a 5′ → 3′ exoribonuclease of saccharomyces cerevisiae: size and novel substrate specificity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 252, No. 2, February 1, pp. 339-347, 1987

A 5’ --* 3’ Exoribonuclease of Saccharomyces cerevisiae: Size and Novel Substrate Specificity’72

AUDREY STEVENS3 AND MARILYN K. MAUPIN

Biology ZXrisim, Oak Ridge National Lobcwatcny, Oak Ridge, Tennessee 37831

Received July 1, 1986, and in revised form October 7, 1986

The purification scheme for a 5’ --) 3’ exoribonuclease of Saccharmyces wmvisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5’ phosphorylation for activity and that poly(A) with a 5’-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5’-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A) . poly(U) and poly(A) . poly(dT) are hydrolyzed at 60 and 50%) respectively, of the rate of poly(A) at 37°C. The RNase H activity of the enzyme can also be demonstrated using an RNA. Ml3 DNA hybrid as a substrate. When poly(dT) . poly(dA) with a 5’-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) “tail,” removing the last ribonucleotide, but does not hydrolyze the poly(dA). o 1987 Academic press, I,,~.

An exoribonuclease which cleaves by a 5’ --f 3’ mode, yielding nucleoside 5’-phos- phates, has been purified from yeast in this laboratory (1) and, more recently, from nucleoli of Ehrlich ascites tumor cells (2). The unique mode of hydrolysis of RNA by the enzyme makes it a candidate for several important processing and turnover reactions. An enzyme of this type may be involved in the turnover of mRNA as earlier

i This investigation was supported by Public Health Service Grant AI 20982-03 from the National Insti- tutes of Health and by the Office of Health and En- vironmental Research, U.S. Department of Energy, under Contract DE-AC05-84OR21-400 with the Martin

Marietta Energy Systems, Inc. ‘The U.S. Government’s right to retain a nonex-

clusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is ac- knowledged.

3 To whom correspondence should be addressed at the Biology Division, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, TN 37831.

suggested (1). Its location in nucleoli of mouse cells makes it a strong candidate for possible 5’-trimming reactions in rRNA maturation. An enzyme of this type could also function in turnover of introns or in the removal of RNA primers involved in DNA replication.

The previous reports from this laboratory (1,3) dealt with the nature of the reaction using poly(A) and oligo (A)s as substrates. Both poly(A) and rRNA were used to demonstrate that the enzyme hydrolyzes by a processive mechanism. The enzyme was highly purified and preliminary studies suggested that it was a large protein. The results presented here demonstrate that the enzyme from yeast can be purified to near homogeneity by use of poly(A)- agarose chromatography. Using gel electrophoresis and antibody reactivity, its size has been determined. A study of its substrate specificity has been done which demonstrates a new feature-its novel hy-

339 0003-9861/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

340 STEVENS AND MAUPIN

drolysis of double-stranded polymers. Its unique RNase H activity has been examined.

MATERIALS AND METHODS

ModiJied Puti&catim of the Enzyme

Preparation of high-sped pellet fraction Smch- romyces cereuisiae S288C was grown and stored as previously described (1). For preparation of larger

amounts of enzyme, 400 g of yeast were used as the starting material. After washing with water, the cells

were suspended in 1 liter of the buffer previously described (l), but also containing the protease inhibitors,

leupeptin (1 pg/ml) and antipain (0.5 pg/ml). The suspension was passed through a Gaulin press three

times, cooling to 10°C between passages. The extract was then centrifuged in the manner previously

described for preparation of the high-speed pellet fraction (1).

High-salt wash of p&A fraction, DEAE-cellulose chromatography, and hydroxylapatite chrcmzabgraphy.

