(Received for publication, October 4, 1995)
From the
RNase L, the 2`,5` oligoadenylate-dependent ribonuclease, is one
of the enzyme systems important in the cellular response to interferon.
When activated in the presence of 2`,5`-linked oligoadenylates, RNase L
can catalyze the cleavage of synthetic oligoribonucleotides that
contain dyad sequences of the forms UU, UA, AU, AA, and UG, but it
cannot catalyze the cleavage of an oligoribonucleotide containing only
cytosines. The primary site of the cleavage reaction with the substrate
CUUC
has been defined to be 3` of the UU dyad
by labeling either the 5` or the 3` end of the oligoribonucleotide and
by examining the reaction products on polyacrylamide sequencing gels.
Reaction time courses have been used to determine the kinetic
parameters of the cleavage reactions. The effect of the overall length
of the oligomeric substrate as well as the sequence of the bases around
the position of the cleavage site on the kinetics of the cleavage
reaction has been examined. The efficiency with which activated RNase L
catalyzes the cleavage of the substrate C
UUC
is 1.9
10
M
s
. Because the cleavage of the synthetic
oligoribonucleotide can be used to monitor the steady-state kinetics of
catalysis by activated RNase L, this method offers an advantage over
previous methods of assay for RNase L activity.
One of the enzyme systems whose activity is enhanced on treatment with interferon is RNase L, the 2`,5` oligoadenylate-dependent ribonuclease (Kerr and Brown, 1978). Interferons induce the expression of 2`,5` oligoadenylate synthetases, which catalyze the formation of oligomers of adenosine linked 2` to 5` (Hovanessian et al., 1977). These 2`,5` oligoadenylates bind to and activate the latent RNase L to cleave viral and cellular RNAs at the 3` side of UpNp sequences (Floyd-Smith et al., 1981; Wreschner et al., 1981), leading to the inhibition of protein synthesis.
RNase L activity has been shown to be important in the mechanisms of cellular antiviral defense. Overexpression of 2`,5` oligoadenylate synthetase leads to the inhibition of picornavirus replication (Chebath et al., 1987; Rysiecki et al., 1989). Introduction of an inactive mutant of RNase L caused an increased susceptibility to infection by picornavirus and a loss of the inhibition of cell growth caused by interferon treatment (Hassel et al., 1993). Introduction of 2`,5` oligoadenylate into cells (Hovanessian and Wood, 1980) or the expression of 2`,5` oligoadenylate synthetase (Rysiecki et al., 1989) have been found to cause growth arrest, suggesting a role for the RNase L system in the regulation of cell growth.
The genes for human and murine RNase L have been cloned and sequenced (Zhou et al., 1993). Overexpression of RNase L employing baculoviral vectors and insect cell lines (Dong et al., 1994) has supplied enough material for protein chemical experiments to be performed. The recent demonstration of the formation of dimers of RNase L in the presence of activators (Dong and Silverman, 1995) has opened up new areas of investigation regarding the mechanism of action of the activated complex and has created the need for detailed investigation of the kinetics of catalysis by RNase L.
Current methods of assay for RNase L activity rely on the cleavage of radiolabeled poly(U) (Silverman, 1985) followed by quantification of the remaining substrate with precipitation or on the cleavage of ribosomal RNA (Kariko et al., 1987) followed by analysis of specific cleavage products with agarose gel electrophoresis. These methods are useful for the detection of RNase L activity, but detailed examination of the steady-state kinetics of catalysis by RNase L requires a quantitative method. This work examines the cleavage by activated RNase L of synthetic oligoribonucleotides containing one or two sites of cleavage as a means of quantitatively determining the steady-state activity of the enzyme. The effect of overall length of the oligoribonucleotide and the sequence of bases at the site of cleavage on the catalytic efficiency and position of cleavage are examined.
RNase L was
overproduced in insect cell line Tn5B1-4 using a baculoviral vector.
