(Received for publication, March 6, 1997, and in revised form, June 4, 1997)
From the Department of Cancer Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
The 2-5A-dependent RNase (RNase L) is
a tightly regulated endoribonuclease of higher vertebrates that is
catalytically active only after engaging unusual effector molecules
consisting of the 2,5
-linked oligoadenylates,
p1-3A(2
p5
A)
2 (2-5A). Progressive
truncations from either terminus have provided insight into the
structure, function, and regulation of RNase L. We determined that
deletion of the N-terminal 335 amino acids of RNase L, about 45% of
the enzyme, produced a constitutively active endoribonuclease, thus
effectively eliminating the requirement for 2-5A. The truncated nuclease had 6-fold lower catalytic activity against an oligo(rU) substrate than wild type RNase L. However, the two enzymes showed identical RNA cleavage site preferences with an mRNA as substrate. The repressor function required only the last three of a series of nine
ankyrin-like repeats present in the N-terminal part of RNase L. In
contrast, the entire ankyrin repeat region was necessary and sufficient
for 2-5A binding activity. Deletion of a 10-amino acid sequence near
the C terminus of RNase L, between residues 710 and 720, eliminated
both the catalytic and RNA substrate binding functions of the enzyme.
The ability to bind native RNase L in response to 2-5A required amino
acid sequences near both termini of the protein. A bipartite model for
the structure of RNase L emerged in which the regulatory functions of
the molecule are located in the N-terminal half, while the catalytic
domain is present in the C-terminal half.
RNase L provides a unique paradigm of enzyme regulation because it
is the only enzyme known to require the unusual
2,5
-oligoadenylate(s), p1-3A(2
p5
A)
2
(2-5A),1 for its catalytic
activity (1, 2). The activators of RNase L, 2-5A, are produced from ATP
by double-stranded RNA stimulation of the 2-5A-synthetases, a family of
enzymes induced by interferon treatment of mammalian cells. Production
of double-stranded RNA during some virus infections leads to 2-5A
synthesis and activation of RNase L (3, 4). The proposed biological
functions of RNase L includes involvement in the antiviral and
antiproliferative mechanisms of interferon action and in the more
general control of RNA decay (5, 6). The human RNase L is a 741-amino
acid protein that forms homodimers upon binding 2-5A (7, 8). Activation
of human RNase L requires 2-5A molecules containing at least three
2
,5
-adenylyl residues, such as pA(2
p5
A)2, and the
binding of 2-5A to RNase L, KD = 40 pM,
is highly specific (9, 10). Previously, we demonstrated that the 2-5A binding activity of RNase L is located in the N-terminal half of RNase
L, a region containing nine ankyrin-like, macromolecular recognition
domains (2, 6). Within the seventh and eighth ankyrin repeats are two
P-loop motifs. In murine RNase L, substitutions of both of the
conserved P-loop lysine residues with asparagines resulted in a
defect in 2-5A binding activity, while deletion of 89 C-terminal amino
acids caused a loss of ribonuclease activity (2, 6). The truncated
RNase L mutant, RNase LZB1, which binds 2-5A but
lacks ribonuclease activity, functions as a dominant negative inhibitor
of wild type RNase L (6).
To further study the structure and function of RNase L, we have performed both a systematic shortening of RNase L from either terminus and a biochemical analysis of each mutant protein. The results provide information about the location of the structural and functional domains in RNase L. Moreover, these studies provide insight into the mechanism of regulation of RNase L. An internal repressor of ribonuclease function was identified, deletion of which released the ribonuclease domain from its dependence on 2-5A.
