(Received for publication, November 23, 1994)
From the
2-5A-dependent RNase is an interferon-inducible enzyme that requires 5`-phosphorylated, 2`,5`-linked oligoadenylates (2-5A) for its endoribonuclease activity against single-stranded RNAs. We demonstrate here that recombinant, human 2-5A-dependent RNase forms stable homodimers during its stimulation by 2-5A. The protein dimers were observed to form only upon binding to 2-5A, as shown using gel filtration chromatography and chemical cross-linking and after centrifugation in glycerol gradients. A monoclonal antibody to 2-5A-dependent RNase was prepared and used to probe the subunit structure of the enzyme in the presence or absence of 2-5A. Using oligoadenylates of different length, structure, and 5`-phosphorylation states we determined that conversion of 2-5A-dependent RNase from its monomeric, inactive form to its homodimeric, active form required the presence of functional 2-5A. These results demonstrate that the catalytically active form of 2-5A-dependent RNase is a homodimer.
2-5A-dependent RNase is a uniquely controlled endoribonuclease that exists in cells of higher vertebrates as a component of the interferon-regulated 2-5A system (Kerr and Brown, 1978; Clemens and Williams, 1978; Slattery et al., 1979; Zhou et al., 1993). Interferon treatment of cells results in enhanced expression of the 2-5A-dependent RNase and the double-stranded RNA-activated synthetases that produce 5`-triphosphorylated, 2`,5`-linked oligoadenylates (2-5A) from ATP. Because double-stranded RNA is a frequent product of virus infection, the 2-5A system is often active in interferon-treated and virus-infected cells. For instance, the 2-5A system is implicated in the mechanism by which interferon inhibits encephalomyocarditis virus replication (Chebath et al., 1987; Rysiecki et al., 1989; Hassel et al., 1993). In addition, a role for the 2-5A system in cell growth control is suggested from the finding that murine cells expressing a dominant-negative form of 2-5A-dependent RNase are resistant to the anti-cellular activity of interferon (Hassel et al., 1993).
The 2-5A-dependent RNase is converted from its catalytically inactive state to its active form upon binding 2-5A, but little is known about the activation process per se. However, intriguing clues about the mode of regulation have emerged from the molecular cloning and expression of 2-5A-dependent RNase (Zhou et al., 1993; Hassel et al., 1993; Dong et al., 1994). For example, sequence analysis indicates the presence in the amino-terminal half of the ribonuclease of nine ankyrin-like protein-protein interaction domains (Hassel et al., 1993). The 2-5A binding domain requires 2 lysine residues present in P-loop motifs in the seventh and eighth ankyrin repeats (Zhou et al., 1993), whereas the ribonuclease function requires the carboxyl-terminal portion of the protein (Hassel et al., 1993). The presence of ankyrin repeats suggests that the catalytic function of the enzyme could be regulated through intra- or intermolecular protein/protein contacts.
Independent determinations of the size of the 2-5A-dependent RNase from various species, cell types, and tissues have yielded differing findings. Size estimations consistent with a monomeric form of 2-5A-dependent RNase were reported for the enzyme in mouse reticulocytes (Revel et al., 1979), rabbit reticulocytes (Wreschner et al., 1982), murine EAT cells (Floyd-Smith and Lengyel, 1986), human HeLa cells, and murine W7 cells (Nilsen et al., 1981), and for the recombinant human enzyme expressed in insect cells (Dong et al., 1994) (see Fig. 1). In contrast, a 185-kDa complex containing 2-5A-dependent RNase activity was reported from murine EAT cells (Slattery et al., 1979), whereas mouse spleen (Bisbal et al., 1989) was reported to contain 2-5A-dependent RNase activities which migrated as 185- and 40-kDa species. The 40-kDa activity has not been reported elsewhere and could represent a degradation product or novel form of 2-5A-dependent RNase. Salehzada et al.(1993) also reported a 160-kDa heterodimer that contained 2-5A-dependent RNase activity from human Daudi cells. Recently, Bayard et al.(1994) reported that murine 2-5A-dependent RNase covalently attached to a 2-5A analog exists largely as a 160-kDa species. To resolve these apparently contradictory findings, we have determined the effect of 2-5A binding on the subunit composition of 2-5A-dependent RNase. Using homogeneous, recombinant enzyme and a monoclonal antibody we provide clear evidence that the binding of functional 2-5A induces the formation of stable dimers of 2-5A-dependent RNase. These findings suggest a molecular mechanism in which activation and dimerization of 2-5A-dependent RNase are different aspects of the same process.
Figure 1:
2-5A-dependent RNase forms a dimer in
the presence of 2-5A as determined by gel filtration chromatography.
2-5A-dependent RNase was applied to a Superose 12 column in the
presence () or absence (
) of
p
A(2`p5`A)
. 2-5A-dependent ribonuclease
activity (in units) in the column fractions was determined (see
``Materials and Methods''). The positions of the protein
markers and their sizes in kilodaltons are indicated (arrows).
Confirmation that 2-5A induced the
formation of homodimers was obtained by monitoring 2-5A-dependent RNase
after centrifugation in glycerol gradients. Purified RNase was
preincubated in the absence or presence of pA(2`p5`A) prior
to loading on to glycerol gradients. Aliquots of the gradient fractions
were analyzed after SDS-polyacrylamide gel electrophoresis. The
proteins were transferred to nitrocellulose filters and reacted with a
monoclonal antibody against human 2-5A-dependent RNase (see
``Materials and Methods''). In the absence of 2-5A, the RNase
migrated as a monomer (Fig. 2, upper panel). In
contrast, after preincubation with 2-5A, the RNase migrated in two
peaks corresponding in sizes to a monomer and a dimer (Fig. 2, lower panel). The lack of complete conversion to the dimeric
form could be due to the dissociation of 2-5A from a portion of the
RNase because of the absence of 2-5A in the gradient.
