INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

This paper focuses on a ribosome-associated protein with the properties of a c-myc mRNA-degrading ribonuclease. The significance of mRNases stems from the fact that mRNA stability affects gene expression in virtually all organisms (1-4). Moreover, mRNA stability is regulated, because the half-lives of many mRNAs fluctuate as a function of cell growth, environmental factors, and the stage of cell differentiation. mRNases must play a central role in these fluctuations.

Unfortunately, we know little about mammalian mRNases, and no enzyme has yet been proved to be a mammalian mRNase. There are many unresolved issues about these enzymes. How many of them are present in each mammalian cell? In what critical ways do mRNases differ from RNA-processing enzymes? Do mRNases also process RNA or attack other classes of RNAs? How do mRNases distinguish mRNA from rRNA and tRNA? Does each mRNase attack only a subset of mRNAs? Genetic approaches have made it feasible to identify mRNases in lower organisms and to analyze the phenotype of cells with mRNase gene mutations (1, 2). In contrast, vertebrate cells with mRNase gene mutations have not been generated. Several candidate vertebrate mRNases have been identified using other strategies as follows: (i) incubating cell extracts with deproteinized mRNA substrates or (ii) analyzing mRNA decay in polysome-containing cell-free mRNA decay systems (reviewed in Refs. 3-5). Some of these enzymes are endonucleases, and others are exonucleases. Based on the findings in yeast and prokaryotes, it is likely that vertebrate cells contain only a few mRNases.

Our laboratory has been investigating the regulation of c-myc mRNA stability. By using cell-free mRNA decay assays and transfection, we and others (6) have suggested that c-myc mRNA can be degraded by alternative pathways involving at least two mRNases. One mRNase is involved in a 3' to 5' decay pathway in which the poly(A) tail is removed first, and then the body of the mRNA is degraded. The second mRNase is an endonuclease that we initially identified using a polysome-based cell-free mRNA decay assay. This endonuclease attacks the C-terminal coding region of endogenous, polysome-associated c-myc mRNA. We refer to this endonuclease cleavage target as the c-myc coding region determinant or CRD,¹ because it is a major determinant of c-myc mRNA stability in cells (7-12). It is also a binding site for a protein that is thought to shield the mRNA from endonuclease attack (9, 10, 13, 14).² The relationship of the endonuclease to the c-myc CRD and to c-myc mRNA stability is further supported by the finding that c-myc mRNA can be degraded endonucleolytically in cells (16, 17).

Here, we describe the partial purification of a polysome-associated endonuclease with the properties of the c-myc mRNase. The soluble enzyme was isolated from a high salt extract of rat liver polysomes. It is a magnesium-dependent protein that is resistant to the RNase A class of RNase inhibitors. Therefore, it is not a member of the RNase A family. It cleaves deproteinized c-myc CRD RNA with high specificity and preferentially attacks one RNA segment that is rich in A residues. Although these data do not prove that this enzyme is the c-myc mRNase, the enzyme we have purified does share several properties with the polysome-associated c-myc mRNase.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Preparation of Radiolabeled Transcripts-- The subcloning of the c-myc DNA fragments corresponding to all or part of the CRD of c-myc mRNA has been described (9, 13). Transcription of these DNAs generates RNAs corresponding to nucleotides 1705-1792 and 1705-1886 of c-myc mRNA, respectively (diagrammed in Fig. 1A, top). The 1705-1886 RNA is the full-length CRD and is designated FL-CRD RNA in the text. The shielding protein or CRD-binding protein (CRD-BP) binds with high specificity to FL-CRD RNA (10, 13). The RNA corresponding to c-myc nts 1705-1792 is referred to as 5'-CRD RNA. 5'-CRD ³²P-RNA was used as the substrate for purifying and characterizing the liver endonuclease. Unlabeled CRD RNA was synthesized by linearizing the 5'-CRD plasmid or the FL-CRD plasmid with EcoRI and transcribing the DNA with SP6 RNA polymerase. As a control, plasmid pBSmyc, which contains full-length human c-MYC cDNA cloned into pBluescript, was digested with EarI and transcribed with T7 RNA polymerase to generate an RNA corresponding to nucleotides 1-217 of c-MYC mRNA. Transcriptions were performed using Megascript kits (Ambion). Each in vitro transcribed RNA (10 µg) was dephosphorylated with 40 units of alkaline phosphatase (Boehringer Mannheim) for 30 min at 37 °C in a 100-µl reaction according to the maufacturer's instructions. Dephosphorylated RNA was purified by phenol/chloroform extraction and ethanol precipitation. One µg of it was incubated in a 25-µl reaction with 50 µCi of [-³²P]ATP (Amersham Pharmacia Biotech; 6000 Ci/mmol) at 37 °C for 1 h with 50 units of T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. Unincorporated label was removed by a G-50 spin column (Amersham Pharmacia Biotech), and the RNA was extracted with phenol/chloroform and precipitated with ethanol. To prepare 3'-³²P-labeled CRD RNA, 65 pmol of the 88-nt CRD RNA was incubated in a 20-µl reaction containing 90 pmol of [5'-³²P]pCp (Amersham Pharmacia Biotech, 3000 Ci/mmol), 10 mM MgCl₂, 5 mM DTT, 200 ng of BSA, 10% (v/v) Me₂SO, 1 mM ATP, 40 units of RNasin (Promega), 120 units of T4 RNA ligase (New England Biolabs), 50 mM HEPES, pH 8.3, for 18-20 h at 4 °C. The resulting RNA was 3'-end-labeled and 3'-phosphorylated (RNA-³²P-C-P). Unincorporated label was removed, and the RNA was purified as described above.

