3'-untranslated region (3'-UTR) of PAM mRNA that are recognized by cytoplasmic factors. From gel retardation assays, one sequence element is shown to form a specific RNA-protein complex. The protein-binding site of the complex was determined by ribonuclease T1 mapping, by blocking the putative binding site with antisense oligonucleotide, and by competition assays. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 20-nucleotide cis element that interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-binding protein complex. The binding activity was redox sensitive. Tissue distribution of the protein in the rat showed a marked tissue-specific expression, with ovary, testis, lung, heart septum, anterior pituitary and hypothalamus containing large amounts compared with liver, ventricle, atrium, and neurointermediate lobe. No binding activity was detectable in pancreas, intestine, or kidney extracts. Northwestern blot analysis of AtT-20 (mouse corticotrope tumor cell line) cytoplasmic extracts revealed a protein of 46 kDa. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 3'-UTR of PAM mRNA from many animal species. Although these data suggest that cis element-binding activity could be a cytoplasmic regulator of PAM mRNA metabolism, the functional consequences of this binding remain to be determined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN NEURONS and endocrine cells, biologically active peptides are stored in secretory granules and undergo regulated release in response to external stimuli. En route through the secretory pathway, inactive propeptides are activated through the sequential action of specialized, neuroendocrine-specific, peptide-processing enzymes. These modifications include endoproteolysis, exoproteolysis, and, for over half of all known peptides, COOH-terminal {alpha}-amidation (1, 2, 3). Enzymatic {alpha}-amidation is a key step in the biosynthesis of these neuroendocrine peptides. The two-step {alpha}-amidation reaction is catalyzed by the bifunctional enzyme, peptidylglycine {alpha}-amidating monooxygenase (PAM; EC 1.14.17.3). The first enzyme, peptidylglycine {alpha}-hydroxylating monooxygenase catalyzes the formation of the peptidyl-{alpha}-hydroxyglycine intermediate in a process dependent on ascorbate, copper, and molecular oxygen. At physiological pH, peptidyl-{alpha}-hydroxyglycine {alpha}-amidating lyase subsequently catalyzes the formation of an {alpha}-amidated product. Both enzymes are derived from the PAM precursor protein (4, 5). Soluble and membrane-associated PAM activities have been identified, and their distribution is tissue specific (6, 7). Complementary cDNA (cDNA) clones encoding the 110-kDa bifunctional integral membrane protein form of PAM have been isolated from many tissues in several species (2, 8). Tissue-specific alternative splicing of the single copy PAM gene can generate at least seven forms of PAM messenger RNA (mRNA) in the rat (9, 10, 11).

PAM expression has been shown to be regulated in a tissue-specific fashion. Levels of PAM mRNA and PAM activity exhibited distinctly developmental profiles in atrium and ventricle (12). During development of the endocrine pancreas, levels of PAM mRNA and activity rise transiently before declining to the low levels observed in the adult rat (13, 14). In response to endocrine manipulations, we demonstrated that hypothyroidism and ovariectomy caused increases in PAM mRNA and activity levels in rat anterior pituitary gland (15, 16). This regulation often parallels the level of amidated peptides in vitro (17) and in vivo (15). Recently, we demonstrated that thyroid hormone regulation of PAM gene expression in anterior pituitary appears to be primarily posttranscriptional by altering the rate of PAM mRNA degradation in the cytoplasm (18). The elucidation of the mechanisms of regulation of PAM expression should bring new insights into the field of neuropeptide-processing enzyme regulation.

Many eukaryotic mRNAs contain regulatory elements that control their posttranscriptional utilization. These regulatory elements often reside within the 5'- and 3'-untranslated (5'- and 3'-UTR) regions of mRNAs and interact with specific cytoplasmic proteins that modulate the stability or translational competence of mRNAs. There are numerous reports demonstrating the pivotal role of the 3'-UTR in mRNA stability, transport, localization, and translation (19). A posttranscriptional role for the 3'-UTR mRNA sequence(s) and interacting protein(s) has been demonstrated in a number of systems (20; for review, see Ref.21). The degradation rate of many eukaryotic mRNAs appears to be regulated by factors binding to specific sequences critical for mRNA destabilization. Several cis-RNA destabilizing elements have been recently characterized in the 3'-UTR of various transcripts, including cytokines (granulocyte-macrophage colony-stimulating factor) (22), protooncogenes c-myc (23) and c-fos (24, 25), amyloid precursor (26), TSH ß-subunit (27), transferrin (28), and catalase mRNA (29).

Although it is now evident that thyroid hormones and estrogen affect PAM gene expression posttranscriptionally through altering message stability (16, 18, 30), the molecular mechanism of the cis-elements and trans-factors involved in these regulations is unknown. To further analyze the mechanism of PAM mRNA metabolism, we used a mRNA gel mobility shift assay. Labeled PAM RNAs corresponding to the 5'-UTR, 3'-UTR, and coding region were prepared and incubated with a cytoplasmic extracts. We focused on the 3'-UTR of the PAM mRNA, because it was the only region that bound protein(s) to form an RNA-protein complex. These data indicate that the cytoplasmic extract contains a factor(s) that interacts with the 3'-UTR of the PAM mRNA.