These procedures were carried out as previously described with the appropriate increases in buffer vol-

ume and column size. The hydroxylapatite column fractions were concentrated by membrane ultrafil-

tration (Amicon YM-10 filter). Phosphocellulose chromatography. The next two

steps were carried out in duplicate using one-half

portions (about 2 ml) of the concentrated hydroxylapatite fraction. The fraction was dialyzed for 3 h against 500 ml of 20 mM Tris-HCl buffer (pH 7.8) con-

taining 10% glycerol and 0.5 mM dithiothreitol (Buffer

A) plus 0.2 M KCI. It was chromatographed on a 0.9 X g-cm column of phosphocellulose (Whatman Pll), equilibrated by prior washing with 250 ml of the same

buffer. The column was eluted with an 80-ml linear gradient of from 0.3 to 9.65 M KC1 in Buffer A, and 2-

ml fractions were collected. The enzyme activity was eluted in fractions 16-21, and these fractions were

concentrated by membrane ultrafiltration as described above.

Poly(A)-agarose chromatography. The phosphocellulose concentrate was dialyzed for 3 h against Buffer

A containing50 mM NaCl. The concentrate was applied to a column (0.9 X 4.8 cm) of poly(A)-agarose (Sigma P1390) equilibrated with the same buffer, and the eol- umn was washed with 36 ml of the same buffer. It

was then eluted with eight fractions of 3 ml of Buffer A plus 100 mM NaCl, followed by eight fractions of 3 ml of Buffer A plus 0.6 M NaCl. The enzyme was eluted

predominantly with 0.6 M NaCl, and the active fractions were combined and concentrated by membrane ultrafiltration as described above. The enzyme was stored in aliquots at -40°C and exhibited a stability similar to that previously described (1).

Preparation of Substrates

[BHjpoly(A) (60 X 103 cpm/nmol), [eH]poly(C) (100

X lOa cpm/nmol), and rH]poly(U) (65 X 108 cpm/nmol)

were prepared as described previously for poly(A) (3). The poly(A) was about 170 bp long and the poly(U) and poly(C) about 50, from end group analyses.

Poly(A) was used without 5’-end phosphorylation except in the reaction mixture shown in Table II. For phosphorylation of the 5’-end of the polymers, reaction

mixtures (0.3 ml) containing 100 nmol of the polymer,

50 mM Tris-HCl buffer, pH 9.0, 10 mM MgClx, 5 mM dithiothreitol, 5 mM ATP, and 30 units of T4 polynucleotide kinase (Bethesda Research Laboratories)

were incubated for 1 h at 37°C. The polymers were

recovered by ethanol precipitation. Yeast 18s rH]rRNA (36 X 1Oa cpm/nmol) was prepared as de-

scribed (4). [8H]poly(A). poly(U) and l?Hlp~l~(A). poly(dT) hybrids were prepared by incubating the

polymers in 20 mM Tris-HCl (pH 8.0), 50 mM NH&l, and 2 mM MgClz for 15 min at 6O”C, and then at 37°C

for 1 h. The 5’-terminal phosphate of poly(dT) was removed in reaction mixtures (0.6 ml) containing 270 nmol of poly(dT), 100 mM Tris-HCl buffer, pH 8.0, and

100 pg of Eschtichia coli alkaline phosphatase

(Sigma). After 45 min at 37”C, the poly(dT) was recovered by phenol extraction and ethanol precipita-

tion. rHJp~ly(A) with a 5’-triphosphate end group was prepared with E. coli RNA polymerase (Boehringer

Mannheim) using poly(dA) . poly(dT) as a template.

The reaction mixture (1 ml) contained poly(dA). poly(dT) (500 nmol), PH]ATP (125 nmol, 66 x 103 cpm/nmol), 20 mM Tris-HCl buffer (pH 7.7), 10 mM

MgClz, 10 mM 2-mercaptoethanol, and 20 units of RNA polymerase. After 15 min at 37’C, the reaction mixture

was made 20 mM in EDTA and 0.2% in sodium dodecyl sulfate and extracted with phenol. After centrifuga-

tion, the aqueous layer was made 400 mM in ammo- nium acetate, and the product was precipitated with ethanol and dissolved in 0.5 ml of 10 mM Tris-HCI

buffer (pH 7.7) containing 10 mM MgClz. RNase-free DNase (5 pg; ICN) was added and the reaction mixture

was incubated for 15 min at 37°C. It was then applied to a Bio-Gel A-5m column (2 X 25 cm) and eluted with

20 IIIM Tris-HCl buffer (pH 7.0) containing 100 mM KCl. The [‘H]poly(A) was collected in the excluded

volume and precipitated with ethanol.