A
full-length coding sequence DNA for human RNase L in plasmid pZC5 (2)
was subcloned downstream (3) of the coding sequence for glutathione
S-transferase (GST) in expression vector pGEX-4T-3 (Pharmacia Biotech Inc.). The cloning strategy involved creating a
BamHI site immediately upstream of the RNase L coding
sequence. This was done using an oligonucleotide primer for PCR
containing the BamHI site. The PCR product containing the
BamHI site at the 5
terminus and the natural
NcoI site at the 3
terminus was subcloned into
BamHI/NcoI digested pZC5. The complete RNase L
coding sequence was subcloned as a BamHI/XhoI
fragment into BamHI/XhoI-digested pGEX-4T-3. The
deletion mutants of RNase L were constructed with PCR by creating new
BamHI restriction sites for the 5
-truncations and new
XhoI sites for the 3
-truncations. To generate the
N-terminal deletions of RNase L, the 5
-primers contained 4-6
nucleotides and GGATCC for creating BamHI sites followed by
18-20 nucleotides of the RNase L sequences. To generate the C-terminal
truncations, 5
-GGCGGCCTCGAGTCA (underlined sequence is the
XhoI site followed by a sequence complementary to a stop
codon sequence), attached to the 16-24 nucleotides of different RNase
L sequences was used as the 3
-primers for PCR. Specific RNase L
mutants were constructed with the primers and restriction enzymes
indicated in Table I. All mutants were
confirmed by DNA sequence analysis.
|
The cDNAs for the wild type and mutant forms of RNase
L in plasmid pGEX4-T-3 were transformed into Escherichia
coli strain DH5. The transformed bacteria were grown at
30 °C to A595 = 0.5 before being induced with
0.1 mM isopropyl-1-thio-
-D-galactpyranoside for 3 h. Harvested cell pellets were washed with PBS, resuspended in 3-5 volumes of PBS-C (PBS with 10% glycerol, 1 mM
EDTA, 0.1 mM ATP, 5 mM MgCl2, 14 mM 2-mercaptoethanol, 1 µg/ml leupeptin, and 1 mM PMSF) supplemented with 1 µg/ml lysozyme, and
incubated at room temperature for 20 min. The suspended cells were
sonicated on ice for 20 s four times, Triton X-100 was added to a
final concentration of 1% (v/v), and the cell lysates incubated at
room temperature for 20 min. The supernatants were collected following centrifugation at 16,700 × g for 20 min at 4 °C.
Purification of fusion proteins was performed as described by the
manufacturer of the glutathione-Sepharose 4B (Pharmacia) with
modifications. Briefly, glutathione-Sepharose 4B (200 µl of a 50%
slurry in PBS-C) was added to extract from 200-ml cultures of bacteria
at room temperature for 20 min with shaking. After washing the
protein-bead complexes three times with 3 ml of PBS-C, the fusion
proteins were eluted with 20 mM glutathione in 50 mM Tris-HCl, pH 8.0, containing 1 µg/ml leupeptin, with
shaking at room temperature for 20 min. Expression and purity of the
protein preparations was determined by SDS-PAGE and Coomassie Blue
staining and by Western blots with monoclonal antibody to RNase L
(7).
2-5A binding activity was
determined by a filter binding method (11, 12). Purified fusion
proteins (2 µg/assay) were incubated with about 10,000 cpm of a
32P-labeled and bromine substituted 2-5A analog,
p(A2p)2(br8A2
p)2A3
[32P]pCp
(3,000 Ci/mmol) in buffer A (20 mM Tris-HCl, pH 7.5, 10 mM magnesium acetate, 8 mM 2-mercaptoethanol,
90 mM KCl, 0.1 mM ATP, 10 µg/ml leupeptin) on
ice for 2 h. The reaction mixtures were transferred to
nitrocellulose filters (Millipore; 0.45 µm Type HA). Filters were
washed twice with distilled water and dried. The amount of the
32P-labeled 2-5A probe bound to protein on the filter was
measured by scintillation counting.
Extracts (50 µg)
containing GST-RNase L or GST-RNase L mutants were incubated with
extract of recombinant, native human RNase L (25 µg) (not a fusion
protein) from insect cells (9) in the presence and absence of 0.8 µM pA(2p5
A)3 in buffer A on ice for 2 h. Subsequently, 5 µl of 20% (v/v) glutathione-Sepharose 4B was
added and the mixture was incubated with platform shaking at room
temperature for 20 min with gentle vortexing every 5 min, followed by
washing three times with 0.3 ml of PBS-C. Analysis of the bound protein
was by SDS-PAGE and Western blot analysis probed with a monoclonal
antibody to RNase L (7) using the enhanced chemiluminescence (ECL)
method (Amersham Corp.).