In Vitro Assay for Soluble Liver Endonuclease Activity-- The pH of all buffers for these experiments was determined at room temperature. The standard 20-µl reaction mixture included 2 mM DTT, 1 unit of RNasin (Promega), 2 mM magnesium acetate, 50 mM potassium acetate, 0.1 mM spermidine, 1 ng of 5'-end-labeled ³²P-RNA (~5 × 10⁴ cpm), 11 mM Tris-HCl, pH 7.6. These components were first assembled on ice, and enzyme was added last. Unless otherwise noted, reactions were incubated for 10 min at 37 °C, placed in dry ice for 10 min, and then lysophilized. Five µl of loading dye (80% formamide, 0.1% xylene cyanol, 12.5 mM EDTA, pH 8.0) were added, and the samples were denatured at 65 °C for 10 min and electrophoresed at 250 V for 2 h in an 8% polyacrylamide, 7 M urea gel. Gels were fixed in 10% acetic acid, 10% methanol for 15 min, dried, and exposed to a PhosphorImager screen (Molecular Dynamics). One major endonuclease decay product of ~30 nt was generated (see Fig. 1B). One endonuclease unit was defined as the amount of enzyme required to cleave 30% of the input RNA substrate into this decay product in a 10-min reaction at 37 °C. Under these conditions, the amount of decay product formed was linear with enzyme concentration.

Preparation of Ribosomal Salt Wash (RSW) from Rat Liver-- All procedures were performed at 4 °C. Seventy male Sprague-Dawley rats (Harlan Sprague-Dawley) each weighing 175-200 g were sacrificed. Their livers (average weight 11 g) were excised. One ml of Buffer A per 0.4 g of tissue was added (Buffer A, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM DTT, 10% (v/v) glycerol, leupeptin (1 µg/ml), pepstatin A (1 µg/ml), phenylmethylsulfonyl fluoride (100 µg/ml), 0.1 mM EGTA, 10 mM Tris-Cl, pH 7.4). The livers were homogenized for 30 s using a Polytron and were further hand-homogenized with 15 strokes to increase the percentage of broken cells. Nuclei were removed by centrifugation at 24,500 × g for 10 min. The post-nuclear supernatant was layered over a 10-ml cushion of 30% (w/v) sucrose in Buffer A and centrifuged in the SW28 rotor at 96,500 × g for 2 h to pellet the polysomes. The polysomes were resuspended in Buffer A, and 4 M potassium acetate was added slowly with gentle mixing to a final concentration of 0.5 M. Gentle mixing was continued for an additional 15 min, avoiding bubbles. The material was then ultracentrifuged as described above. The supernatant above the sucrose pad contained salt-eluted polysomal proteins and is designated ribosomal salt wash (RSW). This RSW was used for endonuclease purification as described below. Typically, between 50 and 75 mg of RSW protein were obtained from one liver. RSW can be stored at 80 °C for at least 6 months without loss of endonuclease activity.

Purification of the Solubilized Liver Polysomal Endoribonuclease-- All procedures were performed at 4 °C. Approximately 4 g of RSW protein were mixed with 6 volumes of 25% (v/v) glycerol. Three hundred mg of RSW protein were loaded at a flow rate of 1 ml/min onto a 2.5 × 12 cm phosphocellulose column (Sigma) equilibrated with 0.05 M KCl, 2 mM DTT, 10% (v/v) glycerol, 25 mM potassium phosphate, pH 6.0. The column was washed until the absorbance at 280 nm (A280) returned to base line. Bound proteins were then eluted with a linear gradient from 0.05 to 0.5 M KCl in the same buffer. Seven-ml fractions were collected, and an aliquot of each was assayed for endonuclease activity as described above. For reasons we do not understand, RNase recovery was higher on smaller phosphocellulose columns than on larger ones. Therefore, to deal with 4 g of RSW from 70 livers, 14 separate chromatography runs were performed using 2.5 × 12 cm phosphocellulose columns. The results were highly reproducible. To plot the data, the fraction containing the highest activity was set at 100%, and the relative activity of all other fractions was calculated (Fig. 2).