This study was intended to define the unique cis element in the 3'-UTR that leads to formation of the PAM mRNA-protein(s) complex. Using a mRNA gel mobility shift assay, we showed that a cytoplasmic protein binds specifically to a short segment of the PAM mRNA 3'-UTR. The core cis element is 20 bases in length, approximately 409 bases from the stop codon, and highly conserved among species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of tissue and cell extracts
Cells (GH4 and AtT20) from a confluent 175-cm2 flask were detached from plastic by incubation in 0.25% trypsin-EDTA (Life Technologies, Grand Island, NY) at 37 C. The dissociation was stopped by 10% FCS (Life Technologies), and cells were pelleted at 400 x g for 10 min. The cell pellet was washed with PBS without Ca2+ or Mg2+ and homogenized on ice in lysis buffer containing 10 mM HEPES (pH 7.5), 40 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol (DTT), 0.2% Nonidet P-40, 5% glycerol, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonylfluoride with a Teflon homogenizer (27). Nuclei and cell debris were removed from the lysate by centrifugation at 600 x g for 10 min at 4 C. The supernatant was centrifuged at 12,000 x g for 10 min, and the cytoplasmic extracts were frozen rapidly and stored at -70 C. Rat tissues were homogenized in 5 vol ice-cold buffer containing 15 mM HEPES (pH 8), 10% glycerol, 1 mM DTT, 20 mM phenylmethylsulfonylfluoride, 2 µg leupeptin/ml, 1 µg pepstatin/ml, 2 µg aprotinin/ml, and 16 µg benzamidine/ml. Homogenates were centrifuged at 12,000 x g for 15 min at 4 C. The supernatant was carefully removed, snap-frozen, and stored at -70 C until assay (31).

Protein concentrations were determined by using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) and BSA as standard.

Plasmid constructs
Plasmid PAM 3'-UTR was constructed by subcloning the 3'-UTR sequence of the rat PAM gene (3243–3976 bp) (11) into SmaI and EcoRI sites of the pBluescript SK-II (pBS SK-II, Fig. 7Go). The plasmid was linearized with EcoRI for transcription with T3 RNA polymerase to produce a 783-nucleotide (nt) sense transcript including 59 nt of pBS SK-II and 724 bases of PAM 3'-UTR [S1 (3243–3967)]. S2 (3243–3848), S3 (3243–3707), S4 (3243–3614), and S5 (3243–3408) were obtained after linearization of PAM 3'-UTR cDNA template with TfiI, DraI, BanI, or HphI, respectively (Fig. 7Go).



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  		Figure 7. Summary of deletion analysis of the PAM 3'-UTR band shift complex. The thick line is a representation of a complete 724-nt PAM 3'-UTR. RNA probes were prepared that contain portions of the PAM 3'-UTR RNA and polylinker sequence from plasmid vector as indicated. Band-shift assays were performed with AtT-20 cytoplasmic extracts and each RNA. Results of band shifts with PAM RNA3243–3967 probe (S1) and unlabeled PAM RNA competitors are shown. The PAM RNA covering the entire 3'-UTR is 724 nt in length. The first and last nucleotides of each transcript are indicated.

 
Plasmid PAM {Delta}-3'-UTR contained the 3614–3967 fragment issued from PAM 3'-UTR plasmid digestion by BanI and EcoRI rendered blunt ended and subcloned into the EcoRV site of the pBS SK-II. The plasmid was linearized with HindIII for transcription with T3 RNA polymerase to produce a 429-nt transcript (S6(3614–3967)) including 76 nt of pBS SK-II and 353 bases of PAM 3'-UTR. S7 (3614–3848) and S8 (3614–3707) were obtained from further endonuclease digestions of PAM {Delta}-3'-UTR plasmid with TfiI and DraI, respectively (Fig. 7Go).

Alternatively, 3'-UTR PAM cDNA templates were prepared by PCR (5 min at 94 C, 35 sec at 50 C, and 50 sec at 72 C for 35 cycles) from the plasmid described above, using the following oligonucleotide primers: oligonucleotide H30 (PAM cDNA3675–3691; 5'-CGA-CGA-AAA-GCT-GCT-AA-3') and a 3'-primer BE21 (PAM cDNA3833–3817: 5'-CGC-TTC-CCA-CGC-GTC-GGG-AGC-GGA-C-3'). PCR-amplified fragment was subcloned into EcoRV-digested pBS SK-II to obtain the PAM {Delta}'-3'-UTR construct. The plasmid was linearized with HindIII for transcription with T3 RNA polymerase to produce a 185-nt transcript [S9 (3675–3806)] including 53 nt of pBS SK-II and 132 bases of PAM 3'-UTR. S10 (3675–3785), S11 (3675–3762), S12 (3675–3710), and S13 (3675–3743) were obtained after linearization of PAM {Delta}'-3'-UTR with DdeI, SwaI, DraI, and ApoI, respectively (see Fig. 8Go).