A chemically
synthesized oligouridylic acid, U25 (Midland Certified
Reagent Co.) was labeled at its 3 terminus with
[5
-32P]pCp (3,000 Ci/mmol) (NEN Life Science Products)
with T4 RNA ligase (Life Technologies, Inc.). The
U25-[32P]pCp (80-160 nM) was
incubated with 400 ng of purified GST-RNase L or GST-mutant RNase L in
the presence and absence of 100 nM pA(2
p5
A)3
in a final volume of 25 µl of buffer A at 30 °C for 30 min.
To compare the relative activities of RNase LN335 to
RNase Lwild type, assays were performed with different
amounts of the purified proteins. RNase Lwild type (600 nM) was preincubated in the presence or absence of 10 µM pA(2
p5
A)3 in buffer A at 0 °C for 30 min prior to dilution and addition of 80 nM
U25-[32P]pCp. RNase LN
335 was
preincubated at 0 °C for 30 min without pA(2
p5
A)3
prior to dilution and addition of substrate. The cleavage reactions
were in final volumes of 20 µl for 30 min at 30 °C.
Reaction mixtures were heated to 100 °C for 3 min in loading buffer, and RNA and RNA degradation products were separated in 20% polyacrylamide, 8% urea sequencing gels. The amount of intact U25-[32P]pCp remaining after the incubations was determined from autoradiograms of the gels with a Sierra Scientific high resolution CCD camera (Sunnyvale, CA) and the computer program NIH Image 1.6.
Determination of RNA Cleavage Site PreferencesHuman PKR
cDNA in plasmid pBS (Ref. 13, a gift of B. R. G. Williams, Cleveland Clinic Foundation) was linearized by
AflIII digestion (523 nucleotides downstream from start
codon) and transcribed in vitro with T7 RNA polymerase
(MEGAscript, Ambion) according to the manufacturer's protocol. The PKR
RNA fragment was purified by electrophoresis in 8% polyacrylamide, 8 M urea gels followed by elution. The PKR RNA fragment (200 ng) was incubated with purified RNase LN335 or RNase
Lwild type in the presence or absence of 100 nM pA(2
p5
A)3 in 20 µl of buffer A at
30 °C for 10 min. Reactions were stopped by heating at 75 °C for
15 min and the RNA cleavage products precipitated in 0.3 M
sodium acetate, 70% ethanol at
20 °C, followed by washing with
70% ethanol. The dried cleavage products were dissolved in 4 µl of
diethyl pyrocarbonate-treated water.
A 18-nucleotide primer complementary to PKR nucleotides 263-280 of the
coding sequence of PKR cDNA (5-GGCCTATGTAATTCCCCA-3
) was labeled
with [
-32P]ATP (NEN Life Science Products) and T4
polynucleotide kinase (Life Technologies, Inc.), and purified through
Sephadex G-25 cartridges (Boehringer Mannheim). The
32P-labeled primer was mixed with the RNA cleavage products
at 70 °C for 10 min and then at 37 °C for 1 h. The reaction
mixtures were incubated with dNTPs and reverse transcriptase
(superscript RNase H
, Life Technologies, Inc.) at
40 °C for 90 min. The cleavage sites were determined by
comparing the migration in 10% polyacrylamide, 8 M urea
gels of the primer extension products and the DNA sequencing products
using the same primer on PKR cDNA with Sequenase 2.0 (U. S.
Biochemical) and deoxyadenosine
[5
-
-35S]thiotriphosphate (NEN Life Science
Products).
