©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
5`-Exonuclease-2 of Saccharomyces cerevisiae
PURIFICATION AND FEATURES OF RIBONUCLEASE ACTIVITY WITH COMPARISON TO 5`-EXONUCLEASE-1 (*)

Audrey Stevens (§) , Toni L. Poole (¶)

From the (1)Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

5`-Exonuclease-2 has been purified 17,000-fold from whole cell extracts of Saccharomyces cerevisiae. A 116-kDa polypeptide parallels the enzyme activity when the purified protein is examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Amino-terminal sequencing of the 116-kDa protein shows that the sequence agrees with that encoded by the HKE1 gene, previously reported to encode exonuclease-2. A 45-kDa polypeptide also parallels the enzyme activity upon purification, and Sephacryl S-200 molecular sieve chromatography of the purified enzyme shows a parallel elution of most of the 116- and 45-kDa polypeptides, suggesting a close association of the two. Enzyme instability has precluded a more detailed analysis of their associative properties. The enzyme hydrolyzes RNA substrates to 5`-mononucleotides in a processive manner. Measurements of its substrate specificity and mode of action are compared with 5`-exonuclease-1. Restriction cut single-stranded T7 DNA is hydrolyzed at approximately 5-7% of the rate of 18 S rRNA of yeast by both enzymes. That 5`-exonuclease-2 hydrolyzes in a processive manner and lacks endonuclease activity is shown by the finding that [5`-P]GMP is the only product of its hydrolysis of [-P]GTP-labeled synthetic RNAs. That 5`-exonuclease-2 hydrolyzes by a 5` 3` mode is shown by: 1) its poor hydrolysis of both 5`-capped and triphosphate-ended RNA substrates; 2) the products of its hydrolysis of [5`-P,H](pA); and 3) the accumulation of 3`-stall fragments when a strong artificial RNA secondary structure is present in synthetic RNAs. 5`-Exonuclease-1 hydrolyzes the synthetic RNAs and (pA) in an identical manner.


INTRODUCTION

The first purification and characterization of a 5` 3`-exoribonuclease was described in 1980(1) . The enzyme purified from Saccharomyces cerevisiae hydrolyzed substrates with 5`-phosphate end groups to 5`-mononucleotides by a 5` 3` mode of hydrolysis. Further study of the enzyme in this laboratory led to the cloning of the XRN1 gene encoding it and characterization of cells with a disrupted gene(2, 3, 4, 5) . These characterization studies showed that the 175-kDa protein may be involved in both pre-rRNA processing and mRNA turnover. The yeast cells containing a disrupted XRN1 gene still contained a second 5` 3`-exoribonuclease activity that had been briefly described(1, 6) . In cloning and characterization of the essential HKE1 gene of S. cerevisiae, Kenna et al.(7) found that the gene product, a 116-kDa polypeptide, had significant amino acid sequence homology to Xrn1.()Studies showed that antiserum against the 116-kDa protein immunoprecipitated the second partially purified exonuclease activity and that the level of this protein and the RNase activity were increased on overexpression of the HKE1 gene. The same gene was also cloned as RAT1 by Amberg et al.(8) by analysis of its effect on nuclear cytoplasmic mRNA transport and as TAP1 by De Segni et al.(9) on the basis of its effect on tRNA transcription activation. The different phenotypes found with temperature-sensitive mutants of the gene are reminiscent of the many phenotypes of XRN1 gene-disrupted yeast strains (10) and are discussed below. The pleiotropic effects caused by a modified HKE1 gene make further study of the encoded protein of considerable interest.

Purification of 5`-exonuclease-2 was undertaken so that NH-terminal sequencing of the 116-kDa protein could be done to show that it is indeed the HKE1 gene product and so that its substrate specificity and mode of action could be characterized and compared to Xrn1, the product of the analogous gene, XRN1. Purification of the 116-kDa protein and a co-purifying 45-kDa polypeptide to approximately 90% purity is reported here. The basic properties of the enzyme with poly(A) and RNAs as substrates have been determined with appropriate comparison to Xrn1.


EXPERIMENTAL PROCEDURES

Substrates

[H]Poly(A) (20 10 cpm/nmol) and [H]adenine-labeled yeast RNAs (14 10 cpm/nmol) were prepared as described previously(1, 3) . [H]T7 DNA (11 10 cpm/nmol)was prepared as described by Johnson and Kolodner(11) . The DNA was digested with HaeIII and for the single-stranded DNA, the fragments were heat-denatured. [5`-P,H](pA) was prepared as described previously for labeled (pA)(1) . The synthetic TAT RNA (540 nt) labeled in the RNA chain with [-P]GTP or in the cap structure with [H]methyl was synthesized as described by Stevens (6) using the Riboprobe Gemini II Transcription System (Promega). The P-labeled synthetic RNAs, MFA2 513, MFA2 489, and MFA2 490 were prepared in the same manner using SP6 polymerase and pRP513, pRP489, and pRP490, cut with SmaI. The plasmids contain the MFA2 gene in pGEM-3Z (12) and were obtained from Denise Muhlrad and Roy Parker, University of Arizona.