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  		Figure 8. Summary of deletion analysis of the PAM mRNA 3'-UTR3675–3806. RNA probes were prepared that contain portions of the PAM 3'-UTR RNA and polylinker sequences as indicated. Gel mobility shift assays were performed with AtT-20 cytoplasmic extracts and each RNA. PAM RNA3675–3806 (S9) is 132 nt in length. Results are presented as described in Fig. 8Go. The protein-binding site between nt 3710–3743 is shown by the hatched bar.

 
To generate S15 (3693–3762) and S14 (3693–3735) constructs, we used a 5'-primer that included a T7 RNA polymerase promoter sequence, which is underlined: ShT7 primer [5'-AAT-ACG-ACT-CAC-TAT-AGG-GTC-TCC-TTC-TAT-TTT-TTT-AA-3']. The 3'-primers Sh2 (PAM cDNA3762–3739; 5'-AAA-TAG-AGA-CTT-TTT-AAA-ATT-T-3') and Sh3 (PAM cDNA3735–3714; 5'-ATT-GCA-ATA-TAA-TGT-TAG-TGA-3') were used. PCR-amplified DNA fragments were excised after electrophoresis in low melting agarose, precipitated, and used for in vitro transcription (Fig. 8Go).

Preparation of the RNA transcripts
Radiolabeled PAM RNA fragments were produced by in vitro transcription from linearized templates corresponding to PAM cDNAs constructs obtained as described above. Transcription was performed at 37 C for 45 min in the presence of [{alpha}-32P]UTP (3000 Ci/mmol; ICN Biomedicals, Paris, France) as previously described (32) to produce transcripts with a specific activity of approximately 5 x 107 to 5 x 108 cpm/µg RNA. After transcription, cDNA templates were digested with 1 U RQ-1 deoxyribonuclease/ribonuclease (RNase) free (Promega, Lyon, France), extracted with phenol-chloroform, and precipitated with ethanol. [{alpha}-32P]UTP-labeled RNAs were quantified by liquid scintillation counting. Unlabeled RNA transcripts were synthesized as described above in the presence of 10 mM UTP, ethanol precipitated, resuspended in diethyl pyrocarbonate-treated water, and quantified by absorbance at 260 nm.

Band-shift assays
Band-shift assays were performed as previously described (31). Briefly, cytoplasmic lysate (2–10 µg) proteins were incubated with radiolabeled RNA (2.5 x 105 cpm) in 20 µl of a solution composed of 15 mM HEPES (pH 8), 5 µg yeast transfer RNA, 10 mM KCl, 10% glycerol, and 1 mM DTT at 30 C for 10 min before the addition of 20 U RNase T1 (Boehringer Mannheim, Mannheim, Germany). All experiments were repeated at least three times. For competition studies, unlabeled PAM control RNAs were added 10 min before radiolabeled RNA to cytosolic lysate in the buffer described above. After a 45-min incubation with RNase T1 at 37 C, 4 µl of 6 x native gel loading buffer (30% glycerol, 0.025% bromophenol blue, and 0.025% xylene cyanol) were added, and the RNA-protein complexes were resolved on a 5% polyacrylamide gel. After RNase T1 digestion, UV cross-linking studies were performed by exposing the reaction mixtures on ice for 10 min to 254 nm UV light in a Stratalinker (Stratagene, La Jolla, CA) on automatic settings. Subsequently, the cross-linked samples were either resolved directly on 8% SDS-PAGE gels or previously boiled for 5 min in 1 x SDS gel loading buffer (33). Gels were dried and exposed to x-ray film for 6–15 h with an intensifying screen at -70 C.

In some competition experiments, sense or antisense oligonucleotides were mixed with the radiolabeled RNA in an equimolar ratio, heated at 80 C for 10 min, and left to cool at room temperature for more than 30 min before adding the cytosolic extracts.

Northwestern blot analysis
Cytosolic proteins (25 µg) were fractionated on 10% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes by electroblotting (Biometra, Göttingen, Germany) and renatured as previously described (34) in buffer A containing 10 mM HEPES (pH 7.9), 40 mM KCl, 3 mM MgCl2, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 0.2% Nonidet P-40, and 5 mg/ml BSA (Boehringer Mannheim). Hybridization was carried out essentially as described by Nakagawa et al. in a buffer containing 10 mM HEPES (pH 7.9), 150 mM KCl, 5 mM MgCl2, 0.2 mM DTT, 8% glycerol, and 50 µg/ml yeast transfer RNA in the presence of radiolabeled transcripts corresponding to the 3'-UTR of PAM mRNA (106 cpm/ml). After 2-h incubation at room temperature, unbound RNA were removed by RNase T1 digestion (25 U/ml) for 15 min at 37 C. Unbound RNA probes were washed rapidly at room temperature in 20 mM HEPES (pH 7.9), 40 mM KCl, and 1.5 mM MgCl2. The blot was exposed for 24–72 h with an intensifying screen at -70 C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding of a cytoplasmic protein(s) to the 3'-UTR of PAM mRNA
To determine whether cytoplasmic extracts might contain a protein(s) that binds to the 3'-UTR of PAM mRNA, band-shift assays were performed. PAM expression has been extensively investigated in the AtT-20 mouse tumor corticotropic cell line (3, 17). RNA band-shift analysis of the 3'-UTR of PAM RNA starting 18 nt downstream of the UAG translational stop codon (nucleotide 3225) and extending to the end of the 3'-UTR (nucleotide 3967) is shown in Fig. 1Go. The band-shift RNA-containing complexes were separated on a 5% native polyacrylamide gels and visualized by autoradiography. Treatment of the probe with RNase T1 reduced all of the free probe to a collection of smaller RNAs (Fig. 1Go, lane 1). Incubation of the RNA probe with AtT-20 cytoplasmic extracts (2 µg protein) resulted in most of the probe migrating as large smears (Fig. 1Go, lanes 2 and 3).