Figure 2:
2-5A induces homodimers of 2-5A-dependent
RNase as shown after centrifugation in glycerol gradients.
2-5A-dependent RNase without 2-5A (upper panel) or
preincubated with 5 µM pA(2`p5`A) (lower
panel) was centrifuged in 10-30% glycerol gradients prior to
gel electrophoresis, transfer to nitrocellulose, and detection with
monoclonal antibody against human 2-5A-dependent RNase (see
``Materials and Methods''). The positions and sizes (in
kilodaltons) of the internal protein markers in the glycerol gradients
are shown above the fraction numbers. To the right are indicated the RNase (arrow) and the positions and
sizes of the protein markers in the gel.
Figure 3:
DMS covalently cross-links homodimers of
2-5A-dependent RNase. The cross-linking agent, DMS, was incubated with
2-5A-dependent RNase in the absence (upper panel) or presence (lower panel) of 5 µM pA(2`p5`A) (see
``Materials and Methods''). Centrifugation of the
2-5A-dependent RNase was in 20-40% glycerol gradients followed by
gel electrophoresis, transfer, and incubation with monoclonal antibody.
Labeling of the figure was as described in the legend to Fig. 2.
Figure 4: Dimerization of 2-5A-dependent RNase occurs only in the presence of functional activator. Covalent cross-linking of 2-5A-dependent RNase with DMS was performed after incubation in the presence of different amounts of various oligoadenylates (as indicated). A, Lane 1 was without DMS. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose and probed with monoclonal antibody against 2-5A-dependent RNase. The positions of the monomer and dimer of 2-5A-dependent RNase are shown.
The relative amounts of 2-5A and
RNase required to obtain dimers are apparent from these assays. Nearly
complete conversion of the monomeric form of the enzyme to the dimeric
form was observed when there was a 1:1 molar ratio of RNase to
pA(2`p5`A), i.e. 250 nM of each (lane
5). However, dimerization was also very efficient at a 2:1 ratio
of [RNase] to [pA(2`p5`A)
] (Fig. 4A, lane 4). Similarly, efficient
dimerization was obtained with 1:1 ratios of
[p
A(2`p5`A)
] or
[p
A(2`p5`A)
] to [RNase] (Fig. 4B, lanes 9 and 12,
respectively).
For comparison, we measured the abilities of the same
oligoadenylates to activate the catalytic activity of 2-5A-dependent
RNase against poly(rU)-[P]pCp (Dong et
al., 1994) (Table 1). Under the conditions of the assay,
which included 24 nM 2-5A-dependent RNase, a 50% decrease of
intact poly(U) was obtained with about 0.3 nM pA(2`p5`A)
, p3A(2`p5`A)
, or
p3A(2`p5`A)
. In contrast, 4 nM A(2`p5`A)
and 110 nM A(2`p5`A)
were necessary to
obtain the same level of RNase activity (Table 1). However,
pppA(3`p5`A)
and pppA2`p5`A were completely lacking in the
ability to activate the RNase. Therefore, there was a relationship
between the abilities of various 2`,5`-linked oligoadenylates to
activate and induce homodimers of the 2-5A-dependent RNase.
We demonstrate here by three independent methods that binding
of 2-5A to 2-5A-dependent RNase induces dimerization of the enzyme. The
results are consistent with a model in which only 2-5A-dependent RNase
molecules which are bound to 2-5A can form such dimers (Fig. 4).
These findings may explain results from a number of different
laboratories that the 2-5A-dependent RNase exists as either a 85-
or
170-kDa protein (Revel et al., 1979; Wreschner et
al., 1982; Floyd-Smith and Lengyel, 1986; Nilsen et al.,
1981; Dong et al., 1994; Slattery et al., 1979;
Bisbal et al., 1989; Bayard et al., 1994). In at
least some instances, the larger species of the enzyme may have
resulted from dimerization caused by endogenous 2-5A. In other
situations, however, 2-5A-dependent RNase may interact with other
proteins. In Slattery et al.(1979) addition of 2-5A did not
alter the migration of the 185-kDa peak of 2-5A-dependent RNase
activity during gel filtration. Therefore, in that study perhaps the
enzyme was already in its homodimeric form. The close correlation
between the ability of different oligoadenylates to activate and
dimerize the enzyme suggests that the association of 2-5A-dependent
RNase molecules into homodimers is an essential feature of the
activation process per se. For instance, pppA(3`p5`A)
and pppA2`p5`A fail to either activate or dimerize the RNase. All
of the 2`,5`-oligoadenylates which activate the RNase also induce
dimers to form. In general, the more efficient the ability of the
2`,5`-oligoadenylate to activate the catalytic function of the enzyme,
the greater the ability to induce dimers. How might the binding of 2-5A
to the enzyme lead to dimerization? Perhaps, in the absence of 2-5A,
the nine ankyrin-like repeats interact with the catalytic domain to
block ribonuclease activity. The ankyrin domains could also prevent
dimerization by blocking access to the interaction sites. Binding of
2-5A to the P-loop motifs present in the seventh and eighth ankyrin
repeats (Hassel et al., 1993) could release the ankyrin clamp,
thus allowing the RNase to dimerize into its active form. It is
possible that the catalytic function of the enzyme requires both
subunits of the dimeric enzyme. In this regard, we previously reported
that a carboxyl-terminal truncation in the RNase produced a potent
dominant negative inhibitor of the wild type RNase (Hassel et
al., 1993). Perhaps the inhibition could have resulted, at least
in part, from the formation of inactive heterodimers of the enzyme. In
this model, the principal function of 2-5A could be to alter the
structure of the monomeric protein to allow the active, dimeric form of
the enzyme to form.