Purification of Enzyme

5`-Exonuclease-2 was purified as described below. All operations were carried out at 0-4 °C unless otherwise specified.

Step 1: Growth of S. cerevisiae

S. cerevisiae (YPH500 xrn1 BglII::URA3) was grown at 30 °C in YPD media (13) containing 2.5% dextrose to an OD of 10, collected, and washed with 200 ml of water/100 g of cells.

Step 2: Preparation of Crude Extract and Ribosome High Salt Wash

The yeast cells (300 g) were suspended in 500 ml of 20 mM Tris-HCl buffer (pH 7.6), containing 10% glycerol, 10 mM MgC1, 0.1 mM EDTA, 0.5 mM DTT, and the following protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml of antipain, leupeptin, pepstatin A, and aprotinin, and 2 µg/ml of tosyl-L-lysine chloromethyl ketone. These same protease inhibitors were used in all the buffers up to Step 5. The cells (three portions) were broken in a BeadBeater (Biospec Products) by grinding six times for 0.5-min intervals with brief cooling in between. The suspension was centrifuged at 12,000 g for 10 min, and the supernatant was spun at 27,000 g for 45 min. The resulting supernatant, called the crude extract, was then centrifuged at 28,000 revolutions/min for 4 h using a Spinco 30 rotor. The supernatant solution was poured off, and the ribosome pellets were rinsed quickly with about 2 ml of water. The 70,000 g pellet, being predominantly ribonucleoprotein and, possibly, partially of nuclear origin, is called the ribosome pellet. The ribosomes were then stored at -60 °C. The ribosome pellets were suspended by homogenization in 100 ml of 20 mM Tris buffer (pH 7.8) containing 0.5 M KCl, 0.1 mM EDTA, 0.5 mM DTT, and protease inhibitors. The resulting solution was stirred for 1 h and then centrifuged for 3 h at 45,000 revolutions/min in a Spinco 50 Ti rotor. The supernatant solution was removed by suction.

Step 3: Heparin-Agarose Chromatography

The high salt wash fraction (about 90 ml) was diluted with an equal volume of buffer A (20 mM Tris-HCl buffer (pH 7.6), 10% glycerol, 0.5 mM DTT) containing the protease inhibitors described in Step 2. The diluted fraction was applied to a heparin-agarose column (50 ml, 2.5 10 cm) prewashed with buffer A containing 250 mM (NH)SO. The column was then washed with 60 ml of the same buffer and eluted with a 200-ml linear gradient of 250-600 mM (NH)SO in buffer A. Five-ml fractions were collected every 5 min. The enzyme was eluted between fractions 30 and 40. The active fractions were combined (data shown in are the combined fractions (about 50 ml)) and solid (NH)SO was added to 60% saturation. After further stirring for 30 min, the suspension was centrifuged for 30 min at 27,000 g. The precipitate was dissolved in 3 ml of buffer A containing 200 mM KCl and protease inhibitors.

Step 4: Sephadex G-75 Column Chromatography

The (NH)SO fraction was applied to a Sephadex G-75 column (78 ml, 2 25 cm). The column was eluted with buffer A containing 75 mM KCl, and 1.3-ml fractions were collected and assayed. The enzyme activity was in the excluded fractions, which were combined (about 6.5 ml) and filtered for the Mono Q column.

Step 5: Mono Q HR Chromatography

The Sephadex G-75 fraction was loaded on a Waters Mono Q HR column (10/10) which had been preequilibrated with buffer A containing 50 mM KCl and 0.5 µg/ml of antipain and leupeptin. The column was washed (flow rate, 1 ml/min) with the same buffer (20 ml) and then eluted with a 50-ml linear gradient of 50-360 mM KCl in buffer A. The effluent was collected in 1-ml fractions. The enzyme was eluted at about 210 mM KCl, and the active fractions were combined (about 5 ml).

Step 6: Hydroxylapatite High Performance Chromatography

Fractions obtained from the Mono Q column were pooled and applied (flow rate, 1 ml/min) to a TosoHaas TSKgel hydroxylapatite column (HA-1000, 8/7.5) preequilibrated with buffer A containing 10 mM potassium phosphate buffer (pH 7.5) and 0.5 µg/ml of antipain and leupeptin. The column was washed with 20 ml of the same buffer and then eluted with 40 ml of a linear gradient of 10 mM to 350 mM potassium phosphate in buffer A. One-ml fractions were collected and assayed.

Step 7: Heparin High Performance Chromatography

The hydroxylapatite column fractions were combined (about 5 ml), diluted with 0.6 volume of the 10 mM potassium phosphate buffer described just above, and applied (flow rate, 1 ml/min) to a TosoHaas TSKgel Heparin-5 PW column (8/7.5). The column was then washed with 25 ml of buffer A containing 150 mM (NH)SO and 0.5 µg/ml of antipain and leupeptin and eluted with 40 ml of a linear gradient of 150-500 mM (NH)SO in buffer A. One-ml fractions were collected and assayed.