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  		Figure 1. Formation of specific RNA-protein(s) complexes between the 3'-UTR of PAM mRNA and AtT-20 cytoplasmic extracts. 32P-Labeled-PAM RNA3243–3967 (2.5 x 105 cpm) was incubated at 30 C with AtT-20 cytoplasmic extracts (2 µg) for 10 min before RNase T1 digestion. The binding mixtures were analyzed by electrophoresis on a 5% native polyacrylamide gel. No RNA-protein complex formation can be observed when cytoplasmic lysates were omitted (lane 1). Labeled RNA was incubated with cytoplasmic lysates alone (lanes 2 and 3) or in the presence of increasing amounts of either unlabeled PAM 3'-UTR transcript (lanes 4 and 5; specific competitor; 10- and 100-fold molar excesses, respectively) or unlabeled polylinker sequence (pBS SK-II) RNA (lane 6; nonspecific competitor) as indicated.

 
To confirm that the binding was specific, competition studies with unlabeled RNA were performed. Preincubation of AtT-20 cytoplasmic extracts with 10 and 100 molar excesses of unlabeled cross RNA (Fig. 1Go, lanes 4 and 5, respectively) resulted in nearly complete elimination of the activity, as detected with a radiolabeled PAM RNA3243–3967 probe, whereas preincubation of the extracts with equivalent amounts of unlabeled pBS SK-II competitor RNA did not deplete the activity (Fig. 1Go, lane 6). These data demonstrate that AtT-20 cytoplasmic extracts bind to PAM 3'-UTR to form a RNA-protein complex.

Characterization of PAM 3'-UTR-binding protein(s) (PAM mRNA-BP)
UV cross-linking. To determine the molecular mass of the protein(s) binding specifically to the 3'-UTR of PAM mRNA and characterize the composition of the RNA-protein complex, UV light was used to covalently cross-link radiolabeled PAM 3'-UTR RNA to proteins of AtT-20 cytoplasmic extracts (35). Figure 2Go illustrates the apparent migration of the RNA-protein complex on SDS-polyacrylamide gel (36). Two of the irradiated samples were retained for RNA band-shift analysis (Fig. 2Go, lanes 2 and 3), whereas the remaining samples were boiled in Laemmli buffer (36) (Fig. 2Go, lanes 4 and 5). A single stable complex migrating with a molecular mass of 60 kDa was observed (Fig. 2Go), providing evidence for the existence of a single subunit in the PAM mRNA-BP complex. In the absence of lysate, RNase T1 cleaved radiolabeled PAM 3'-UTR, and no complex was observed (lane 1). No bands were detected in the absence of UV treatment (data not shown).



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  		Figure 2. Identification of protein interacting with labeled PAM mRNA 3'-UTR. RNA-protein binding reactions using 32P-labeled RNA were carried out as described in Fig. 1Go. After incubation with RNase T1, 32P-labeled RNA-protein complexes were UV cross-linked as described in Materials and Methods. The cross-linked products were directly resolved by SDS-PAGE. In the absence of cytoplasmic lysates, no RNA-protein complex can be seen (lane 1). Band-shift assays performed in the presence of cytoplasmic extracts without (lanes 2 and 3) or with (lanes 4 and 5) SDS sample buffer treatment showed a complex at 60 kDa indicated by the arrow. Prestained molecular mass standards (Rainbow marker, Amersham, Les Ulis, France) were used as markers.

 
To determine whether the binding factor is a protein, extracts were digested with proteinase K (100 µg/ml), trypsin (0.5 µg/µg proteins), and pronase (100 µg/ml), respectively, for 15 min at 37 C. Without protease treatment, the mixture RNA probe and extracts showed a shifted band at 60 kDa (Fig. 3AGo, lane 1). Digestion with proteases completely abolished the shifted band (Fig. 3AGo, lanes 2–4). Band-shifted complexes were observed with AtT-20 cytoplasmic extracts that were preheated at 70 C for 10 min before the addition of radiolabeled PAM RNA3243–3967 (Fig. 3BGo, lane 3). Preheating of lysates at 100 C for 10 min prevented RNA-protein complex formation (Fig. 3BGo, lane 4). These data demonstrate that PAM 3'-UTR RNA forms a complex with a heat-sensitive cytosolic factor(s) that is most likely a protein(s).