Glycerol Gradient Centrifugation of Liver Ribosomal Salt Wash (RSW)-- RSW (0.1 ml; 1 mg of protein) was layered onto a 10-30% (v/v) glycerol gradient (4 ml, 11 × 60-mm tubes) made in 0.25 M KCl, 0.1 mM EDTA, 50 mM Tris-HCl, pH 7.4, and was centrifuged for 18 h, 4 °C, 200,000 × g (44, 100 rpm) in a Beckman SW 60 rotor. Fractions of 0.2 ml were collected manually from the top, and 5 µl of each fraction were assayed for endonuclease activity. A separate 10-30% glycerol gradient was centrifuged in the same manner and contained the following proteins as molecular mass standards: -galactosidase (116.4 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and lysozyme (14.3 kDa). The protein standards were identified by SDS-PAGE.

Size-exclusion Chromatography of the Liver RSW Endonuclease-- RSW (0.4 ml; 4 mg of protein) was chromatographed on a SEC 2000 high pressure liquid chromatography column (Beckman) equilibrated with 0.25 M KCl, 10% (v/v) glycerol, 100 mg/ml carrier bovine serum albumin, 20 mM triethanolamine, pH 7.4. The column was run at 0.4 ml/min, and 0.4-ml fractions were collected into tubes containing carrier carbonic anhydrase to give a final concentration of 100 µg/ml protein. A 4-µl aliquot of each fraction was assayed for endonuclease activity. The column was calibrated with Bio-Rad SDS-PAGE low molecular weight markers, the elution of which was determined by SDS-PAGE.

Globin-MYC-Globin Gene Expression and Endonucleolytic Degradation of Endogenous, Polysome-associated c-myc mRNA in Vitro-- We analyze the in vitro decay of endogenous, polysome-associated mRNAs by incubating polysomes in an appropriate buffer at 37 °C (5, 18). To investigate the properties of the polysome-associated c-myc mRNA-degrading endonuclease, HeLa cell polysomes expressing GMG mRNA were used. The construction of the GMG gene and culturing and transfection of HeLa cells have been described (10). Briefly, the GMG gene is driven by the cytomegalovirus immediate-early promoter, and the major features of GMG mRNA are diagrammed in Fig. 1A, bottom. GMG mRNA includes 419 nt from the human -globin mRNA cap site to the EcoRI site in the coding region, 249 nt from the C-terminal coding region of human c-myc mRNA, 6 nt from an EcoRI linker, and 207 nt from the -globin EcoRI site to the mRNA 3' terminus. The c-myc segment is inserted in frame and includes the 182-nt c-myc CRD plus 67 coding nts 5' of the CRD. mRNA decay reactions contained polysomes from GMG-expressing HeLa cells and were performed as described previously (9, 10, 18). Where indicated, excess c-myc CRD competitor RNA was added to the reactions at 1 µg of competitor per 10 µg of polysomal RNA. The CRD competitor RNA activates the c-myc endonucleolytic decay pathway and causes the mRNA to be cleaved within the CRD (9). After incubation at 37 °C for various times, total RNA was prepared by phenol extraction and was blotted to a Hybond-N+ membrane (Amersham Pharmacia Biotech). Blots were hybridized with a [³²P]ApaLI-EcoRI fragment of human -globin cDNA prepared by random priming. This probe anneals to the 5' region of GMG mRNA and recognizes both undegraded GMG mRNA and the 5'-endonuclease decay product.