Glutathione-Sepharose 4B, 10 µl of a 20% (v/v) suspension, or poly(U)-Sepharose 4B, 10 µl of a 60% (w/v) suspension (~0.5 mg of poly(U)/ml of gel; ~100 uridyl residues/chain) (Pharmacia) were added to extracts of the GST fusion proteins, 50 µg/assay, in PBS-C buffer. Reactions were incubated at room temperature for 20 min with shaking and gentle vortexing every 5 min. The protein-bead complexes were washed three times with 0.3 ml of PBS-C, and the proteins bound to glutathione-Sepharose 4B and poly(U)-Sepharose 4B were eluted in gel loading buffer containing SDS at 100 °C for 5 min and separated by electrophoresis on 7.5% polyacrylamide-SDS gels. The proteins were detected after transfer to nitrocellulose membranes (Schleicher & Schuell) by probing with murine polyclonal antibody to human RNase L (sera of immunized mice used for making monoclonal antibody) (7). The relative amounts of the bound proteins were determined from Western blots by enhanced chemiluminescence (Amersham) after capture of the images with a video camera.
To map and study
the functional domains in RNase L, complete or truncated RNase L coding
sequence DNAs were expressed as GST fusion proteins from vector
pGEX-4T-3 (Pharmacia) in E. coli (Fig. 1). Previously, it was shown that GST
fusion proteins of RNase L are fully active in the presence of 2-5A
(14). Because ankyrin repeats are structural as well as functional
units (15), the approach taken for the design of the N-terminal
truncations was to progressively delete the individual ankyrin repeats.
The exception was the first mutant RNase LN23, which
lacks the 23 N-terminal amino acids proceeding the first ankyrin
repeat. The other N-terminal truncations, mutants RNase
LN
56, RNase LN
90, RNase
LN
123, RNase LN
237, and RNase
LN
335, lack 1, 2, 3, 6, and 9 ankyrin repeats,
respectively (Fig. 1). On the other hand, a functional approach was
used in the design of the C-terminal truncations. In essence,
progressive C-terminal mutations were made and the mutant proteins
assayed for loss of various functions (Fig. 1). In addition, one
mutant, RNase LN
23/C
504, was truncated from both termini. Assays were performed, which measured and compared the abilities of wild type and mutant forms of RNase L to bind with
2-5A, native RNase L, or RNA and to degrade RNA.
Localization of the 2-5A Binding Domain
The portion of RNase
L that contains the 2-5A binding function was localized by measuring
the abilities of the mutant proteins to bind to a radioactive 2-5A
analog in a filter binding assay (11, 12). The 2-5A binding activity of
RNase LN23, lacking 23 N-terminal amino acids, was 82%
of that of GST-RNase Lwild type (Fig.
2). However, deletion of amino acid
sequences including the first ankyrin repeat resulted in a complete
loss of 2-5A binding activity (mutant RNase LN
56).
Further truncations from the N terminus also produced mutant proteins
that lacked 2-5A binding activity. In contrast, C-terminal truncations
of RNase L, removing as much as 406 amino acid residues in RNase
LC
406, had little or no effect of 2-5A binding activity.
However, RNase LN
23/C
504, lacking 504 C-terminal amino acids and 23 N-terminal residues, did not bind 2-5A.
These findings show that the 2-5A binding function of RNase L requires
the ankyrin repeat region, but not other portions of the enzyme.
Mapping the RNase L/RNase L Interaction Domains
To map the
RNase L/RNase L interaction domains, the ability of the GST-RNase L
mutants to bind native RNase L was determined in the presence and
absence of 2-5A (Fig. 3). The
2-5A-dependent binding of the 83-kDa native RNase L (not a
fusion protein) to GST-RNase Lwild type protein was
observed after the complex was immobilized on glutathione-Sepharose
(Fig. 3A, lane 3, see arrow). The
proteins that were retained were analyzed in Western blots probed with
a monoclonal antibody to human RNase L, thus distinguishing the
83.5-kDa native RNase L from the 110-kDa GST-RNase L. In the absence of
2-5A, there was little or no interaction between the two proteins (Fig.
3A, lane 4). The removal of the C-terminal 21-amino acid segment did not impair the 2-5A-dependent
binding of the proteins (Fig. 3A, lane 5).