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  		Figure 3. Characterization of the PAM mRNA-BP. Band-shift assays were performed as indicated in Fig. 1Go, using radiolabeled RNA3243–3967 (S1; 2.5 x 105 cpm). A, Without proteases treatment, a complex at 60-kDa can be observed (lane 1). Cytoplasmic extract treatment with proteinase K (PK; 100 µg/ml), trypsin (Tryp; 0.5 µg/µg protein), or pronase (Pro; 100 µg/ml) at 37 C for 15 min before the addition to RNA-binding reaction mixtures containing 32P-labeled RNA abolished the complex formation (lanes 2–4). B, No complex can be observed when cytoplasmic lysate was omitted (lane 1). Incubation with cytoplasmic extracts showed a complex at 60 kDa (lane 2). Heating the protein extracts for 10 min at 70 C did not affect the binding activity to form complex with labeled RNA (lane 3). Incubation for 10 min at 100 C abolished the complex formation (lane 4).

 
Oxidation and reduction of PAM mRNA-BP. Several studies reported that the suggested mechanism of binding (RNA-proteins) involves a nucleophilic attack by a sulfhydryl on the C-6 of a pyrimidine within the target RNA (37). For example, it was shown that the active sulfhydryl sites of the iron response element-binding protein have been implicated as the mediators of binding to the iron response element in the ferritin mRNA (38). We investigated the possibility that there were similar interactions with the PAM mRNA cis element. We carried out a study of the effects of reducing and oxidizing agents on the formation of PAM mRNA-BP in standard gel shift assays. AtT-20 cell cytoplasmic extracts were preincubated for 10 min at room temperature with a reducing agent, ß-mercaptoethanol, or with chloramine-T, an agent that catalyzes the oxidation of free sulfhydryl groups. A gentle oxidation with chloramine-T (1 mM at room temperature) did not interfere with the formation of the mRNA-protein complex (Fig. 4Go, lane 1). ß-Mercaptoethanol had no effect on complex formation at a low concentration (0.5%; Fig. 4Go, lane 2); however, 1% completely inhibited binding (Fig. 4Go, lane 3). The addition of chloramine-T (1 mM) completely reversed this inhibition (Fig. 4Go, lane 4). Pretreatment of the cytoplasmic lysates with 0.02% SDS was sufficient to suppress the binding ability of the PAM mRNA-BP (Fig. 4Go, lane 6).



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  		Figure 4. Effect of reducing and oxidizing agents on protein binding to PAM RNA3675–3806 probe. Gel-shift analyses were performed with cytoplasmic extracts from AtT-20 cells and S9 RNA probe (2.5 x 105 cpm/lane). Preincubations were performed for 15 min at room temperature in the presence of the indicated concentration of ß-mercaptoethanol (ß-ME) and/or chloramine-T (Chlo T); lane 4 represents a preincubation with ß-mercaptoethanol (1%) followed by the addition of chloramine-T (1 mM). Treatment with cytoplasmic extract and 0.02% SDS before binding to the probe prevented the complex formation (lane 6). Molecular mass standards are indicated.

 
Phosphorylation is necessary for the 60-kDa protein to bind RNA. As phosphorylation can influence RNA-protein interactions (39), we sought to determine whether phosphorylation of the 60-kDa protein could play a role in binding by assaying AtT-20 cytoplasmic extracts for RNA binding before and after phosphatase treatment. After phosphatase treatment (0.35 U/µg protein for 30 min at 37 C) of an aliquot of AtT-20 cytoplasmic extract, protein binding to the PAM RNA3243–3967 probe was determined by gel retardation assays. Phosphatase treatment greatly reduced the amount of the specific RNA-protein complex formed compared with that in the untreated AtT-20 extract (Fig. 5Go, lanes 1 and 3). This reduction in binding is probably a result of protein dephosphorylation rather than proteolysis, because addition of phosphatase inhibitor (10 µM ammonium molybdate) prevented any reduction in complex formation (Fig. 5Go, lane 2). The damage to the RNA probe caused by alkaline phosphatase treatment was negligible (Fig. 5Go, lane 4). These results suggest that a relationship exists between the decrease in cellular protein phosphorylation and the PAM mRNA cis element-binding activity.



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  		Figure 5. Dephosphorylation prevents protein binding to PAM RNA probe. Aliquots of AtT-20 cytoplasmic extracts were incubated with alkaline phosphatase for 30 min at 37 C and analyzed by the gel retardation assay. Phosphatase-treated aliquots were incubated with 32P-labeled PAM RNA3243–3967 followed by RNase T1. Lane 1, Control incubation with no phosphatase; lane 2, 0.35 U phosphatase/µg protein and 10 µM ammonium molybdate, a phosphatase inhibitor; lane 3, 0.35 U phosphatase/µg protein; lane 4, PAM RNA3243–3967 probe treated with phosphatase (0.35 U for 30 min at 37 C) before adding the cytoplasmic extracts.