Mapping RNA 3' Ends-- The method of Zaug et al. (19) was used, with slight modifications. Eight ng of 5' ³²P-labeled 5'-CRD RNA (c-myc nucleotides 1705-1792) were cleaved by 1 unit of heparin-Sepharose-purified endonuclease. The reaction was terminated at several time points, and the RNA was extracted with phenol/chloroform. A 1.5-ng aliquot of each RNA sample was electrophoresed in an 8% denaturing polyacrylamide gel to confirm that the endonuclease had cleaved the RNA. The remainder of each sample (6.5 ng) was poly(A)-tailed with yeast poly(A) polymerase (550 units; U. S. Biochemical Corp.) in a 20-µl reaction according to the manufacturer's instructions. The poly(A)-tailed RNA was then reverse-transcribed with 25 units of avian myeloblastosis virus-reverse transcriptase (Boehringer Mannheim) using 0.5 µg of primer T (5'-dAACCCGGCTCGAGCGGCCGC(T₁₈)-3') in a 20-µl reaction containing 8 mM MgCl₂, 30 mM KCl, 1 mM DTT, 1 mM each of the 4 dNTPs, 4 units of RNasin, 50 mM Tris-HCl, pH 8.5. The underlined nucleotides in the primer T sequence denote the XhoI restriction site used for cloning. The reaction was incubated first at room temperature for 15 min and then at 50 °C for 15 min. Reverse transcriptase was inactivated by heating the reaction mix to 95 °C for 5 min, and unincorporated dNTPs were removed with a G-50 spin column (Amersham Pharmacia Biotech). Amplification by PCR was performed using primers T and Myc. The Myc primer (5'-dCTCGGATCCATTTAGGTGACACTATAGACCAGATCCCGGAGTTGG-3') includes c-myc nucleotides 1705-1722. The underlined nucleotides indicate a BamHI restriction site. Two DNAs were detected following PCR. One corresponded to undegraded 5'-CRD RNA and the other to the endonucleolytic degradation product (see Fig. 10B). Each DNA was gel-purified, cleaved with XhoI and BamHI, cloned into pGEM7Z, and sequenced using a T7 primer.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Identification and Purification of a Polysomal, c-myc mRNA-degrading Endonuclease-- The existence of a polysomal c-myc mRNA-degrading endonuclease was revealed in previous studies using a cell-free mRNA decay assay (9). The assay reaction includes polysomes from tissue culture cells, and the decay of endogenous, polysome-associated mRNAs such as c-myc and histone is monitored. Under certain reaction conditions, polysome-associated c-myc mRNA is cleaved endonucleolytically in a c-myc segment designated the coding region determinant or CRD. The CRD is the last 180-250 nt of the coding region (Fig. 1A; Ref. 9). Two additional findings strengthened the connections among an endonuclease, the CRD of c-myc mRNA, and c-myc mRNA stability. (i) The CRD is a major determinant of c-myc mRNA expression and stability in vivo (7, 8, 10-12). (ii) c-myc mRNA is cleaved endonucleolytically in at least some cells (16, 17). In view of these findings, we undertook to purify and characterize the responsible enzyme. The strategy was to solubilize polysomal proteins using high salt extraction and to incubate the extract with a deproteinized c-myc CRD ³²P-RNA substrate. Since endonucleolytic cleavage of endogenous c-myc mRNA occurred in the 5' one-half of the CRD (9, 10, 13), RNA from this segment of the CRD was used as substrate (designated 5'-CRD RNA; Fig. 1A).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   c-myc CRD RNA-degrading endoribonuclease activity in rat liver ribosomal salt wash. A, top, endonuclease RNA substrate. Human c-MYC mRNA is diagrammed at the top, with its CRD indicated by the hatched box. In the text, this 182 c-myc RNA segment is designated FL-CRD. FL-CRD RNA corresponds to c-myc nucleotides 1705-1886. The 88-nt RNA corresponding to c-myc nts 1705-1792 is from the 5'-half of FL-CRD RNA. In the text, it is designated 5'-CRD. Unless otherwise noted, 5'-CRD RNA 32P-labeled at its 5'-end was the substrate for all liver endonuclease assays. A, bottom, diagram of GMG mRNA. The gene encoding GMG mRNA is described under "Experimental Procedures" and in Herrick and Ross (10). GMG mRNA consists of the FL-CRD of human c-MYC mRNA plus an additional 67 c-myc coding nts subcloned in frame into human -globin. GMG mRNA was expressed in HeLa cells and was used as an endogenous, polysome-bound mRNA substrate to assay the polysome-associated c-myc mRNase. B, endonuclease activity in crude rat liver RSW. 5'-CRD 32P-RNA was incubated with or without 10 µg of liver RSW protein at 37 °C for the indicated times. RNA was extracted and electrophoresed in an 8% polyacrylamide, 7 M urea gel. Unfilled arrowhead, undigested 5'-CRD RNA. Filled arrowhead, major endonucleolytic decay product, which migrates at 30-40 nts. In gels with greater resolving power, this band migrates at ~30 nt (Fig. 6). The marker is 5'-end-labeled pBR322 32P-DNA cleaved with HaeII. The length of each marker fragment is noted in nts on the left.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Purification of the rat liver endonuclease. The starting material was ~4 g of ribosomal salt wash protein from 70 male Sprague-Dawley rat livers. The endonuclease was assayed as under "Experimental Procedures," using PAGE to assess the cleavage of the 5'-CRD 32P-RNA substrate, as per Fig. 1B. A, phosphocellulose cation exchange. B, reactive blue-3 dye affinity. C, Q-Sepharose anion exchange. D, reactive green-19 dye affinity. E, heparin-Sepharose. Solid lines connecting the unfilled circles, relative endonuclease activity measured as percent of maximal RNA decay product generated (see "Experimental Procedures"). Broken lines, absorbance at 280 nm. Bars indicate fractions that were pooled for subsequent steps.

View larger version (71K): [in this window] [in a new window]   Fig. 3.   SDS-PAGE of purified endonuclease. 2000 units of endonuclease from each activity peak of each column except Q-Sepharose (Fig. 2) were precipitated with acetone and air-dried. The pellets were resuspended in 10 µl of SDS-PAGE protein loading buffer containing 5 mM -mercaptoethanol, boiled for 5 min, and electrophoresed in a 12% SDS-polyacrylamide gel. Proteins were visualized by silver staining. Marker, low molecular mass standards (Bio-Rad); molecular mass is indicated in kDa on the left. RSW: starting material. The next 4 lanes contain endonuclease from each column except Q-Sepharose, as noted. Protein in the green-19 and heparin-Sepharose lanes included carrier carbonic anhydrase, which was added to maintain endonuclease activity. Carrier lane, 10 µg of carrier carbonic anhydrase alone. Arrow, the ~39-kDa protein that co-purifies with endonuclease activity (see text).