Recombinant virus was produced using the Baculogold system (Pharmingen,
San Diego, CA) according to the supplier's instructions. Tn5B1-4
cells in spinner flasks in Ex-Cell 401 medium (JRH Bioscience, Lenexa,
KS) at 1-2 10
cells/ml at 27 °C were
infected at a multiplicity of infection of 5 and were harvested 72 h
post-infection. The purification of RNase L was accomplished according
to published procedures (Dong et al., 1994) with the following
exception. Instead of chromatography on Superose 12, the protein was
chromatographed on Biospin-6 columns (Bio-Rad) that had been
equilibrated with 25 mM Tris, pH 7.5, 100 mM KCl, and
5.8 mM magnesium acetate. Approximately 4 mg of RNase L were
purified from 3.7
10
cells. The RNase L was
90% pure as judged with SDS-polyacrylamide gels and Coomassie Blue
staining.
Protein concentrations were determined using amino acid analysis. Attempts to sequence the RNase L with automated Edman degradation were unsuccessful, suggesting that the N terminus of the enzyme was blocked. Sequencing of peptides generated by trypsin-catalyzed hydrolysis confirmed the presence of the published amino acid sequence. Purified RNase L was stored in 25 mM Tris, 100 mM KCl, 5 mM magnesium acetate, pH 7.5, and 50% glycerol at -70 °C.
Oligoribonucleotide
substrates were radiolabeled at the 5` position with
[-
P]ATP (NEN, 6000 Ci/mmol) and
polynucleotide kinase (U. S. Biochemical Corp.). 5` end-labeled
oligoribonucleotides were purified from the kinasing reaction with
ethanol precipitation. Radiolabeling at the 3` position of
oligoribonucleotides was carried out using
5`-[
P]-3`,5` cytidine bisphosphate (NEN, 3000
Ci/mmol) and RNA ligase (New England Biolabs), according to the
supplier's protocol, followed by ethanol precipitation.
For product analysis, an aliquot of the quenched solution was electrophoresed on 20 or 25% acrylamide/7 M urea/TBE sequencing gels. The presence of HEPES in the reaction buffer caused smearing of the bands on 20% acrylamide gels in the region of the gel where oligomers of lengths of 8-10 bases migrate. The bands corresponding to substrate and product were quantified with the use of a PhosphorImager (Molecular Dynamics). Conversion of the raw PhosphorImager counts to the concentration of product was accomplished by integrating the product band as a fraction of the total of the counts in the substrate and product bands and multiplying by the initial substrate concentration, thus eliminating errors due to loading different volumes of the samples on the gel.
Cleavage of substrate
CUUC
by RNase Phy M (Sigma) was carried out
according to the supplier's instructions using 3 units of enzyme
in a 30-µl reaction. Base-catalyzed hydrolysis of
oligoribonucleotides was accomplished by incubating the 5`-labeled
oligoribonucleotide in 100 mM Na
CO
, pH
9, at 85 °C for 1 h.
Figure 1:
Identification of
the site of cleavage for RNase L substrate
CUUC
. 5` end-labeled substrates were incubated
under various conditions and then subjected to electrophoresis on 20%
acrylamide/7 M urea/TBE gels and then autoradiographed. Lane 1, 5`-
P-C
UUC
was
incubated in 100 mM Na
CO
at 85 °C
for 45 min. Lane 2,
5`-
P-C
(2`-fluoroU)UC
was
incubated in 100 mM Na
CO
at 85 °C
for 45 min. Lane 3,
5`-
P-C
(2`-fluoroU)UC
. Lane
4, 5`-
P-C
UUC
. Lane
5, 5`-
P-C
UUC
and RNase L
(300 pM) incubated in reaction buffer without activator
p2,5A
for 30 min. Lane 6, 400 nM 5`-
P-C
UUC
incubated in
reaction buffer in the presence of 500 nM p2,5A
without RNase L for 30 min. Lane 7, RNase L (300
pM) incubated in reaction buffer in the presence of 500 nM p2,5A
and 400 nM 5`-
P-C
UUC
for 30 min. Lane 8, RNase L (600 pM) incubated in reaction buffer
in the presence of 500 nM p2,5A
and 400 nM 5`-
P-C
UUC
for 30 min. Lane 9, RNase Phy M-catalyzed cleavage of substrate
5`-
P-C
UUC
. Lane 10,
5`-
P-C
U. Lane 11,
P-C
UU. Lane 12,
3`-
P-C
UUC
. Lane 13,
3`-
P-C
. Lane 14, RNase L-catalyzed
cleavage of 3`-
P-C
UUC
. Lane
15, RNase Phy M-catalyzed cleavage of
3`-
P-C
UUC
.