 
Tissue distribution of PAM mRNA-BP
We have assayed several tissues for the presence of PAM mRNA-BP. Figure 6Go shows binding assays performed with 6 µg cytoplasmic extracts prepared from various tissues isolated from mature male rats; female rats were only used for ovary tissue. The highest levels of binding activity were found in ovary, testis, lung, septum (heart), and anterior pituitary, and low activity was found in the other tissues, i.e. liver, ventricle, atrium, and neurointermediate lobe extracts. No binding activity was detectable in pancreas, intestine, or kidney extracts (Fig. 6Go). These levels of expression did not appear to correlate with known PAM activities or mRNA levels in these rat tissues (6); however, it is noteworthy that the rat atrium, which expresses very high levels of PAM mRNA and enzyme activity constitutively, contained barely detectable PAM mRNA-BP activity in this assay.



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  		Figure 6. Gel shift analysis using PAM RNA3243–3967 probe and cytoplasmic extracts from various rat tissues. RNA gel shift analyses were performed using S1 RNA probe (2.5 x 105 cpm) and 6 µg cytoplasmic extracts from various rat tissues. The major RNA-protein complex with 60 kDa is indicated. The hypothalamus tissue showed two complexes at 60 and 54 kDa. The lanes containing probe alone are labeled S1-RNase T1 and S1+RNase T1. The same results were obtained in six independent experiments.

 
In the hypothalamus, a 60-kDa RNA-protein complex was observed; however, a second RNA-protein complex with a molecular mass of 54 kDa was detected (Fig. 6Go). This complex could be another brain-specific PAM mRNA-BP or reflect modification of the previously described complex. Cytosolic preparations from the brain contain a variety of generic nucleases, such as RNase Y, A, and R, and several other brain-specific nucleases (40, 41). These activities may further cleave the RNase T1-protected PAM RNA and generate complexes with different mobilities. The absence of a shift with kidney, intestine, and pancreas lysates further suggests that the shift observed with hypothalamic extracts is not due to nonspecific RNA-protein interactions.

The binding activity detected in the anterior pituitary extracts is 2-fold less intense than that observed with AtT-20 cells extracts for the same amount of proteins (based on densitometer scan analysis), suggesting that corticotrope cells are a major source of production of PAM mRNA-BP (data not shown). Indeed, the anterior pituitary tissue is composed of different cell types, including corticotroph, lactotroph, somatotroph, gonadotroph, and thyrotroph cells. To gain insight into the cell type population expressing PAM mRNA-BP, we performed gel-shift binding assays with GH4 somatolactotrope cell cytoplasmic extracts. GH4 cells produced PAM mRNA-BP as well, but the binding activity was lower than that in AtT-20 corticotrope cells (data not shown).

Mapping of the binding region in the 3'-UTR of PAM mRNA
To map the binding region for the activity referred to as PAM mRNA-BP, additional band-shift analyses were performed with deletion variants of the PAM RNA 3'-UTR. PAM RNAs of various lengths (S1 to S5) starting 18 nt downstream from the stop codon were transcribed in vitro after linearization of the PAM 3'-UTR cDNA template with EcoRI, TfiI, DraI, BanI, or HphI, respectively. These PAM RNAs were assessed for their binding capacity to PAM mRNA-BP and as competitors of radiolabeled RNA3243–3967 (S1), derived from EcoRI-digested PAM cDNA template. Band-shift assays of radiolabeled S1 were carried out with AtT-20 cytoplasmic extracts, as described in Fig. 2Go. A summary of these data are shown in Fig. 7Go, unlabeled PAM RNA3243–3848 (S2) prevented the complex formation, and labeled S2 showed the same binding capacity as S1. PAM RNA3243–3707 (S3), PAM RNA3243–3614 (S4), and PAM RNA3243–3408 (S5) did not show any binding activity. These data demonstrate that PAM mRNA-BP interacts with a region common to S1 and S2, corresponding to a region of PAM RNA spanning nt 3707–3848. Furthermore, this interaction was specific, as other regions of PAM RNA failed to suppress binding. Identical results were obtained when labeled and unlabeled S6, S7, and S8 PAM RNAs were used (Fig. 7Go).

To further map the protein-binding site within nucleotides 3707–3848, we produced radiolabeled PAM RNA3675–3806 (S9), PAM RNA3675–3785 (S10), PAM RNA3675–3762 (S11), PAM RNA3675–3710 (S12), and PAM RNA3675–3743 (S13) (Fig. 8Go). These PAM RNAs were transcribed in vitro after linearization of the PAM 3'-UTR cDNA3675–3806 template with TfiI, DdeI, SwaI, or ApoI, respectively (Fig. 8Go). PAM RNA3693–3735 (S14) and PAM RNA3693–3762 (S15) probes were obtained from PCR amplification (see Materials and Methods). The data obtained are summarized in Fig. 8Go and suggest that the protein-binding site is located within 33 nt of the PAM 3'-UTR between nt 3710 and 3743.