[aH]poly(A)-$P]poly(dA) . poly(dT) was prepared with the Klenow fragment (Bethesda Research Lab-

oratories) using poly(dT) (treated with alkaline phosphatase as described above) as a template, @lpoly(A) as a primer, and [a-“PjdATP as a substrate. The re-

action mixture (0.3 ml) contained [aH]poly(A) (17.8 nmol), poly(dT) (80 nmol), @P]dATP (75 nmol, 5.3 X lo6 cpm/nmol), dTTP (25 nmol), 80 ml Tris-HCl buffer (pH 8.6), 80 mM NaCl, 13 mM MgClz, and 5 units of the Klenow fragment. The mixture was incubated

A 5’ + 3’ EXORIBONUCLEASE OF YEAST 341

for 45 min at 37°C and sodium dodecyl sulfate was

then added to 0.1% and EDTA to 6.6 mhi. The mixture

was extracted with phenol and the product was precipitated with ethanol. Approximately 1 nmol of

dAMP was incorporated per 5 nmol of [aHlpoly(A). A fH]RNA *Ml3 DNA hybrid was prepared with

E. wli RNA polymerase using Ml3 DNA as a template

and pCpA (prepared by phosphorylation of CpA (Pharmacia) with T4 polynucleotide kinase) as a

primer. The reaction mixture (0.2 ml) contained Ml3

DNA (20 nmol), 0.21 mM pCpA, 10 mM MgClz, 20 mM Tris-HCl buffer (pH 8.1), 20 rnEd 2-mercaptoethanol,

UTP, CTP, and GTP (2 nmol each), [BHJATP (2 nmol, 7 X lo6 cpm/nmol), and 4 units of RNA polymerase.

The reaction mixture was incubated for 30 min at 3’7”C, and the product was extracted with phenol and precipitated with ethanol. Approximately 2 nmol of

RNA was formed.

Assay of Exoribonuclease For measurements of the activity of the enzyme

during purification, the assay measuring acid solubilization of [3H]poly(A) as previously described (1) was used. One unit of enzyme was the amount needed

to release 5 nmol of CaH]AMP from 150 nmol of

[3H]polyA per 30 min at 37°C. For the determinations of substrate specificity, the reaction mixtures (50 ~1)

contained 33 mM Tris-HCI buffer (pH 8.0), 2 mM MgClz, 50 mM NH&I, 0.5 mM dithiothreitol, 30 pg of acetylated bovine serum albumin (Bethesda Research

Laboratories), and the substrate and amount of en-

zyme described in the figure and table legends. The mixtures were incubated for 10 min at 37”C, then 50 pl of 7% perchloric acid was added, and after 5 min

the mixtures were centrifuged for 5 min in an Ep- pendorf centrifuge. Radioactivity was determined

with 50 pl of the supernatant solution.

Preparation of Antisera

The high molecular weight (162,000 Da) band from polyacrylamide gels was excised and used as an an-

tigen for immunization of a rabbit as described by

Eberle and Courtney (5). Four booster immunizations

were administered at 2-week intervals and 10 days after the fifth injection, the rabbit was bled and

the sera were prepared as described by the same authors (5).

Protein and Polynucleotide Determinations

Protein was determined by uv absorbance at 230 nm. The concentrations of polynucleotides were de-

termined by uv measurements at 260 nm using the appropriate EM and are expressed as nanomoles of

nucleotide.

Other Materials

Unlabeled synthetic polynucleotides were obtained

from Pharmacia. T5 DNA and Ml3 DNA were the

kind gifts of Robert Fujimura and Sankar Mitra, respectively. Marker proteins as well as leupeptin and antipain were obtained from Sigma. Sephadex G-200

was from Pharmacia, and Sl nuclease from Nzymes.