View larger version (86K): [in this window] [in a new window]   Fig. 4.   Chromatography of the liver endonuclease on heparin-Sepharose. A, SDS-PAGE of heparin-Sepharose column fractions. Thirty-µl aliquots from fractions 14-23 of the heparin-Sepharose column (Fig. 2E) were separated by 12% SDS-PAGE, and proteins were visualized by silver staining. All fractions contained carrier carbonic anhydrase to maintain endonuclease activity. Marker, low molecular mass standards (Bio-Rad); molecular mass is indicated in kDa on the left. Arrow, the ~39-kDa band that comigrates with endonuclease activity. B, endonuclease activity in heparin-Sepharose fractions. A 1-µl aliquot of fractions 14-23 from heparin-Sepharose was assayed for endonuclease activity by incubation with 5'-CRD 32P-RNA at 37 °C for 30 s or for 2 min, as indicated. RNA cleavage was determined by denaturing gel electrophoresis. Unfilled arrowhead, undigested RNA; filled arrowhead, major endonucleolytic decay product.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Analysis of the liver endonuclease by size-exclusion chromatography and glycerol gradient centrifugation. Filled circles, endonuclease activity measured as percent of maximal endonucleolytic decay product generated. Unfilled circles, low molecular mass standards (Bio-Rad). A, SEC 2000 size exclusion high pressure liquid chromatography. The column was calibrated with molecular mass markers before chromatography of 4 mg of rat liver RSW. B, glycerol gradient centrifugation. One mg of rat liver RSW was centrifuged in a 10-30% glycerol gradient. A parallel gradient contained molecular mass marker proteins.

The Enzyme Is an Endonuclease-- To prove that the enzyme is an endonuclease, it was incubated with 88-nt CRD RNA that was ³²P-labeled at either the 5' or 3' terminus. 5'-Labeled substrate was cleaved primarily at a single site located ~30 nt 3' of its 5' terminus (Fig. 6, left 3 lanes). 3'-Labeled substrate was also cleaved at a single major site located ~60 nt from its 3' terminus (Fig. 6, lanes denoted 3' Label). Therefore, the combined sizes of these cleavage products correspond to the size of the full-length substrate. Moreover, in this and other experiments, the amounts of radioactivity in each cleavage product plus the remaining full-length substrate were equal to the amount of substrate added at time 0 (data not shown). This result further confirms that a single cleavage event generates the two cleavage products. In summary, the enzyme is an endonuclease.

View larger version (62K): [in this window] [in a new window]   Fig. 6.   The polysomal enzyme is an endonuclease. The 88-nt CRD RNA substrate was 32P-labeled at its 5' or its 3' terminus. Each RNA was then incubated under standard conditions for the indicated times, when the reactions were stopped and the RNA was harvested. Each RNA was electrophoresed, and the bands were visualized by autoradiography. They were also quantified by PhosphorImager. Lane M, 5'-end-labeled pBR322 32P-DNA cleaved with HaeII. The marker bands from top to bottom are 83, 60, and 53 nt, respectively.

Properties of the Purified Polysomal Endonuclease-- The heparin-Sepharose-purified endonuclease was active between 0 and 100 mM KCl but lost activity at 200 mM or higher KCl (Fig. 7A). It remained active when pre-heated to 42 °C but was completely inactivated at 50 °C (Fig. 7B). It did not require a nucleic acid cofactor, because its activity was unaffected by pretreatment with micrococcal nuclease (60 units; data not shown). It was completely inactivated by proteinase K treatment (0.02 mg/ml; data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Salt and temperature sensitivity of the heparin-Sepharose-purified endonuclease. One unit of endonuclease purified through the heparin-Sepharose step (Fig. 2) was incubated with 5'-CRD 32P-RNA as under "Experimental Procedures." The RNA cleavage product was quantitated by electrophoresis and PhosphorImaging. A, effect of salt. Reactions contained different concentrations of KCl. At the times noted, reactions were terminated, and the endonucleolytic decay product was quantitated. B, effect of temperature. Endonuclease was first incubated for 5 min at 4, 30, 42, 50, 60, or 70 °C prior to the start of the reaction, which was incubated at 37 °C for 10 min.