However, deletion of 80 or 399 C-terminal amino acid residues results
in a loss of the ability to bind native RNase L, as observed by the
absence of the 83-kDa RNase L (Fig. 3A, lanes 7 and 9). These results show that the C-terminal 22-80-amino
acid portion is necessary for RNase L/RNase L binding activity. The
monoclonal antibody used to detect RNase L recognizes an epitope in the
C-terminal half of RNase L, between residues 342 and 661; therefore,
RNase LC399 could not be detected on the Western blot
with this antibody. However, the RNase LC
399 was clearly
visualized by staining the protein in the gel with Coomassie Blue dye
(data not shown). Despite repeated attempts, we were unable to obtain
clearly interpretable data on the RNase L binding activities of the
C-terminal mutants lacking 31, 41, or 51 amino acid residues. This was
due to the relatively low levels of expression of these mutant proteins
compared with the amount of protein required for the assay. In
addition, some of the GST fusion proteins were less stable than others.
The N-terminal mutants were also assayed for the ability to bind native
RNase L. Removal of the N-terminal 23-amino acid segment had no effect on the binding to native RNase L in response to 2-5A (Fig.
3B, lanes 9 and 10). However, an
N-terminal deletion including the first ankyrin repeat resulted in the
loss of this function (see RNase LN
56 in Fig.
3B, lane 7). Interestingly, RNase
LN
56 is also defective for 2-5A binding (Fig. 2).
Further N-terminal truncations in RNase L also resulted in proteins
that lacked the ability to bind with native RNase L (Fig.
3B, lanes 1-6). Therefore, amino acid residues
near both termini of RNase L were required for the binding to native
RNase L.
The Ribonuclease Domains Maps to a C-terminal Fragment of RNase L
To measure the endoribonuclease activity of the mutant
proteins, a radiolabeled, short RNA consisting of 25 uridylate residues linked at the 3 terminus to [32P]pCp,
U25-[32P]pCp, was synthesized as substrate.
In addition, shorter fragments of oligo(U)-[32P]pCp were
present as a result of premature terminations during the chemical
synthesis of the U25 (Fig.
4A, lane 1).
Analysis of RNA cleavages after incubation with the wild type and
mutant forms of RNase L was performed in the presence or absence of
2-5A (Fig. 4A). Incubations with RNase Lwild
type produced potent 2-5A-dependent ribonuclease
activity (Fig. 4A, lanes 2 and 3). The
cleavage products were
4 nucleotides in length. Deletion of the
N-terminal 23 residues had no effect on 2-5A-dependent
RNase activity (lanes 4 and 5). However, RNase
LN
56, missing sequence including the first ankyrin
repeat, lacked ribonuclease activity in the presence or absence of 2-5A
(lanes 6 and 7). RNase LN
90, RNase
LN
123, and RNase LN
237 (missing 2, 3, and
6 ankyrin repeats, respectively) also lacked the ability to degrade RNA
(Fig. 4A, lanes 8-11, and data not shown).
Surprisingly, however, deletion of all nine ankyrin repeats, produced a
mutant protein, RNase LN
335, with unregulated ribonuclease activity (Fig. 4A, lanes 12 and
13), i.e. ribonuclease activity was observed with
RNase LN
335 even in the absence of the activator,
2-5A.
To further localize the ribonuclease domain, the C-terminal mutant
proteins were also analyzed. RNase LC21, lacking 21 C-terminal residues, displayed wild type 2-5A-dependent
RNase activity (Fig. 4A, lanes 14 and
15). However, mutant RNase LC
31, lacking an
additional 10 residues was inactive (Fig. 4A, lanes 16 and 17). All of the mutants containing further
C-terminal truncations, beyond residue 710, were similarly inactive
(Fig. 4A, lanes 18-29). Quantitation of the
intact RNA remaining after the incubations confirmed that
2-5A-dependent RNase activity required the N-terminal segment that included the first ankyrin repeat and the 10-amino acid
segment located between residues 710 and 720 (Fig. 4B).
The unregulated mutant, RNase
LN335, caused a complete loss of intact RNA, but the
appearance of some incomplete RNA digestion products show that its
ribonuclease activity was diminished compared with wild type enzyme
(Fig. 4A, compare lane 3 to lanes 12 and 13). To directly compare the relative activities of the two proteins, the effects of protein concentration on RNA cleavage was
measured with U25-[32P]pCp as substrate.