Comments on Purification Procedure

The enzyme could be frozen with only minor losses in activity at all steps up to the Mono Q HR chromatography. At that stage and at further stages, as much as 30-50% of the activity was often lost on freezing. Some stabilization could be obtained if albumin (37.5 µg (in 7.5 µl) of acetylated albumin (Bethesda Research Laboratory)) was added to the tubes in the fraction collector for the Mono Q, hydroxylapatite, and heparin-gel chromatography. The final enzyme fractions were frozen in aliquots suitable for further use.

Assay of Enzyme

Exonuclease-2 was assayed during the purification by using [H]poly(A) as the substrate and measuring the release of acid-soluble radioactivity. The procedure was similar to that described for Xrn1 (1) with slight modifications. The reaction mixtures (150 µl) contained: 4 nmol of [H]poly(A) (80 10 cpm), 90 nmol of unlabeled poly(A) (Takamine), 2 mM MgCl, 33 mM sodium glycinate buffer (pH 9.4), 50 mM NHCl, 25 µg of acetylated albumin, 0.33 mM DTT, and enzyme. After incubation at 37 °C for 15 min, the reactions were stopped by the addition of 100 µl of 7% HClO and allowed to stand on ice for 10 min. After centrifugation for 10 min in a microcentrifuge, radioactivity was determined with 100 µl of the supernatant solution. A reaction mixture lacking enzyme was used as a control. One unit of enzyme activity was defined as the amount needed to release 1 10 cpm of label in the 100-µl aliquot. This unit of exonuclease-2 activity can be correlated with a unit of Xrn1 previously reported (1, 14) by multiplying the exonuclease-2 units by 1.1. For enzyme assay, approximately 0.5-3 units of exonuclease-2 were used.

For the determinations of substrate and reaction specificity, the reaction mixtures (50 µl) contained 33 mM Tris-HCl buffer (pH 8.0), 2 mM MgCl, 50 mM NHCl, 0.5 mM DTT, 30 µg of acetylated albumin, and the amount of substrate and enzyme described in the figure and table legends. The mixtures were incubated for 10 min at 37 °C. For measurement of activity by release of acid-soluble label, 50 µl of 7% HClO were added, and after 10 min the mixtures were centrifuged for 5 min in a microcentrifuge. Radioactivity was determined with 50 µl of the supernatant solution.

Protein Sequencing

A heparin gel exonuclease-2 fraction (fraction 50, Fig. 1B) was precipitated with 10% trichloroacetic acid, and the pellet was washed with 100% ethanol. The sample was then electrophoresed on a 0.75-mm 8% SDS-polyacrylamide gel(15) . Following transfer to an Immobilon P membrane by electroblotting, the proteins were visualized with Coomassie Blue and the 116 and 45 kDa bands were excised(16) . The samples were submitted to the City of Hope Micro Sequencing and Mass Spectrometry Core Facility, Beckman Research Institute of City of Hope, Duarte, CA, for NH-terminal sequencing.


Figure 1: SDS-PAGE of purified enzyme fractions. Aliquots of the enzyme fractions from a hydroxylapatite column (A) (the aliquots contained 2.5 µg of albumin used for stabilization during purification) and from a heparin gel column (B) were prepared for electrophoresis by precipitation with trichloroacetic acid at a final concentration of 10%. The pellets were washed with 500 µl of ethanol, dissolved in a reducing buffer (Laemmli (15)), heated 4 min in a boiling bath, and loaded on 8% SDS-polyacrylamide gels (15). Protein was stained with Coomassie Blue. The units ( 10) of exonuclease-2 loaded were as follows: A, 0.3, 1.5, 2.3, and 0.9, in lanes 43-46, respectively; B, 0.3, 2.5, 7.0, 7.2, 2.4, and 0.2 in lanes 47-52, respectively.



Protein and Polynucleotide Determinations

Protein was determined by uv absorbance at 280 nm. The concentrations of polynucleotides were determined by uv measurements at 260 nm using the appropriate E and are expressed as nanomoles of nucleotide.

Paper Chromatography and Electrophoresis

Descending paper chromatography using Whatman No. 3MM paper was carried out for 18-24 h with the solvent n-propyl alcohol/NHOH/HO, 60:30:10 for separation of mono- and oligonucleotides and with the solvent isopropanol, NHOH, 0.1 M boric acid, 70:10:20, for the separation of 5`-AMP from 3`-AMP. High voltage paper electrophoresis was carried out at 2000 V for 75 min using pryridine-acetate buffer (pH 3.5).

Other Materials

The protease inhibitors and protein molecular mass standards were obtained from Sigma. The heparin-agarose, Sephacryl S-200, and Sephadex G-75-120 were also from Sigma.


