From the
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`-
The first purification and characterization of a 5`
Purification of 5`-exonuclease-2 was undertaken so that
NH
[
5`-Exonuclease-2 was purified as described below. All
operations were carried out at 0-4 °C unless otherwise
specified.
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.
Exonuclease-2 was assayed during the purification by using
[
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
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
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
Descending paper chromatography using Whatman No. 3MM paper
was carried out for 18-24 h with the solvent n-propyl
alcohol/NH
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.
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.
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.
Monovalent cation stimulates at 50-100 mM and becomes
inhibitory above 150 mM. ATP does not stimulate the reaction.
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 m
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 (
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`
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
The catalytic
properties of Xrn1 and exonuclease-2 with RNA are also very similar, as
reported above. Exonuclease-2 has a higher specific activity
(
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)
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`
Studies of 5`
The starting material was 300 g of yeast cells (S.
cerevisiae YPH500 xrn1
The reaction mixtures (50 µl) were as described under
``Experimental Procedures.'' In A., 1.5-1.85 nmol of
each
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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
-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.
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
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
Assay of Enzyme
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
NH
Cl, 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.
, 50 mM NH
Cl, 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
-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
and are
expressed as nanomoles of nucleotide.
Paper Chromatography and Electrophoresis
OH/H
O, 60:30:10 for separation of
mono- and oligonucleotides and with the solvent isopropanol,
NH
OH, 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
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'').
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.
NH
An enzyme fraction (fraction 50) shown in Fig. 1B was used for NH-terminal Sequencing of the 116- and 45-kDa
Protein Bands
-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.
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.
GpppG upon paper electrophoresis at
pH 3.5 and paper chromatography using n-propyl
alcohol/NH
OH/H
O 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) .
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).
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.
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.
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.
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) .
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) .
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
BglII::URA3). All
fractions were assayed as described under ``Experimental
Procedures.''
Table: Substrate specificity of exonuclease-2
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.
-terminal sequencing.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.