Reactions with RNase Lwild type were performed by
preincubating with 2-5A prior to addition of substrate to avoid an
initial lag that occurs when 2-5A and substrate are added at the same
time (16). The concentrations of protein required to obtain 50% loss
of intact RNA in 30 min at 30 °C was 20 nM RNase Lwild type and 115 nM RNase
LN
335 (Fig. 5). Therefore,
RNase LN
335 was about 6-fold less active than RNase
Lwild type. In the absence of 2-5A, no RNA cleavage was
seen with 300 nM RNase Lwild type, whereas 300 nM RNase LN
335 caused an 87% decrease in
the level of intact RNA (Fig. 5).
To compare the RNA cleavage site preferences of RNase Lwild
type and RNase LN335, the proteins were incubated
in the presence or absence of 2-5A with a fragment of PKR mRNA
(13). The cleavage sites were mapped by primer extension assays in
comparison with the products of DNA sequencing reactions of PKR
cDNA (Fig. 6). RNase Lwild
type and RNase LN
335 cleaved the RNA at precisely the same sites. In both cases, the two nucleotides immediately 5
to
the cleavage sites were UA (products 1 and 3), AU (products 2 and 4),
and UG (product 5) (Fig. 6). With PKR mRNA as substrate, RNase
LN
335 appear to be somewhat less active compared with RNase Lwild type than with oligo(rU) as substrate. RNA
cleavage by RNase Lwild type was dependent upon addition of
2-5A, whereas the ribonuclease activity of RNase LN
335
was identical in the presence or absence of 2-5A.
Localization of the RNA Binding Domain
To map the RNA binding
domain, the affinities of the various GST-mutant proteins for
poly(U)-Sepharose and glutathione-Sepharose were compared (Fig.
7). The RNase L mutants truncated only
from the N terminus showed similar affinities for poly(U)-Sepharose and
glutathione-Sepharose (Fig. 7A, lanes 1-12).
Analysis of the C-terminal truncated mutants showed that RNase
LC21 bound poly(U)-Sepharose but with reduced activity
(Fig. 7A, lane 14). However, further deletions
from the C terminus, beginning with RNase LC
31, result
in a loss in poly(U) binding activity (Fig. 7A, lanes
15-28). These results were measured and expressed as percentage
of the activities of the mutant proteins compared with the wild type
protein (Fig. 7B). The data show that the substrate binding
activity and the RNase function both require the amino acid sequence
between residues 710 and 720.
Here
we have studied the structure, function, and regulation of RNase L
through a series of truncations from either terminus (Fig. 1). The 2-5A
binding activity of the protein is localized in the N-terminal half of
RNase L. For example, RNase LC406, which lacks 406 of
the 741 amino acid residues of RNase L, binds 2-5A nearly as well as
the complete, wild type enzyme (Fig. 2). Previously, we showed that the
2-5A binding domain of the murine RNase L was expressed in a
polypeptide consisting of residues 1-342 (2). Furthermore,
substitution of the P-loop motif lysines, at residues 240 and 274 in a
truncated murine RNase L resulted in a defect in 2-5A binding activity.
In this study, we show that the N-terminal 23 amino acid residues do
not contribute to 2-5A binding activity. However, the first ankyrin
repeat, residues 24-56, is required. Although RNase
LC
399 contains the complete ankyrin repeat region, a
presumptive protein-protein interaction domain with 2-5A binding
activity, it fails to bind with native RNase L in the presence or
absence of 2-5A (Figs. 1 and 3). Results show that both termini are
necessary for RNase L binding activity. For instance, RNase
LC
80 also lacks the ability to bind native RNase L. Therefore, while the ankyrin repeats may contribute to RNase L/RNase L
binding, they are insufficient for this function.