RESULTS

Purification of 5`-Exonuclease-2

The results of the purification scheme for 5`-exonuclease-2 are summarized in . The ribosome high salt wash fraction contained about 50% of the activity of the crude extract, but the remainder of the activity was not completely recovered in either the pelleted ribosomes upon resuspension or in the supernatant fraction from the ribosome spin. These two fractions each had about 10% of the activity of the crude extract. The enzyme activity eluted as a single peak from all the columns. Because of the instability of the enzyme on freezing, the last three steps were sometimes done on the same day. The low levels of enzyme obtained made further manipulations very difficult. High level purification of 5`-exonucleases of higher eukaryotic cells has not been possible because of protein instability (see ``Discussion'').

Fig. 1shows an analysis of the hydroxylapatite (A) and heparin-gel (B) fractions by SDS-PAGE. Paralleling the enzyme activity from both columns were two protein bands of 116 and 45 kDa. (A parallel elution was also found on Mono S columns.) Densitometer scanning of the gel bands showed that the 45-kDa band was about 52% of the 116-kDa band. The scanning also showed that other minor bands (Fig. 1B) account for approximately 10% (fractions 50 and 51) to 15-20% (fractions 48 and 49) of the protein. The minor bands are not consistently found in the same amounts.

The enzyme fraction shown in Fig. 1B (fractions 48 and 49) was supplemented with albumin for stabilization, and the preparation was then chromatographed on a Sephacryl S-200 column. The results are shown in Fig. 2. Fig. 2(top) shows the profile of exonuclease activity eluted from the Sephacryl column in fractions 18-36. Approximately 70% of the enzyme activity was recovered. At the bottom of Fig. 2is shown an SDS-polyacrylamide gel analysis of the even-numbered fractions from the column. The peak of enzyme activity (fractions 20-24) contains both the 116- and 45-kDa polypeptides; however, approximately 15% (determined by densitometer scanning) of the 45-kDa protein eluted separately in the size range of 43-kDa (with ovalbumin as protein marker). By comparison with the protein standards (see elution positions of 150-, 68-, 43-, and 29-kDa polypeptides at top (arrows)), the main activity peak has a mass of about 150-160 kDa. The results suggest that the 45-kDa protein is a closely associating polypeptide. Fraction 30, which has a higher 45-kDa/116-kDa polypeptide ratio than the main activity peak showed no higher RNase or DNase activity.


Figure 2: Sephacryl S-200 chromatography of purified exonuclease-2. Aliquots of the exonuclease-2 fractions 48 (650 µl) and 49 (650 µl), shown in Fig. 1B, were combined and supplemented with 100 µg of acetylated albumin. The combined fraction was then applied to a 25 ml (1.5 14 cm) Sephacryl S-200 column, prewashed with buffer A containing 200 mM KCl, and leupeptin, 1 µg/ml. Acetylated albumin (25 µg in 5 µl) was added to collection tubes 15-23 since these tubes would not contain albumin from the load. The column was eluted with the same buffer, and 0.52-ml fractions were collected. Suitable aliquots were assayed, and fractions 18-36 (even-numbered tubes) were prepared for SDS-PAGE as described in Fig. 1. The lane marked Albumin on the gel contained 25 µg of the carrier albumin and the Load lane, 100 µl of the enzyme fraction loaded. The lane marked M is protein standards with the kDa values shown at the right. The Sephacryl column was calibrated using alcohol dehydrogenase (150 kDa), serum albumin (68 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa) as protein standards. The elution position of each of these polypeptides is shown at the top of the activity profile (arrows).



Both the 116- and 45-kDa polypeptides were seen in all final preparations of the enzyme in the same relative amounts, both paralleling the exonuclease activity. Due to the harsh conditions of SDS-PAGE, it was not possible to show renaturation of activity after elution of the bands from the gel. Non-reducing PAGE showed both polypeptide bands in the positions expected, so the two polypeptides are not linked by disulfide bonds. Nondenaturing PAGE and antibody immunoprecipitation studies were unsuccessful because of the instability of the enzyme. It will require further study possibly using a different experimental approach to determine if both polypeptides are required for exonuclease activity.

The specific activity of the highly purified exonuclease-2 using poly(A) as a substrate is about 10 times higher than Xrn1, highly purified in our laboratory(14) . Exonuclease-2 requires a 17,000-fold purification and Xrn1, a 1000-fold purification, so exonuclease-2 is present at about 6% of the protein level of Xrn1 in yeast cells.

NH-terminal Sequencing of the 116- and 45-kDa Protein Bands

An enzyme fraction (fraction 50) shown in Fig. 1B was used for NH-terminal sequence analysis as described under ``Experimental Procedures.'' The following NH-terminal sequence for the 116-kDa polypeptide was obtained: Gly-Val-Pro-Ser-Phe-Phe-Arg-Trp. The same NH-terminal sequence is encoded by the HKE1 gene. The NH-terminal sequence of the 45-kDa polypeptide did not match sequences in data bases.