RESULTS

Puri$cation and Size of the Exwibmuclease

Several modifications, described under Materials and Methods, have been made in the purification scheme previously reported for a 5’ + 3’ exoribonuclease of yeast (1). The results of a typical preparation from the hydroxylapatite column chromatography through the poly(A)-agarose chromatography are summarized in Table I. The earlier steps show protein and activity values similar to those reported previously (l), except that the specific activity of the

TABLE I

PURIFICATIONOFTHEYEAST~'+~'EXORIBONU~LEASE

Fraction Volume Protein Total activity Specific activity

(ml) (w/ml) (units X lo-‘) (units X 10m3/mg)

Hydroxylapatite

concentrate Phosphocellulose

concentrate Poly(A)-agarose

concentrate

4 4.6 480 26

3.2 0.75 192 80

2.0 0.2 80 200

Note. The purification data are for 400 g of yeast cells. The enzyme was assayed with [3H]poly(A) as a

substrate as previously described (1).


different fractions is about 2.5- to 3-fold higher, apparently because the poly(A) preparation used in the assays is a better substrate. (The influence of modification of the 5’-end of poly(A) on its activity is described below.) The overall purification of the enzyme is lOOO-fold.

To determine the apparent molecular weight of the exoribonuclease, the enzyme was subjected to molecular-sieve chromatography to determine the Stokes radius, and to gradient centrifugation to determine the sedimentation coefficient. The enzyme was chromatographed on a Se- phadex G-200 column standardized with proteins of known molecular weight (cat- alase, d-globulin, bovine serum albumin, and ovalbumin). A single peak of RNase activity was eluted close to the position of elution of &globulin. A Stokes radius of 50 A was determined from the elution volume of the nuclease according to method of Sie- gel and Monty (6). A sedimentation coefficient of ‘7.5 S for the exoribonuclease was estimated according to the method of Mar- tin and Ames (7) using d-globulin, bacterial alkaline phosphatase, bovine serum albumin, and ovalbumin as markers on sucrose gradients. Using the Svedberg equation (6) with the values for Stokes radius and sedimentation coefficient, an apparent molecular weight of 158,000 was calculated. (A partial specific volume of 0.73 was as- sumed.)

The highly purified enzyme was also subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Equal amounts of activity (1000 units) of a phosphocellulose column fraction and a poly(A)-agarose fraction were electrophoresed on an 8% polyacrylamide gel. The results of the electrophoresis are shown in Fig. 1. A major band of high molecular weight with the same intensity is found with the two enzyme fractions. Only minor other bands (40%) are found with the poly(A)-agarose enzyme fraction. The small band just below the major band is found with all preparations and is a more dominant band when the protease inhibitors antipain and leupeptin are not in- cluded in the buffers used in the purification procedure. An M, of 162,000 can be

103 units 10’units P-cellulose IA)“-agorose

kOa

- 200

- 92

- 68

- 43

- 25

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis of the exoribonuclease. 1000 units of

the phosphocellulose concentrate and 1000 units of the poly(A)-agarose concentrate were electrophoresed

on an 8% acrylamide gel according to the procedure of Laemmli (8).

calculated for the major band. Antiserum has been prepared by injection of the major gel band into a rabbit (see Materials and Methods), and the antiserum leads to inhibition of the exoribonuclease activity (Fig. 2).

Substrate Specificity Studies

Hydrolysis rates of dgerent polyribmu- cleotides and efect of $-end groups, Differ- ent synthetic polyribonucleotides (before and after 5’-phosphorylation) and rRNA were compared as substrates for the highly purified exoribonuclease. Table II(A) shows that poly(A) and poly(C) containing 5’- phosphate end groups are the best substrates while poly(U) after phosphorylation is hydrolyzed at only 12% of the rate of poly(A). Untreated poly(U) and poly(C)

A 5’ -* 3’ EXORIBONUCLEASE OF YEAST 343

2 8

FIG. 2. Effect of antisera against the major gel band

on exoribonuclease activity. Reaction mixtures with

[aH]poly(A) (1 nmol) were as described under Mate- rials and Methods and contained 1.76 units of enzyme. Normal rabbit sera was used to make each reaction

mixture contain 8 ~1 of total sera.