The C-terminal 31 residues of RNase L are critical for the catalytic
function of the enzyme. RNase LC31, lacking residues 711-741, has neither ribonuclease nor substrate binding activities. In
contrast, RNase LC
21 has full activity in the presence of 2-5A. These findings clearly show that residues 711-720,
EYRKHFPQTH, are essential for the RNA binding and ribonuclease
activities of the enzyme. However, it is likely that portions of the
catalytic domain are located further upstream (toward the N terminus)
or that deletion of the C-terminal segment results in conformational alterations that impair function. Interestingly, the C-terminal region
of the yeast Ire1p protein, a putative ribonuclease that functions in
the splicing of the HAC1 mRNA during the unfolded protein response,
has significant homology with the residues 587-706 of RNase L (17,
18). The region of homology with Ire1p suggests that amino acid
residues that are N-terminal to residue 706 are also important for the
catalytic function of RNase L.
Results of this study show
that the ankyrin repeat region of RNase L functions as a potent
repressor. Binding of 2-5A could cause a conformational change in the
enzyme that releases the inhibitory effect of the ankyrin repeats
leading to both an unmasking of the ribonuclease and interaction
domains near the C terminus. Previous studies involving multiple
biochemical and biophysical methods established that the binding of
2-5A to monomers of RNase L induces the formation of RNase L homodimers
(7, 8). However, additional studies will be required to determine if
dimerization is actually required for ribonuclease activity. Dimer
formation could be a mechanism for maintaining the enzyme in an active
conformation by preventing the ankyrin clamp from reforming. The
failure of RNase LN56 to bind to either 2-5A or to
native RNase L was consistent with the requirement of 2-5A for
dimerization (7, 8). Presumably, the protein-protein interaction domain
in RNase LN
56 cannot be unmasked because the protein
lacks the capacity to bind with 2-5A. It is unknown if the ankyrin
repeats directly interact with the catalytic domain. However, the
failure of the complete ankyrin repeat region in RNase
LC
80 and RNase LC
399 to bind with native
RNase L in the presence of 2-5A argues against a direct interaction
(Fig. 3).
The repressor function requires only three, or possibly fewer, of the
nine ankyrin repeats. RNase LN335, lacking all nine
ankyrin repeats was a constitutive ribonuclease whereas RNase LN
237, lacking ankyrin repeats 1-6, was in a repressed
state. Therefore, ankyrin repeat domains 7-9 by themselves are capable of causing repression. RNase Lwild type and RNase
LN
335 produced identical RNA cleavage sites, providing
further evidence that the RNA binding and catalytic sites are localized
in the C-terminal half of the enzyme (Fig. 6). Cleavages occurred 3
of
UA, UG, and AU dinucleotide sequences in the PKR mRNA fragment. The
first two of these RNA cleavages site preferences have been previously reported for RNase L, while cleavages 3
of AU have not been reported (16, 19, 20). 2-5A binding to a complete RNase L releases the
inhibition by the ankyrin repeat regions of the protein. However, because mutants RNase LN
56, RNase LN
90,
RNase LN
123, and RNase LN
237 lack the
ability to bind 2-5A, addition of 2-5A fails to release of the
repression (Fig. 1). To date, we have not observed a trans
inhibition of ribonuclease activity by mixing RNase
LC
399 with RNase
LN
335.2
However, further studies are required to determine if the ankyrin repeats must be present in the same polypeptide chain as the catalytic domain to silence the ribonuclease activity.
The results of this study show that the 2-5A binding and repression functions of RNase L map to different regions than the RNA binding and ribonuclease domains. Additional features in the C-terminal half are a high cysteine content segment and limited homology to protein kinases, both regions are of unknown function (1, 2, 17). A binary model of RNase L emerges from these studies in which the regulatory functions map to the ankyrin repeat region in the N-terminal half, while the ribonuclease portion localizes to the C-terminal part of the enzyme (Fig. 1).
We thank Bryan Williams (Cleveland) for
comments and for the gift of PKR cDNA, Paul F. Torrence (Bethesda)
for the generous gift of the bromine-substituted 2-5A analog, Guiying
Li (Cleveland) for synthesizing p(A2p5
A)3, to Aimin Zhou
(Cleveland) for radiolabeling the 2-5A and RNA.