Properties of Exonuclease-2

The basic requirements of the highly purified enzyme were analyzed using [H]poly(A) as a substrate. The optimum pH for enzyme activity is 8-8.8, but 85 and 90% of the activity are found at pH 7.0 and 9.4, respectively. Divalent cation is required for the reaction since 10 mM EDTA inhibits completely. Mg stimulates optimally at 2 mM, and Mn is about 75% as good at 100 µM.

Monovalent cation stimulates at 50-100 mM and becomes inhibitory above 150 mM. ATP does not stimulate the reaction.

Nature of the Product

The products of poly(A) hydrolysis by the purified enzyme are the same as reported for the partially purified enzyme(7) . 5`-AMP was identified by paper electrophoresis at pH 3.5 and distinguished from 3`-AMP by paper chromatography as described under ``Experimental Procedures.'' With [H]adenine-labeled 18 S rRNA, the labeled hydrolysis products are 5`-AMP and 5`-GMP as determined by paper electrophoresis. No oligonucleotide products were detectable. With synthetic [P]RNAs, prepared with SP6 RNA polymerase using [-P]GTP, [5`-P]GMP is the only product.

Substrate Specificity of Exonuclease-2 and Comparison to Xrn1

The substrate specificity of exonuclease-2 with RNA and DNA substrates and some of the same values with Xrn1 are shown in . Exonuclease-2 hydrolyzes yeast 25 S rRNA, 18 S rRNA, and tRNA at 16, 21, and 44%, respectively, of the rate of poly(A). Experiments with synthetic RNAs (see below) also show that the rate of poly(A) hydrolysis is about 2-5 times better than an RNA when examined at the same concentration of polynucleotide. Xrn1 hydrolyzes 18 S rRNA at about 77% of the rate of poly(A) (see also, reference(14) ). As the results in A show, both exonucleases hydrolyze HaeIII cut ssT7 DNA at about 5-7% of the rate of 18 S rRNA. The hydrolysis of poly(A) by exonuclease-2 is also strongly inhibited by poly(dC) and poly(dI), as has been previously described for exonuclease-1(14), showing an affinity of the enzyme for polydeoxynucleotides.

B shows the rates of hydrolysis of TAT RNA with a 5`-phosphate end group, a 5`-cap structure, and a 5`-triphosphate end group as compared to poly(A). The RNA with a 5`-cap structure is hydrolyzed at 2.1% of the rate of RNA with a 5`-phosphate by exonuclease-2. The same value is 0.5% for Xrn1. The 5`-triphosphate end group also restricts the hydrolysis rate with both enzymes. It has not yet been determined if the effect of the cap structure or 5`-triphosphate end group differs with different RNAs. The product of the cleavage of the RNA with a 5`-cap structure (with both enzymes) migrated with mGpppG upon paper electrophoresis at pH 3.5 and paper chromatography using n-propyl alcohol/NHOH/HO as the solvent (see ``Experimental Procedures'').

Mode of Hydrolysis of RNA by Exonuclease-2

Bio-Gel A-5m chromatography of reaction mixtures with poly(A) showed that the hydrolysis of 20 and 80% of the substrate follows a processive mechanism since there was no change in the size of the poly(A) remaining, and the only product detectable eluted at the position of 5`-AMP (data not shown). The data were the same as previously reported for Xrn1(1) .

To examine more closely the processivity of the enzyme using RNA substrates and to detect possible endonucleolytic activity, two synthetic RNAs were prepared, TAT RNA and MFA2 513 RNA. Analysis of the products of hydrolysis of the two RNAs with both exonuclease-2 and Xrn1 are shown in Fig. 3. Fig. 3A shows reaction mixtures with TAT RNA using two levels of each exonuclease (Xrn1, lanes 1, 3, and 5; exo-2, lanes 2, 4, and 6). Reaction mixtures 1 and 2 (lanes 1 and 2) contained 20 mM EDTA, resulting in no enzyme activity. Lanes 3 and 4 show reaction mixtures with a low level of each enzyme resulting in about 5% hydrolysis, and lanes 5 and 6, higher levels (40) of each enzyme resulting in about 90% hydrolysis. A similar analysis is shown in Fig. 3B for MFA2 513 RNA (no enzyme, lane 1; Xrn1, lanes 2 and 3; exo-2, lanes 4 and 5). No accumulation of intermediates of the hydrolysis above the low level found plus EDTA (Fig. 3A) or with no enzyme (Fig. 3B) is found, showing a highly processive reaction. The results also show that the enzyme has little or no endonucleolytic activity.