apparently lack 5’-phosphate end groups and are essentially inactive as substrates. Untreated poly(A) is hydrolyzed at about one-third the rate of phosphorylated poly(A). The poly(A) preparations apparently contain a somewhat variable amount of 5’-phosphate ends, but even after treatment with alkaline phosphatase, 20% of the maximal activity is found (3). Poly(A) with a 5’-triphosphate end group also was prepared and as shown in Table II(A), it is a much poorer substrate. The overall studies as reported here and in Ref. (3) show that polyribonucleotides with a 5’-OH, 5’-triphosphate, or 5’-capped end are hydrolyzed at less than 15% of the rate of the same polymers with a 5’-phosphate end group. Poly(I) is not a substrate for the exoribonuclease as measured by acid solubilization of either 260-nm absorbing material or 5’- 32P label incorporated with T4 polynucleotide kinase (data not shown). Yeast 18s rRNA is hydrolyzed, under the conditions shown in Table II, at about 22% of the rate of poly(A). Phosphorylated poly(A) has a l&fold higher KM (200 PM, as nucleotide than 18s rRNA (13 PM, as nucleotide).

Inhibitory properties of synthetic poly- dmxyribonucleotid Villadsen et al. (9) described a DNase of S. cerewkiae that hydrolyzes DNA by a 5’ -+ 3’ mode, producing

5’-mononucleotides, and the results raised the possibility that the 5’ + 3’ exoribonuclease and the DNase may be the same enzyme. Both double- and single-stranded T5 DNA were again tested as substrates of the highly purified exoribonuclease under conditions similar to those used in the assay of the DNase (9). As Table II(B) shows, no hydrolysis was observed. That a chain of poly(dA) at the 3’-end of a segment of poly(A) is also not hydrolyzed is described below.

It was found during the studies of substrate specificity that poly(dI), poly(dT), and poly(dC) are strong competitive inhibitors of the hydrolysis of the noncomplementary ribopolymers, showing that the enzyme has an affinity for these deoxypo- lymers. Figure 3(A) shows the inhibition of poly(C) hydrolysis by different polymers. Poly(dT) and poly(dC) are very inhibitory, while poly(d1) is less so. The substrate ribopolymers, poly(A) and poly(U), also were tested for their effect on the hydrolysis of poly(C). Poly(A) is not inhibitory while poly(U), a poorer substrate, is slightly inhibitory. Figure 3(B) shows the same type

TABLE II

FEATURESOFTHE SUBSTRATE SPECIFICITY

OFTHEENZYME

Relative Substrate activity

A. [3H]poIy(A), untreated 1.00 [3H]p~ly(A), 5’-phosphate end 3.50 [3H]p~ly(A), 5’-triphosphate end 0.43

[aH]poly(C), untreated <.lO [3H]poly(C), 5’-phosphate end 1.21

[‘H]poly(U), untreated <.lO [3H]poly(U), 5’-phosphate end 0.42

[‘HIyeast 18s rRNA 0.77

B. r3H]T5 DNA, native <.Ol [3H]T5 DNA, heated <.Ol

Note. The reaction mixtures (50 ~1) were as de-

scribed under Materials and Methods and contained

0.9-1.1 nmol of each polynucleotide and 1 unit of enzyme.


0% a------ .--- - _ ____ --.

0.2 0.6 I.0 0.2 0.6 I.0

POLYNUCLEOTIDE ADDED (nmol)

FIG. 3. Inhibition of poly(C) and poly(A) hydrolysis by synthetic polydeoxyribonucleotides. The reaction mixtures were as described under Materials and Methods with 2 nmol of [%Ilpoly(C) in (A)

or 2 nmol of rHlp~ly(A) in (B) as substrate, 1.5 units of enzyme, and the concentrations of competing

polynucleotide shown.

of effect with labeled poly(A) as the substrate. The noncomplementary polymers, poly(d1) and poly(dC), are very inhibitory, while poly(dT), a complementary polymer is less so. Unphosphorylated poly(C), which is not hydrolyzed, is not inhibitory. Line- weaver-Burk plots showed that the inhibition of hydrolysis of poly(A) by poly(dC) or poly(d1) is competitive. The inhibition studies were done at substrate concentrations less than the KM. The hydrolysis of yeast RNA, which has a much lower KM, is inhibited only slightly by the synthetic polydeoxyribonucleotides and also only slightly by single-stranded or double- stranded DNA.