Similarities between the Soluble Rat Liver Endonuclease and the Polysome-associated c-myc mRNase-- We have exploited two assays to identify a c-myc mRNA-degrading endonuclease. (i) Polysomes from cultured cells are incubated under conditions that activate the endonuclease (see below). This enzyme is designated the polysomal c-myc mRNase. (ii) Solubilized enzyme from liver polysomes is incubated with deproteinized c-myc CRD ³²P-RNA (Fig. 1B). The goal of the experiments described below was to compare several properties of the polysomal c-myc mRNase with the solubilized liver enzyme.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Liver endonuclease activity in the presence of RNasin, VRC, or different concentrations of magnesium. One unit of heparin-Sepharose-purified endonuclease was incubated with 5'-CRD 32P-RNA at 37 °C for the times indicated. RNA was extracted and electrophoresed in a denaturing gel. Unfilled arrowhead, undigested 5'-CRD RNA. Filled arrowhead, major endonucleolytic decay product. A, effect of RNase inhibitors. Lane 1, pBR322 32P-DNA marker cleaved with HaeII; sizes in nts are noted on the left. Lanes 2-5, endonuclease incubated under standard conditions, except that RNasin was omitted. Lanes 6-9, 1 unit of RNasin added to the reactions. Lanes 10-13, RNasin omitted but 20 mM VRC added. B, magnesium dependence of the endonuclease. Reactions were incubated for the indicated times under standard conditions. Lanes 1 and 2, no endonuclease. Lanes 3-6, endonuclease added; reactions contained 10 mM EDTA and no magnesium. Lanes 7-10, endonuclease added; reactions contained 2 mM magnesium acetate and no EDTA.

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Endonucleolytic degradation of endogenous, polysome-associated globin-MYC-globin mRNA in cell-free mRNA decay reactions in the presence or absence of RNasin, VRC, or magnesium. A GMG gene was transfected into HeLa cells, and permanent transfectants were selected (10). The GMG gene is driven by a cytomegalovirus promoter and generates GMG mRNA, which is diagrammed in Fig. 1A. Polysomes were made from this cell line and were incubated in the presence or absence of 1 µg of competitor c-myc CRD RNA. The competitor RNA activates the endonucleolytic decay pathway for c-myc and GMG mRNAs, and the responsible enzyme is designated the polysome-associated c-myc mRNase (9, 10). Reactions were incubated for the indicated times. Total RNA was isolated, electrophoresed, and analyzed by Northern blotting using a human -globin 32P-DNA probe that anneals to the 5' region of GMG mRNA. Therefore, this probe recognizes both undegraded GMG mRNA and the 5'-endonuclease decay product. Unfilled arrowheads, undegraded GMG mRNA; filled arrowheads, the 5' decay product resulting from endonucleolytic cleavage of GMG mRNA within its c-myc CRD segment. RNA molecular weight markers are indicated in kilobases on the left. A, effect of RNase inhibitors. Polysomes were incubated in reactions containing either 1 unit of RNasin or 20 mM VRC. Some reactions were supplemented with CRD competitor RNA, as noted, to activate the endonuclease decay pathway. B, magnesium requirement for endonucleolytic cleavage. In the 1st six lanes, the reactions contained no magnesium acetate but did contain 10 mM EDTA. In the remaining lanes, reactions did not contain EDTA but did contain the indicated amount of magnesium acetate and, where noted, competitor CRD RNA.

Substrate Specificity of the Purified Polysomal Endonuclease-- We challenged the purified endonuclease with three deproteinized 5'-³²P-RNA substrates as follows: c-myc 5'-CRD RNA (nts 1705-1792), c-myc FL-CRD RNA (nts 1705-1886), and nts 1-217 from the c-myc mRNA 5'-UTR. The following observations were made (Fig. 10). (i) Both CRD RNAs were attacked in the same region and generated the same prominent decay product (Fig. 10, asterisk). No other prominent CRD RNA decay products were detected. (ii) A discrete decay product was not observed with c-myc 5'-UTR (nts 1-217) RNA. However, this RNA was degraded by the liver enzyme, as determined by quantitating the amount of full-length (nts 1-217) RNA destroyed by the enzyme (Fig. 10, numbers at bottom). Approximately 49, 57, and 37% of the 5'-CRD, FL-CRD, and (nts 1-217) RNAs were degraded, respectively. Either the (nts 1-217) RNA was not cleaved in a single region or was cleaved so close to its 5' terminus that the resulting 5' decay product was electrophoresed off of the gel. In either case, these data indicate that the purified endonuclease cleaves other deproteinized RNAs besides c-myc CRD RNA. Moreover, when present in excess, it probably cleaves CRD RNA at sites other than the major one (for example, see results with fraction 18, Fig. 4B, 2-min reaction). Therefore, the liver endonuclease not is a "restriction RNase" in the sense that it cleaves deproteinized RNA at only one specific site or that it cleaves only one RNA substrate. On the other hand, it does exhibit considerable specificity. When challenged with the 182-nt FL-CRD RNA substrate, it clearly prefers to cleave in one region (Fig. 10).