Figure 3: PAGE of the products of hydrolysis by exonuclease-2 and Xrn1 of synthetic RNAs. P-Labeled TAT RNA and MFA2 513 RNA were prepared as described under ``Experimental Procedures.'' The 5`-triphosphate terminus of each transcript was hydrolyzed to a 5`-monophosphate terminus by treatment with tobacco acid pyrophosphatase (see Table II). For A, TAT RNA (0.32 nmol, 26 10 cpm) was incubated in reaction mixtures (50 µl) as described under ``Experimental Procedures'' with 11 units of Xrn1 (lane 1), 26 units of exonuclease-2 (lane 2), 0.27 units of Xrn1 (lane 3), 0.65 units of exonuclease-2 (lane 4), 11 units of Xrn1 (lane 5), and 26 units of exonuclease-2 (lane 6). Reaction mixtures in lanes 1 and 2 contained 20 mM EDTA. For Fig. 3B, MFA2 513 RNA (0.25 nmol, 10 10 cpm) was incubated with no enzyme (lane 1), 0.22 and 4.4 units of XRN1 (lanes 2 and 3, respectively, and 2.6 and 52 units of exonuclease-2 (lanes 4 and 5, respectively). Following a 10-min incubation at 37 °C, the mixtures were extracted with phenol saturated with 10 mM Tris-HCl buffer (pH 7.6), 1 mM EDTA, and the RNAs were recovered from the aqueous phase by ethanol precipitation. The pellets were dissolved in loading buffer and examined by PAGE using a 6% sequencing gel (1.5 mm) (17).



An analysis of the direction of hydrolysis of an RNA chain by partially purified exonuclease-2 was previously presented in the paper of Kenna et al.(7) . Using yeast [5`-P,H]rRNA (25 S) as a substrate at 0 °C, release of label upon incubation with the enzyme was measured, and the results showed a much faster release of 5`-P label than H label, demonstrating that a 5` 3` mode of hydrolysis is a predominant one. That exonuclease-2 hydrolyzes the 5`-capped and 5`-triphosphate-ended RNAs poorly () also suggests a 5` 3` mode of hydrolysis. Further evidence for a 5` 3` direction of hydrolysis was obtained by determining label in the products of hydrolysis of [H](pA) containing 5`-terminal P label. If the hydrolysis proceeds in a 5` 3` direction, no P label should be found in (pA) if it is a detectable intermediate. The results are shown in Fig. 4. Little or no P label was found in (pA). The results are similar to those reported for Xrn1 and suggest that, as with Xrn1, (pA) is poorly hydrolyzed.


Figure 4: Paper chromatography of the products of hydrolysis by exonuclease-2 of [5`-P, H](pA). [5`-P, H] (pA) (0.5 nmol, 1600 cpm of H label, and 8400 cpm of P label) was incubated for 30 min at 37 °C in a 50-µl reaction mixture as described under ``Experimental Procedures'' with 20 units of exonuclease-2 in the presence and absence of 20 mM EDTA. The reaction mixtures were chromatographed as described under ``Experimental Procedures.'' The paper was cut into 1/2-inch segments from the origin, and the segments were eluted with water and counted. (-, + EDTA; - - - - , no EDTA).



Using synthetic RNAs containing a (G) tract, which introduces a strong secondary structure (see Ref.(18) ), a 3`-stall fragment is found with both exonucleases. This finding is of considerable interest in view of recent in vivo studies of Vreken and Raué (19) and Decker and Parker (20) using S. cerevisiae. These studies showed that turnover of the encoded mRNAs is stalled when poly(G) tracts are inserted into specific genes due to blockage of 5`-exonucleases. mRNA fragments which are trimmed at the 5`-end to the site of the poly(G) tract accumulate in the yeast cells. Our results with both exonucleases are presented in Fig. 5. The products of the hydrolysis of MFA2 489 RNA are shown in lanes 1-3. This RNA contains a (G) tract inserted at nt 178 in the 3`-untranslated sequence of MFA2 mRNA. The (G) tract is about 170 nt from the 3`-end of the RNA. MFA2 490 RNA is shown in lanes 4-6, and this RNA contains two (G) tracts at nt 28 and nt 178 of the MFA2 RNA. The (G) tract at nt 28 is about 360 nt from the 3`-end of the RNA. The control RNA, MFA2 513, containing no (G) sequence is shown in lanes 7-9. Lanes 1, 4, and 7 were reaction mixtures with no enzyme, lanes 2, 5, and 8 were the same with Xrn1, and lanes 3, 6, and 9 show the results with exonuclease-2. With both exonucleases, the accumulation of an RNA fragment is found and the length of the stall fragment is close to the length of the RNA from just 5` of the (G) tract to the 3`-end of the RNA. With each RNA, the size of the accumulated fragment is changed with alteration of the 3`-end of the RNA by use of a different restriction site to cleave the DNA for transcription. The only other detectable product of the hydrolysis is 5`-GMP. The nature of the secondary structural element involved in the blockage of the enzymes is under further investigation so that suitable stall sequences can be introduced into genes to further delineate the pathways in which the exonucleases are involved in RNA metabolism.