Hydrolysis of dmble-stranded polyribonucleotides. The 5’ + 3’ exoribonuclease of yeast shows a novel hydrolysis of the double-stranded polymers, poly(A) * poly(U) and poly(A) . poly(dT) (Table III). Poly(A) * poly(U) is hydrolyzed at 60% of the rate of [3H]poly(A) at 37°C. Poly(A) l poly(dT) is poorly hydrolyzed (15%), but if the poly(dT) used to form the complex is first treated with E. coli alkaline phosphatase to remove the terminal 5’-phosphate, the double-stranded molecule becomes a better substrate (hydrolyzed at 50% of the rate of poly(A)). The presence of a 5’-terminal phosphate on the poly(dT) apparently in-

TABLE III

ACTIVITY OF THE EXORIBONUCLEASE WITH

DOUBLE-STRANDED POLYMERS

Polymer

Exoribo- Sl

nuclease Nuclease hydrolysis hydrolysis

(So) (%)

VHIP~WA) [3J4~ol~(A) * PO~YW) [aH]poly(A). poly(dT); no

phosphatase treatment of poly(dT)

[‘Hlpoly(A) . poly(dT);

poly(dT) treated with alkaline phosphatase

[3H]RNA + Ml3 DNA [8H]RNA. Ml3 DNA;

heated

25 12 15 <2

3.7 12

12.5 <2

40 10

22 70

Note. The reaction mixtures with the synthetic polymers contained 1.2 nmol of poly(A) and in the

case of the double-stranded polymers, 1.2 nmol of the complementary polymer. One unit of enzyme was used. The [8HlRNA. Ml3 DNA reaction mixtures contained

10 pmol of [SH]RNA and 3.3 units of enzyme. The Sl n&ease reaction mixtures (50 al) contained 30 mM sodium acetate buffer (pH 4.5), 1 mM ZnSO,, 50 mM NaCl, the polymers as shown above, and 1.25 units of Sl nuclease. Acid-soluble label was determined after 15 min at 37°C as described for the exoribonuclease.

A 5’ + 3’ EXORIBONUCLEASE OF YEAST 345

:: 24- 0

10 20 30 40

TEMP., OC

FIG. 4. Effect of temperature on the reactivity of poly(A), poly(A) . poly(U), and poly(A) . poly(dT). The

reaction mixtures were as described under Table III and were incubated for 10 min at the temperatures

shown. [3W~ol~(A) (0); PHl~oly(A) * poly(U) (0); [‘H]poly(A) . poIy(dT) (---).

hibits the hydrolysis of the poly(A) of the double-stranded complex by 60-70%. As Table III shows, the same double-stranded molecules are not hydrolyzed by Sl nuclease. Poly(A) is not hydrolyzed at pH 4.5 because a double-stranded molecule is formed. If the Sl nuclease treatment is carried out at pH 7.0 with 10 times more Sl nuclease, poly(A) is hydrolyzed lOO%, while poly(A) - poly(U) and poly(A) . poly- (dT) are still hydrolyzed at less than 2% (data not shown). It has not been possible to show a significant (greater than 10%) hydrolysis of either poly(C) . poly(1) or poly(C) - poly(d1). It is possible that the inhibitory properties of the complementary polymers, poly(1) and poly(dI), may inter- fere. Figure 4 shows the effect of temperature on the reactivity of poly(A), poly(A) - poly(U), and poly(A) * poly(dT). There is little effect of temperature on the relative rates of hydrolysis from 30 to 42°C. At 22”C, the hydrolysis of the double- stranded molecules is reduced in propor- tion to the poly(A). Local “melting” of the structures may not play a big role in the hydrolysis of the double-stranded molecules. The activities with the double- stranded molecules parallel the poly(A) hydrolytic activity on purification and on heat inactivation of the enzyme. Figure 5

shows the effect of time of heating the enzyme at 43°C on the activities.

Using E. coli RNA polymerase, an RNA *DNA hybrid was synthesized with Ml3 DNA as a template and pCpA as a primer to generate RNA with a 5’-phosphate end group. As shown in Table III, the RNA * DNA hybrid is hydrolyzed by the exoribonuclease, showing 40% hydrolysis as compared to 22% hydrolysis after heating to generate single-stranded RNA. Sl nuclease in excess showed only 10% hydrolysis of the double-stranded molecule.