View larger version (51K): [in this window] [in a new window]   Fig. 10.   Substrate specificity of the purified liver endonuclease. One unit of heparin-Sepharose-purified liver endonuclease was incubated for 10 min at 37 °C with the following deproteinized, 5'-end-labeled 32P-RNAs: 5'-CRD (c-myc nts 1705-1792), FL-CRD (c-myc nts 1705-1886), and RNA corresponding to nts 1-217 from the 5'-UTR of c-myc mRNA. Asterisks indicate the cleavage product generated from 5'-CRD or FL-CRD RNA. Numbers at the bottom indicate the percentage of input RNA that was degraded. These numbers were obtained using the PhosphorImager to quantitate the amount of undegraded RNA in reactions without and with endonuclease. Marker, 5'-end-labeled pBR322 32P-DNA cleaved with HaeII; sizes are noted in nts on the left.

Mapping the Major Endonuclease Cleavage Site within c-myc CRD RNA-- A PCR-based method was exploited to map precisely the endonuclease cleavage site within the 5'-CRD RNA substrate (19). First, the RNA was incubated with heparin-Sepharose-purified endonuclease between 12 s and 10 min. During this time the amount of the expected decay product increased (Fig. 11A, filled arrowhead), and the substrate RNA decreased (unfilled arrowhead). An aliquot from each time point was poly(A)-tailed using yeast poly(A) polymerase. cDNA was prepared by reverse transcribing the tailed RNA using an oligo(dT) primer. Then, the cDNA was amplified by PCR, and PCR products were visualized following electrophoresis in a 2% agarose gel (Fig. 11B). As expected, no PCR product was detected with non-tailed RNA (Fig. 11B, lane 2). An ~170-bp band was observed with poly(A)-tailed RNA that was not incubated with the endonuclease (Fig. 11B, lanes 3 and 4, unfilled arrowhead). As documented below, this band corresponds to undegraded 5'-CRD RNA. The DNA was larger than the RNA substrate because of the poly(A) tail and the extra nucleotides derived from the PCR primers ("Experimental Procedures"). The intensity of the 170-bp DNA decreased with time as the RNA was attacked by the endonuclease. Concurrently, an ~90-bp DNA was generated (filled arrowhead). The ~90-bp DNA corresponds to the major endonucleolytic decay product.

View larger version (48K): [in this window] [in a new window]   Fig. 11.   PCR amplification of c-myc 5'-CRD RNA that was cleaved by purified liver endonuclease. One unit of heparin-Sepharose-purified endonuclease was incubated with 5'-end-labeled 5'-CRD 32P-RNA under standard conditions for the indicated times. A, time course. RNA was harvested at the indicated times, and one portion was electrophoresed in an 8% polyacrylamide, 7 M urea gel. Unfilled arrowhead, undegraded RNA substrate; filled arrowhead, endonucleolytic cleavage product. Marker, 5'-end-labeled pBR322 32P-DNA cleaved with HaeII; sizes are noted in nts on the left. B, PCR amplification of the endonucleolytic cleavage product. An aliquot of total RNA from the reactions described in A was tailed with poly(A) and reverse-transcribed. The resulting cDNAs were then amplified by PCR ("Experimental Procedures"). The PCR products were electrophoresed in a 2% agarose gel and visualized with ethidium bromide. Unfilled arrow, PCR product corresponding to undegraded 5'-CRD RNA; filled arrow, PCR product corresponding to the endonucleolytic cleavage product. Each PCR product was further characterized by sequencing as per Fig. 12. In Fig. 12, the PCR products corresponding to the unfilled and filled arrowheads are designated Large and Small, respectively.