Figure 5: PAGE of the products of hydrolysis by exonuclease-2 and Xrn1 of MFA2 489 RNA, MFA2 490 RNA, and MFA2 513 RNA. P-Labeled MFA2 RNAs were prepared as described under ``Experimental Procedures.'' The 5`-triphosphate terminus was hydrolyzed to a 5`-monophosphate terminus as described in Table II. The RNAs were then purified by electrophoresis on a polyacrylamide-urea gel followed by excision and elution of the desired band from the gel with Probe Elution Buffer (Ambion). Reactions mixtures (50 µl) were as described in Table II and contained no enzyme (lanes 1, 4, and 7), 4.4 units of Xrn1 (lanes 2, 5, and 8), and 52 units of exonuclease-2 (lanes 3, 6, and 9). After 10 min at 37 °C, the reactions were stopped by the addition of EDTA to 20 mM, and 10 µl of each reaction mixture were added to 10 µl of loading buffer, heated 4 min at 80 °C, and analyzed by PAGE using a 6% sequencing gel (1.5 mm) (17).




DISCUSSION

Exonuclease-2 of S. cerevisiae was highly purified so that the basic features of its RNase activity could be determined and compared to those of Xrn1. Preliminary studies of the enzyme had been done previously in this laboratory, when upon purification of both Xrn1 and an mRNA decapping enzyme, two peaks of 5` 3`-exonuclease activity were detected using [H]poly(A) as a substrate (1, 6). These two peaks were routinely separated by hydroxylapatite chromatography. When the gene for Xrn1 was cloned and disrupted, the xrn1 yeast cell extracts still contained the poly(A)-exonuclease activity that eluted first from the hydroxylapatite columns(2) . The earlier studies of the enzyme with poly(A) as a substrate are summarized briefly in the paper of Kenna et al.(7) describing the cloning of the HKE1 gene. In the same studies it was shown that antiserum against the 116-kDa polypeptide immunoprecipitated exonuclease-2 activity. A finding in the antibody studies was that the partially purified, overexpressed exonuclease-2 had a lower specific activity (activity/amount of immunoreactive 116-kDa polypeptide) than exonuclease-2 from control yeast cells (see B, Ref. 7). It was mentioned in the paper that the overexpressed protein might differ in a structural modification(s) affecting RNase activity or immunoreactivity. This result could possibly be due to the 45-kDa copurifying polypeptide described here limiting the amount of active enzyme. The contribution of this polypeptide to the activity and properties of exonuclease-2 will be investigated further when larger amounts of the enzyme can be obtained.

The amino acid sequences of Xrn1 (175-kDa) and exonuclease-2 (116-kDa polypeptide) show significant homology as reported previously(8, 21) . A block of homology is found close to the NH terminus of both proteins and is composed of seven short stretches of homology. Matches of 18, 14, and 11 amino acids are found. This region is separated from a second homologous region by a sequence predicted to be -helical in both proteins. In the most COOH-terminal portion conserved, amino acids 626-728 of the 116-kDa polypeptide and 532-634 of Xrn1, there is a sequence similarity of 50%. No analogies of the gene sequences to reported functional domains are found. It will be of interest to also compare the sequence of the 45-kDa associative polypeptide when the gene encoding it is cloned.

The catalytic properties of Xrn1 and exonuclease-2 with RNA are also very similar, as reported above. Exonuclease-2 has a higher specific activity (10) with poly(A) as a substrate, but with the RNAs examined, its activity is the same or only two to three times better than Xrn1. This difference with exonuclease-2 may result from either primary or secondary structural features of the polynucleotides affecting the rate of cleavage. At similar substrate concentration, both enzymes hydrolyze 18 S rRNA about 15-20 times better than restriction enzyme cut single-stranded DNA. Xrn1 has been found by others to catalyze DNA strand exchange (11) and endonucleolytic cleavage of DNA oligonucleotides containing G sequences that can form a G-tetraplex structure(22) . These activities have not been examined at this time for exonuclease-2. Functionally, it appears that both Xrn1 and exonuclease-2 are, at least in part, involved in RNA metabolism as RNases, and this aspect of their activity is the focus of this laboratory.

Functionally, the HKE1 gene was also cloned in two other laboratories. Cloning and characterization of the RAT1 gene by Amberg et al.(8) showed that the gene affected nucleocytoplasmic export of mRNA. As visualized with an RNA localization assay, a rat 1-1 allele conferred temperature-sensitive accumulation of poly (A) RNA in one of several intranuclear spots at the nuclear periphery. They found that pre-rRNA processing was unaffected in rat 1-1 strains except for an inducible defect in trimming the 5`-end of the 5.8 S rRNA. TAP1 was cloned using a genetic selection system based on nonsense suppression in S. cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III(9) . The gene activated the expression of a tRNA gene with a defective internal promoter. Results of the studies suggested that the gene product has a role involving yeast chromatin structure and chromosome-associated functions(21) .