Further evidence to pinpoint the site of cleavage of
CUUC
comes from comparison of base-catalyzed
hydrolysis of 5`-
P-labeled oligoribonucleotides.
Base-catalyzed hydrolysis of 5`-
P-C
UUC
and of 5`-
P-C
(2`-fluoroU)UC
creates a ladder of 3`phosphate-terminated oligos (Fig. 1, lanes 1 and 2). The hydrolysis product that is
present in the base-catalyzed hydrolysis of C
UUC
but is absent in the hydrolysis of
C
(2`-fluoroU)UC
corresponds to
C
U(3`-p). Because the major cleavage product of
C
UUC
by RNase L is one nucleotide longer than
this position, the product corresponds to C
UU(3`-p).
That a single cleavage event leads to the production of 5`
end-labeled CUUp is demonstrated by examination of the
reaction products from RNase L-catalyzed cleavage of 3` end-labeled
C
UUC
cytidine 3`,5`-bisphosphate (Fig. 1, lane 14). A single radiolabeled product is
formed that migrates to the same position as the smaller of the two
products generated by cleavage of C
UUC
cytidine 3`,5`-bisphosphate by RNase Phy M (Fig. 1, lane 15). This radiolabeled product migrates with the same
mobility as authentic C
cytidine 3`,5`-bisphosphate
produced by RNA ligase-catalyzed ligation of C
and cytidine
3`,5`-bisphosphate (Fig. 1, lane 13).
The lengths of
the 5` cleavage products of the other substrates used in this study are
determined by comparison with the product from the cleavage of
CUUC
and are shown in schematic form in Fig. 2. Substrates containing the dyad sequence UU with overall
lengths of 11, 20, and 32 nucleotides are cleaved in the presence of
activated RNase L at the same position relative to the UU dyad. The
addition of a third U, as in substrate C
UUUC
,
does not change the primary cleavage site. Activated RNase L catalyzes
the cleavage of C
AAC
and
C
AUC
between the nucleotides of the dyads.
Activated RNase L catalyzes the cleavage of C
UC
and C
UGC
in two positions.
Figure 2: Cleavage sites of the oligoribonucleotide substrates of RNase L studied. The arrows indicate the primary site(s) of cleavage.
Figure 3:
Reaction time course with and without
preincubation of RNase L and p2,5A. RNase L (1.5
nM) was incubated on ice in the presence (
) or the
absence (
,
) of 500 nM p2,5A
for 30
min on ice. Cleavage reactions were then initiated in the presence
(
,
) or the absence (
) of 500 nM p2,5A
and time points from 0.5 to 5 min were obtained
by quenching an aliquot with formamide load buffer. Product formation
was monitored with gel electrophoresis and a
PhosphorImager.
Figure 4:
Reaction rate as a function of the
concentration of RNase L. Enzyme (2.5 nM) was incubated with
p2,5A (800 nM) in reaction buffer in the absence
of substrate for 30 min on ice. The preactivated enzyme was used to
initiate reactions at the final enzyme concentrations as indicated. The
amount of product formed was monitored as described under
``Materials and Methods.'' The line drawn represents
a linear fit to the data through the
origin.
As shown in Fig. 5, the rate of cleavage
of CUUC
increases with increasing
concentrations of p2,5A
and approaches a maximal rate at
about 10 nM with half-maximal activation at a concentration of
p2,5A
of 1 nM. For this experiment reactions were
initiated by the addition of RNase L that had been incubated with the
same concentration of p2,5A
that was included in the
subsequent cleavage reaction. Reaction time courses were monitored for
product formation for 2.5 min. Higher concentrations of
p2,5A
, up to 1 µM, gave the same maximal
reaction rate.