Using poly([3H]rA-[32P]dA) * poly(dT) as a substrate, it was possible to show that the last ribonucleotide linking the ribo- polymer to the deoxyribopolymer is removed upon hydrolysis by the yeast exoribonuclease. The [32P]poly(dA) portion of the substrate is not degraded. The substrate before hydrolysis and after 50% hydrolysis with the exoribonuclease was examined for the amount of 3H and 32P released in 3’(2’)-AMP after alkaline hydrolysis (Table IV). If the phosphodiester bond joining the final rH]AMP to the first [32P]dAMP had not been cleaved by the en-

1

2 4 6 0 TIME (min) AT 43O C

FIG. 5. Heat inactivation of the exoribonuclease. The phosphocellulose concentrate enzyme (100 units) was diluted to 1 ml in buffer A containing 50 mre KCl, 0.2

rnre dithiothreitol, and 100 pg of acetylated albumin and heated at 43°C. Aliquots (150 ~1) were removed at 0,1.5,3,6, and 10 min and 10 pl was assayed with

l?Hlpol~(A) (Oh [3W~ol~(A)*pol~W) 0, ad l%l- poly(A) . poly(dT) (---).


TABLE IV

CLEAVAGE OF PoLv(A)-PoLY(dA) . PoLY(dT) BY THE EXORIBONUCLEASE REMOVES pA LINKED TO PoLY(dA)

[‘H](pA), - [“P](pdA), * (pdT),uz [‘H,=Pj3’(2’) - AMP

Before exoribonuclease hydrolysis After exoribonuclease hydrolysis (50%)

‘H/%P in 3’(2’)-AMP 1.42 1.43

Note. The reaction mixtures (50 ~1) were as described under Materials and Methods and contained the labeled polymer which had 0.2 nmol of [3H]poly(A) (12 X l@ cpm) and 40 pmol of [3zP]poly(dA) (190 X lo3 cpm). No enzyme was added to the first reaction mixture (before exoribonuclease hydrolysis) and 5 units was added to the second (after exoribonuclease hydrolysis). After 12 min at 37°C 50 ~1 of 7% perchloric acid was added and the precipitates resulting from centrifugation were suspended in 30 pl of 0.4 N KOH. After 18 h at 37°C 50 ~1 of 7% perchloric acid was added and ‘70 ~1 of the supernatant solution resulting from centrifugation was neutralized with KOH, centrifuged, and applied to Whatman 3MM paper. Paper electrophoresis in pyridine acetate (pH 3.5) was carried out for 1.5 h at 2000 V and the segments of the paper containing the 3’(2’)-AMP spot were counted after suspension in water.

zyme, all the [“PI label in that dAMP should still appear in 3’(2’)-AMP after alkaline hydrolysis, while 50% of the label in the AMP would have been removed by the 50% hydrolysis of the tritium label. That the ratio of 3H to 32P remains the same before and after exoribonuelease hydrolysis shows that the final AMP residue is removed. It has previously been shown that degradation of poly(A) and oligo(A)s is not complete, but leaves (PA), and (PA)~ as 3’- terminal products (1). The phosphodiester linkage of the final 3’-terminal AMP to poly(dA) in the doubly labeled substrate apparently makes complete cleavage possible.

DISCUSSION

The first evidence for the presence of a 5’ + 3’ exoribonuclease in cells was pro- vided by results of Furuichi et al. (10) and Shimotohno et ~2. (11) who found that 5’- terminally capped mRNAs are more stable to degradation in crude extracts than mRNAs with unblocked termini. The studies of the purified 5’ + 3’ exoribonuclease described here indeed have shown that the yeast enzyme has a high level of specificity for polyribonucleotides with B-monophosphate end groups. Previous studies showed that poly(A) with a 5’-phosphate end group was five times better as a substrate than poly(A) treated with alkaline phosphatase (3). (Ap),A was a very poor substrate com-