Henry et al.(23) have shown that the 5`-end of yeast 5.8 S rRNA can be generated by exonucleases from an upstream cleavage site in the pre-rRNA. In cells mutant for either the XRN1 gene or the RAT1 gene, the pathway is inhibited. The authors conclude that Xrn1 and Rat1 likely function as 5` 3`-exonucleases in vivo and that the apparent diversity of phenotypes found with mutants arises from the fact that the enzymes are catalytic subunits of different complexes involved in RNA metabolism. The XRN1 gene is not essential, while the HKE1 gene is an essential gene. The results of Kenna et al.(7) showed that overexpression of the XRN1 gene does not rescue HKE1 gene disruptants. Xrn1 is predominantly a cytoplasmic protein,()and the data of Kenna et al.(7) and Amberg et al.(8) suggest that exonuclease-2 can be found in the nucleus. It is possible that exonuclease-2 also acts as a cytoplasmic protein. The results of Muhlrad et al.(12) which show that the primary mRNA decay pathway in yeast involves 5` 3`-exoribonuclease digestion suggest that exonuclease-2 may substitute for Xrn1 in xrn1 cells. They find that the decapped mRNA that accumulates in the xrn1 cells is less stable than the fragments that are found when a poly(G) tract is present in the 5`-untranslated region of a specific mRNA. We find that xrn1 yeast cells containing a multicopy HKE1 plasmid have a lower level of both deadenylated mRNA species and the internal transcribed spacer fragment of pre-rRNA than xrn1 cells without the plasmid.()The accumulation of these RNAs was described previously in the xrn1 cells(3, 4, 5) .

Studies of 5` 3`-exoribonucleases of higher eukaryotic cells lag behind the studies with the yeast enzymes, and only partially purified enzymes have been described. Lasater and Eichler (24) described a 5` 3`-exoribonuclease isolated from Ehrlich ascites tumor cell nucleoli and suggested that it could be involved in pre-rRNA processing. In this laboratory(25) , a 5` 3`-exoribonuclease was purified from human placental nuclei. The RNA substrate specificity was similar to that of exonuclease-2. Murthy et al.(26) described a nuclear exoribonuclease of HeLa cells that degraded uncapped but not capped RNA substrates. Coutts and Brawerman(27, 28) have described a 5`-exoribonuclease from cytoplasmic extracts of mouse sarcoma 180 ascites cells. It will be very interesting to compare the higher eukaryotic enzymes with the yeast enzymes when the eukaryotic ones are more highly purified. Both Murthy et al.(26) and Coutts and Brawerman (27, 28) found that a high level of purity could not be attained because of enzyme instability.

  
Table: Purification of S. cerevisiae exonuclease-2

The starting material was 300 g of yeast cells (S. cerevisiae YPH500 xrn1 BglII::URA3). All fractions were assayed as described under ``Experimental Procedures.''


  
Table: Substrate specificity of exonuclease-2

The reaction mixtures (50 µl) were as described under ``Experimental Procedures.'' In A., 1.5-1.85 nmol of each H-labeled substrate was used and in B., about 0.32 nmol of each substrate was used. For the poly(A) reactions in A., 1.3 units of exonuclease-2 and Xrn1 were used and a suitably larger amount to give measurable hydrolysis of the RNAs and DNAs. In B., 0.2 unit of each enzyme was used for the poly(A) and 5`-phosphate TAT RNA and 11 units were used for the capped and triphosphate-ended RNAs. The TAT RNA with a 5`-triphosphate terminus was labeled in the chain using [-P]GTP in the synthesis reaction (see ``Experimental Procedures''). The TAT RNA with a 5`-phosphate terminus was prepared from the triphosphate-ended RNA by incubation of 2 nmol with 30 units of tobacco acid pyrophosphatase (Epicentre Technologies) in a 150-µl reaction mixture according to the manufacturer's directions. The hydrolysis of these two RNAs was measured by release of acid-soluble P label. The TAT RNA with a 5`-cap structure was prepared with [H]adenosylmethionine to label the m G of the cap structure. Its hydrolysis was measured by acid-solubilization of H label and by determination of H label released from RNA by paper electrophoresis at pH 3.5.



FOOTNOTES

*
This work was supported by the Office of Health and Environmental Research, U. S. Department of Energy Contract DE-AC05-84OR21400 with the Martin Marietta Energy Systems, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 615-574-1196; Fax: 615-574-1274.

Supported by National Cancer Institute postdoctoral Training Grant CA 09336.

The abbreviations used are: Xrn1, 5`-exonuclease-1; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; nt, nucleotide; cpm, counts/min.

A. Johnson and R. Kolodner, personal communication.

A. Stevens, unpublished observations.


ACKNOWLEDGEMENTS

We thank Denise Muhlrad and Roy Parker for sending plasmids pRP513, pRP489, and pRP490. We are also very indebted to Arlen Johnson and Richard Kolodner for a gift of purified Xrn1, used for the experiments in Fig. 3and Fig. 5. We thank Lorraine Symington for communicating results on the DNase activity of 5`-exonuclease-2. Dev. K. Niyogi, K. Nandagopal, and Eric Larsen provided assistance and advice in the early stages of the purification of the enzyme. We thank Stephen J. Kennel and Patricia Lankford for help and advice with preparing the 5`-exonuclease-2 sample for NH-terminal sequencing.


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