Figure 5:
Activation of RNase L as a function of
concentration of p2,5A. RNase L (500 pM) was
incubated in the presence of p2,5A
(0.5-20
nM) in reaction buffer on ice for 30 min. The preactivated
enzyme was then used to initiate cleavage reactions containing the same
concentration of p2,5A
that was used in the preincubation
and that included 1500 nM C
UUC
as
substrate. Reaction time courses were monitored for 2 min, and the rate
of each reaction was determined relative to the rate of the reaction
that included 15 nM p2,5A
. The line represents a saturation curve as dictated by the data
points.
In the presence of an activator, RNase L is capable of efficiently catalyzing the cleavage of oligoribonucleotide substrates containing dyads of bases other than cytosine. The ability of RNase L to cleave synthetic RNA substrates has been utilized to develop a quantitative assay for enzyme activity.
The precise position of the site(s) of cleavage of the RNA substrates is determined at least in part by the sequence of the bases surrounding the cleavage site. For the substrates containing dyads of UU or UA, the primary cleavage site is 3` of the second nucleotide of the dyad. Substrates containing either an AA or AU dyad are cleaved between the nucleotides of the dyad. Substrates with a UG or a single U are cleaved 3` of the U and 3` of the next base to the 3` side of the U.
For most of the
experiments, RNase L and the activator p2,5A were
preincubated on ice prior to initiating subsequent cleavage reactions.
The preactivation of the enzyme was carried out to eliminate the
possibility of any rate associated with the activation of RNase L from
contributing to the observed rate of cleavage of substrates. The
subsequent reaction time courses displayed a linear increase in the
amount of product and a replot of the rate of the reactions, as a
function of enzyme concentration, was also linear over the range of
enzyme concentrations tested. These conditions are satisfied to allow
the use of steady-state equations to determine the kinetic parameters, k
and K
. Reactions that
were initiated by the addition of RNase L that had not been
preincubated with activator resulted in time courses displaying a lag
in the first few minutes of the reaction (Fig. 3). One possible
reason for the lag is a slow formation of active dimerized enzyme on
the time scale of the reactions. A more detailed investigation of the
kinetics of dimerization of RNase L and the effect of dimerization on
enzyme activity is currently under way.
The efficiency with which
activated RNase L catalyzes the cleavage of a substrate depends on the
length of the oligoribonucleotide and the number of non-C bases around
the cleavage site. The efficiency of cleavage of the
oligoribonucleotide substrates containing one UU dyad increases by a
factor of 3 with an increase in the length of the substrate from 11 to
20 nucleotides. When the length of the substrate is increased from 20
to 30 nucleotides, a further 2-fold increase in efficiency of cleavage
is found. The increases in efficiency of catalysis for cleavage of the
longer oligoribonucleotides are primarily due to decreases in the
values of K.
Activated RNase L cleaves substrates containing a UU dyad approximately 20-fold more efficiently than a substrate with a single U. A substrate with three consecutive Us is cleaved 2.3 times more efficiently than the substrate with the UU dyad. The increases in the efficiency of cleavage are primarily due to increases in the rate of catalysis. Thus, for a given length, the information determining the rate of cleavage of an oligoribonucleotide containing two or more non-C bases primarily resides in two bases of the substrate. Substrates containing the dyad sequences UU, UA, or UG are cleaved by RNase L with high efficiency relative to the substrate containing a single U, in agreement with published reports of the cleavage of viral RNAs (Wreschner et al., 1981; Floyd-Smith et al., 1981).
In vivo, secondary structural
characteristics associated with individual RNA sequences may mask
potential cleavage sites or possibly enhance the efficiency of cleavage
of specific sites. However, the value of k (7.6
s
; see Table 1) for the cleavage of
C
UUUC
is comparable with the rate of cleavage
of a specific mRNA catalyzed by RNase L that has been activated by a
2`,5` adenosine oligomeric DNA chimera (7 s
; Maitra et al., 1995).
Recent investigations of the quaternary structure of the activated RNase L have indicated that the enzyme is dimerized in the presence of activator (Dong and Silverman, 1995; Cole et al., 1996). The existence of a quantitative assay for RNase L activity will make possible the determination of the dynamic relationship between the oligomeric/liganded states of the activated complex and its catalytic activity.