Identification of a novel cis element in the 3'-UTR of PAM mRNA
To identify the binding element more closely, we designed a series of short antisense DNA oligonucleotides (Sh1, Sh2, and Sh3) complementary to the putative binding region of the 3'-UTR and analyzed their effects on complex formation. The antisense oligonucleotides were annealed in equimolar concentrations to the labeled PAM 3'-UTR, as described in Materials and Methods. One corresponding sense oligonucleotide (Sh4) was used as a control. Figure 9AGo shows the sequence of the PAM 3'-UTR with the oligonucleotides and some probes depicted. Band-shift assays with AtT-20 cytoplasmic extracts and labeled PAM 3'-UTR probe formed a typical complex (Fig. 9BGo, lanes 1 and 6). Interestingly, Sh3 antisense oligonucleotide was sufficient to abolish completely the RNA-protein complex formation (Fig. 9BGo, lane 4). The presence of either Sh1 or Sh2 antisense oligonucleotides did not prevent the complex formation, except for a slight decrease in the relative intensity with Sh1 (Fig. 9BGo, lane 2) and when a combination of Sh1 and Sh2 was used (data not shown). These data suggest that the immediate 5'-flanking sequences of Sh3 are important (see Discussion). As expected, a sense oligonucleotide, Sh4, did not interfere in the complex formation.



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  		Figure 9. Mapping of protein-binding site with competing antisense and sense oligonucleotides. A, Sequence of the 3'-UTR of rat PAM gene encompassing nt 3675–3806, illustrating the sequence and position of oligonucleotides used in gel-shift competition studies. Vertical arrows denote the restriction enzyme used to generate the corresponding probe as indicated. B, Antisense or sense oligonucleotides were hybridized to 32P-labeled PAM RNA3675–3806 (S9) at a molar ratio of 1, and binding assays were performed as described in Fig. 2Go, using AtT-20 cytoplasmic extracts. A RNA-protein complex without any competitor is shown (lanes 1 and 6).

 
These results together with the previous observation (Fig. 8Go) indicate that 20 nt of the 3'-UTR contain the major protein binding recognition site.

A conserved sequence within the 3'-UTR of PAM mRNA is important for protein-binding activity
A sequence search of the PAM genes cloned from human (42), rat (11), bovine (43), and equine (Lida, T., T. Kaminuma, M. Tajima, H. Okamoto, and M. Yanagi; GenBank accession no. D29625) revealed that the 20-base motif [(CAC-UAACAUUAUAUUGCAAU/C) that is within PAM RNA3675–3806] is highly conserved among these species, suggesting some biological significance of this cis-acting element in terms of regulation (Fig. 10Go).



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  		Figure 10. Conservation within species of the 3'-UTR consensus sequence Sh3 shown to interact with rat PAM mRNA-BP. Portions of the 3'-UTR of PAM mRNA from rat (11), human (41), bovine (42), and equine (Lida, T., T. Kaminuma, M. Tajima, H. Okamoto, and M. Yanagi; GenBank accession no. D29625) were aligned to demonstrate conservation of sequence motif in mRNAs from all species (bold box). The motif is highly conserved within species.

 
Northwestern analysis
Cytosolic extracts from AtT-20 cells were analyzed by Western blot. Proteins blotted on nitrocellulose were renatured (see Materials and Methods) and further hybridized to PAM RNA3675–3806 (S9) probe to detect the PAM mRNA-BP. With 25 µg AtT-20 cytosolic proteins, three products at 46, 36, and 32 kDa were detected (Fig. 11Go, lane 1). PAM RNA3675–3710 (S12), which did not form any complex with AtT-20 cytoplasmic extracts, was further used as an unspecific competitor. Preincubation of the blot with unlabeled S12 RNA prevented the detection of the 36- and 32-kDa forms and pointed out the specificity of the 46-kDa protein observed (Fig. 11Go, lane 2).



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  		Figure 11. Northwestern blot of AtT-20 cytoplasmic extracts. Western blots containing 25 µg AtT-20 cytoplasmic extracts were probed with {alpha}-32P-labeled PAM RNA3675–3806 (S9). Lane 2, The blot was hybridized for 2 h with unlabeled unspecific RNA before adding the probe. The positions of molecular mass markers are denoted for reference.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a putative novel 20-nt cis element, 5'-CACUAACAUUAUAUUG CAAU-3', in the 3'-UTR of PAM mRNA, which interacts with a cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-BP. The binding activity of the cis element was sequence specific. Similar to several previously reported mRNA cis elements (44), there is only one copy of the 20-nt cis element within the PAM message. Except in the hypothalamus, where two RNA-protein complexes at 60 and 54 kDa were observed, the cis element-binding site showed a similar band-shift complex at 60 kDa in a variety of cells and tissues, suggesting a common pathway for the regulation of PAM mRNA. Northwestern studies demonstrated that this protein has an approximate molecular mass of 46 kDa in mammals. Proteins with similar binding characteristics were found in a variety of cells and tissues from several animal species.