pared to (PA), (1). A capped mRNA (globin mRNA) was hydrolyzed poorly (3). The results presented here show that poly(C) and poly(U) preparations made with polynucleotide phosphorylase of Micrococcus lu- teus apparently lack a 5’-phosphate end group and are very poor substrates. These polymers are about 50 bp long and are active as substrates following treatment with T4 polynucleotide kinase. A poly(A) preparation containing a 5’-triphosphate end group is hydrolyzed at about 12% of the rate of a poly(A) preparation with a 5’- monophosphate end group. The results suggest that the yeast enzyme could be involved in trimming reactions or turnover reactions involving 5’-phosphate-ended se- quences. The nucleolar enzyme described by Lasater and Eichler (2) was found not to be influenced by the nature of the 5’-end of the polyribonucleotide substrate. How- ever, that a similar enzyme can be detected in nucleoli suggests that the enzyme could be involved in 5’-trimming reactions during rRNA maturation. The heterogeneous 5’- termini of 5.8s RNA of yeast (12) suggest that a trimming reaction may be involved in their formation. Preliminary localiza- tion studies (unpublished data) suggest that the yeast enzyme is also predominantly a nuclear enzyme.

The yeast enzyme hydrolyzes the double- stranded polymers, poly(A) . poly(U), poly(A) . poly(dT), and an RNA * Ml3 DNA

A 5’ -e 3’ EXORIBONUCLEASE OF YEAST 347

hybrid. The nuclear exoribonuclease of Ehrlich ascites tumor cells hydrolyzed poly(A) 9 poly(U) at only 8% of the rate of poly(A) (2). Data on poly(A) . poly(dT) were not presented. The RNase H activity of the yeast enzyme makes it a possible candidate for the enzyme involved in removal of RNA primers during DNA replication. That the enzyme removes the last ribonucleotide as shown here linking poly(A) to poly(dA) means that it meets a necessary require- ment of a deprimase. RNases H of eukary- otes have been found to remove the last ribonucleotide linking an RNA segment to a DNA chain rather poorly (13,14) or not at all (14). A 5’ --f 3’ exoribonuclease showing little RNase H activity in vitro could conceivably hydrolyze RNA primers in ~ivo, since DNA binding proteins may dis- rupt the double-stranded structure of the RNA primer. DNA molecule, thereby making the RNA primer more accessible.

That an antibody is now available for the yeast enzyme may make cloning and mu- tagenesis of the exoribonuclease gene possible so that the essentiality and function of the enzyme can be studied.

REFERENCES

1. STEVENS, A. (1980) J. Biol Chem. 255,3080-3085.

2. LASATER, L. S., AND EICHLER, D. C. (1984) Bie chemistry 23,4367-4373.

3. STEVENS, A. (1978) Biochem. Biophys. Res. Co-m-

mun 81,656-661. 4. STEVENS, A. (1985) J. Bacterial 164,57-62.

5. EBERLE, R., AND COURTNEY, R. J. (1980) J. Vi~ol. 35,902-917.

6. SIEGEL, L. M., AND MONTY, K. J. (1966) B&hem. Biophys. Acta 112,346-362.

‘7. MARTIN, R. G., AND AMES, B. N. (1961) J. BioL Chem 236,1372-1379.

8. LAEMMLI, U. K. (1970) Nature (London) 227,680- 685.

9. VILLADSEN, I. S., BJORN, S. E., AND VRANG, A. (1982) J. BioL Chem. 257,8177-8182.

10. FURUICHI, Y., LAFIANDRA, A., AND SHATKIN, A. J. (1977) Nature &on&n) 266.235-239.

11. SHIMOTOHNO, K., KODAMA, Y., HASHIMOTO, J., AND MIURA, K. (1977) Proc. NatL Acd Sci. USA 74,

2734-2738. 12. RUBIN, G. M. (1974) Eur. J. Bio~hem 41,197-202.

13. DIFRANCESCO, R. A., AND LEHMAN, I. R. (1985) J. BioL Chem 260,14764-14770.

14. CROUCH, R. J., AND DIRKSEN, M. L. (1982) in Nu- cleases (Linn, S. M., and Roberts, R. J., Eds.),

pp. 211-241, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

a 5′ → 3′ exoribonuclease of saccharomyces cerevisiae: size and novel substrate specificity

Documents