View larger version (36K): [in this window] [in a new window]   Fig. 12.   Sequences of cDNA clones of endonuclease decay products: location of the major endonucleolytic cleavage region. 5'-CRD RNA (c-myc nts 1705-1792) was cleaved with heparin-Sepharose-purified liver endonuclease as per Fig. 11A. Poly(A) was added enzymatically to the RNA, which was then reverse-transcribed and amplified by PCR. Two major products were observed and were designated Large and Small (unfilled and filled arrowheads in Fig. 11B, respectively). Each DNA (Large and Small) was purified separately and cloned into pGEM7Z. Clones were then picked and sequenced. A, DNA sequence of all sequenced clones. The complementary strand was sequenced, but the mRNA strand is shown in a 5' to 3' direction for clarity. The EcoRI sequence that was added when subcloning the 5'-CRD RNA is shown in italics as part of the poly(A) segment. Tail indicates part of the 3'-terminal poly(A) that was added to the RNA by poly(A) polymerase. Nucleotide 1792 corresponds to the 3' c-myc nt of the 5'-CRD RNA used as the endonuclease substrate. B, location of the major endonucleolytic cleavage sites in c-myc 5'-CRD RNA determined as per A. The major cleavage region is double underlined. There is ambiguity as to some of the actual cleavage sites, because cleavage occurred at or 3' of a C residue (nt 1727) or a G residue (nt 1731), each of which is followed by 2-4 A residues. These A residues in the RNA cannot be distinguished from the A residues added by the poly(A) polymerase.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Three convergent observations highlight the significance of the liver endonuclease and its potential role as a c-myc mRNase. (a) The CRD of c-myc mRNA is cleaved in a specific region by the liver endonuclease. The c-myc CRD is also an instability determinant and potential endonuclease target site in cells, as indicated by the following findings. (i) c-myc mRNA is unstable even when its 5'- and 3'-UTRs are both deleted (32-34). Therefore, the c-myc coding region contains an instability determinant. (ii) The c-myc CRD is required for down-regulating c-myc mRNA post-transcriptionally when myoblasts fuse to form multinucleated myotubes (7, 8, 11, 12). Down-regulation does not occur when the CRD is deleted. (iii) The coding region is also required to regulate c-myc mRNA post-transcriptionally in liver cells. c-myc mRNA levels increase transiently and then decrease during the liver regeneration process that occurs following partial hepatectomy in adult rodents (20, 21). These fluctuations in c-myc mRNA abundance are regulated post-transcriptionally and are dependent on the c-myc mRNA coding region, not the c-myc gene promoter (22-25). (iv) The CRD is an mRNA instability element in cells independent of other c-myc mRNA regions. When the c-myc CRD is inserted into the coding region of globin mRNA to generate globin-MYC-globin mRNA (Fig. 1A, bottom), this mRNA is destabilized in cells (10). (b) In cells, c-myc mRNA is degraded by alternative pathways, one of which is endonucleolytic. Like many other mRNAs containing AU-rich elements in their 3'-UTRs, c-myc mRNA can be degraded 3' to 5' in cells (16, 35). However, it can also be degraded endonucleolytically (16, 17). (c) Under appropriate conditions in polysome-based cell-free mRNA decay assays, c-myc mRNA is degraded by endonucleolytic cleavage within its CRD. The responsible endonuclease is associated with polysomes, because polysomes are the only cellular constituents in the assay mix (9).

Based on these findings, we sought to solubilize and characterize an endonuclease whose properties reflect those of the polysome-associated c-myc mRNase. The soluble liver enzyme, like the polysomal mRNase, is insensitive to RNasin, sensitive to VRC, and dependent on magnesium (Figs. 8 and 9). We have not proved that the purified enzyme is the only liver polysomal endonuclease with these properties. However, it is clear that the RSW from which the purification began does not contain multiple RNasin-insensitive endonucleases (Fig. 2).

Two findings suggest that the soluble endonuclease might be the ~39-kDa protein band marked by the arrow in Fig. 3. (i) The presence of this band correlates with endonuclease activity in heparin-Sepharose fractions (Fig. 4). (ii) The endonuclease migrates in the 35-40-kDa range by size exclusion chromatography and in a glycerol gradient (Fig. 5). It might be significant that higher molecular weight endonuclease activities were also detected in these assays. Perhaps this endonuclease, like other RNases, multimerizes or associates with other proteins (27-30).

The substrate specificity of the liver enzyme is not absolute but is nevertheless striking. Cleavage occurs in one major region of deproteinized 5'-CRD RNA (Figs. 6 and 12). The same region is also preferentially cleaved when FL-CRD RNA is the substrate (Fig. 10). This region is A-rich, and cleavage might occur at the A residues themselves. If so, it is unclear why nts 1727-1736 are preferentially cleaved, whereas other A-rich or purine-rich sites are spared (for example, nts 1758-1763). Two other sites in FL-CRD RNA (nts 1801-1804 and 1833-1835) are also A-rich but are not cleaved preferentially by the liver endonuclease. Perhaps the enzyme recognizes primary sequence and secondary structure, thereby limiting its access to most sites, even A-rich ones. We have postulated that unique features of the c-myc CRD make it an avid binding site for a shielding protein, the CRD-BP (9, 13). If so, it would be of interest to determine whether mutant CRD RNAs that bind poorly to the CRD-BP are also poor substrates for the endonuclease.

To our knowledge, the polysomal endonuclease is a novel enzyme. Several other vertebrate endonucleases have been reported by other labs. These include a 13.3-kDa human endonuclease that shares several core functional properties with the E. coli enzyme RNase E (36), a 65-kDa enzyme that also shares some properties with E. coli RNase E (37), an ~60-kDa Xenopus liver polysomal endonuclease that preferentially attacks albumin mRNA (38, 39), an ~120-kDa endonuclease from Xenopus and Drosophila cells that preferentially attacks maternal homeobox mRNAs (40, 41), and an ~68-kDa endonuclease from human T cells that preferentially attacks interleukin-2 mRNA in vitro (15). The endonuclease we have purified differs in at least one respect from all of these enzymes. None of these enzymes, including the one reported here, has yet been proved conclusively to be mRNase. This is unfortunate, because we will not fully understand how vertebrate mRNAs are degraded until we identify vertebrate mRNases (3, 4). By having partially purified a candidate c-myc mRNase, however, it should now be feasible to obtain cDNA for the enzyme and to analyze how the enzyme affects mRNA stability in cells.