Our results indicate that the cis element-binding activity is redox sensitive; the inhibition of RNA-protein complex formation by ß-mercaptoethanol is reversed by 1 mM chloramine-T, suggesting a possible sulfhydryl switch mechanism. Changes in phosphorylation have also been implicated in binding activities of certain other trans-acting factors (39, 45). Because alkaline phosphatase reduces the RNA-binding activities of cytoplasm extracts, it is possible that the 46-kDa protein (and/or a cofactor) exists in a phosphorylated state, suggesting that the cis element-binding activity may be regulated via a phosphorylation pathway. The determination of PAM mRNA-BP function should clarify the consequences of such modulations. Phosphorylation has been proposed to regulate translation in Xenopus oocytes (46, 47, 48). One of the RNA-BPs in Xenopus oocytes, the phosphoprotein pp60, forms a stable complex with mRNA and inhibits translation of in vitro assembled messenger ribonucleoprotein particles (mRNPs), whereas after dephosphorylation, the binding affinity of this protein is reduced, and translation occurs. Phosphorylation of mRNPs and protein kinase activity associated with free mRNP particles have been reported for a variety of cell types and are believed to be important for the interaction of mRNPs with ribosomes (49).

To identify the location of the PAM mRNA cis element, a combination of deletion mapping and antisense oligonucleotide analysis was used. We were able to map the precise binding site to a region of PAM mRNA 3'-UTR between nucleotides 3715 and 3734. However, the failure of PAM RNA3693–3735 (S14) and PAM RNA3693–3762 (S15) to produce any shift suggests that the element spanning nucleotides 3715–3734 is necessary, but not sufficient, for binding. We presume that the 5'-flanking sequences of the binding site are necessary for binding. Such data imply that the folding of the RNA may be important for successful binding. Alternatively, the entry point for PAM RNA-protein interactions could be between nucleotides 3715–3734, and the protein subsequently moves upstream. Detailed biophysical studies must wait purification of the RNA-BP.

The identified cis element of PAM mRNA is highly conserved among species and is located at roughly the same distance from the stop codon: 477 nt in human, 484 nt in bovine, and 409 nt in rat. The surroundings are well conserved among species (92% identity for rat/human, 91% identity for rat/equine, 90% identity for rat/bovine). Band-shift assays with murine or rat cytoplasmic extracts and rat PAM RNA demonstrated identical reactivities, suggesting that these proteins may subserve similar functions. It is interesting to specify that the cis-element described in this report is not included in the well conserved 86-bp segment in the 3'-UTR of PAM cDNA previously described by Stoffers et al. (11). Based on the high degree of conservation (98%), the researchers predicted a specific function for the 86-bp fragment. The present study shows that this fragment is not necessary for complex formation with cytoplasmic extracts.

The presence of PAM mRNA-binding site-like sequences in other mRNAs was examined to determine the eventual identity with potential trans factor-binding sites described to date. It seems that the AU-rich cis element in PAM mRNA is unique and presents no homology to previously described cis elements, such as the AUUUA motif described as the instability determinant located in the 3'-UTR of many labile cytokine, lymphokine, oncogene, or inducible growth factor mRNA (22, 44, 50) the 5'-GAGUUUGAG-3' in the ribonucleotide reductase 3'-UTR (51), the iron-responsive element (28), the ornithine decarboxylase 5'-UTR conserved sequence (44), or the amyloid precursor protein mRNA 29 nt stability element (26). Interestingly, we found the TSHß 3'-UTR conserved sequence in PAM 3'-UTR (nt 3790–3800) with no binding affinity or implication in the RNA-protein interactions (27). This suggests that the flanking sequences of the TSHß 3'-UTR cis element or perhaps the secondary structure have some effect on binding affinity.

Multiple AU-specific mRNA-BPs have recently been described (52, 53, 54) with molecular masses between 32–50 kDa. These activities are thought to mediate the rapid decay of c-myc mRNA in a cell-free mRNA decay system (53) or APP mRNA stabilization in human neuroglioma H4 cells (26, 31).

The tissue-specific expression, sequence specificity, high affinity, and cytoplasmic localization suggest that the cis element-binding activity could regulate PAM mRNA metabolism within the cytoplasm. To date, however, we have not been able to ascribe a function to this protein. Recently, we demonstrated in hypothyroid and ovariectomized rats, a posttranscriptional regulation of PAM expression with an increase in mRNA stability in anterior pituitary gland (16, 18). However, in preliminary experiments, we have been unable to detect the effects of ovariectomy and thyroidectomy on RNA-protein complex formation with anterior pituitary cytoplasmic extracts compared with those in control animals (not shown). Then, the protein may well have a function unrelated to mRNA stability. Possible functions proposed, but not demonstrated, for several other mRNA-BPs include regulation of internal initiation (55), regulation of transcript stability (56), transport of mRNA from nucleus to cytoplasm (56), membrane partitioning of viral RNA (57), and developmental expression of mRNA (58).

To further define the function of PAM mRNA-BP, we are now involved in purifying PAM mRNA-BP and cDNA cloning to determine whether it has effects on the mRNA stability or translation in in vitro systems.


   Acknowledgments
 
We thank R. M. Saura and F. Youssouf for their technical assistance. We are grateful to Prof. Charles Oliver for his critical reading of the manuscript.

Received June 2, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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