Figure is a schematic depiction of the RNA probes used in the present study; details of their construction are in Materials and Methods. All of the probes contained the same 5′ sequence of 58 (or in one case 61) ribonucleotides transcribed from the multiple cloning sites (MCS) of vector SK− (Stratagene), which permitted the comparison of the degradation profiles of related probes. All probes contained a 5′-cap. Probe ARE-A50 (Fig. ) consisted of 58 bases transcribed from the MCS of SK− and 24 bases of the core ARE from the mouse TNF-α mRNA (bp 1309 to 1332 of GenBank accession no. X02611), followed by 50 A's; the 3′ end of the RNA did not contain any non-A ribonucleotides (see Materials and Methods). Probe A/C ARE-A50 consisted of the same components as described for ARE-A50 except that the flanking A's of the AUUUA motif were replaced by C's. Probe ARE was identical to ARE-A50 except that it did not contain a poly(A) tail. In probe V, the RNA transcript only consisted of the 58 bases transcribed from the MCS of SK−. Probe A50 (Fig. ) contained 61 bases of the SK− MCS 5′ of 50 A's. Probe g668-775A50 (Fig. ) contained 58 bases of the SK− MCS, followed by the 3′-most 108 bases of the mouse GM-CSF mRNA (bp 3399 to 3506 of GenBank accession no. X03020), followed again by 50 A's. This region of the GM-CSF mRNA contains the ARE, as indicated in Fig. . Probe g668-775 was identical except that it lacked the poly(A) tail.
Cell-free deadenylation of polyadenylated, ARE-containing probes.
These probes were then used to characterize the ability of 293 cell extracts to promote RNA deadenylation in a TTP-dependent manner. These cells do not express endogenous TTP; the extracts used for these experiments were from cells transiently transfected with CMV.hTTP.tag, its derivatives, or vector alone (20
). Similar results were obtained with extracts derived from a 20-min, 12,000 × g
centrifugation after cell lysis with 0.5% NP-40, as with extracts homogenized in the absence of detergent and centrifuged for 100,000 × g
for 45 min at 4°C. Therefore, all of the results shown here were obtained with the detergent-free, high-speed extracts. Similar results were also obtained with capped and uncapped RNA substrates; all data shown in the present study were obtained with capped substrates.
When probes A50, ARE, ARE-A50, and V were incubated with extracts from cells transfected with vector alone, all three probes showed similar patterns of slight degradation after 1 h at 37°C (Fig. , compare lanes 1 and 2, lanes 5 and 6, lanes 9 and 10, and lanes 13 and 14). However, when these probes were incubated in parallel with the same amount of extract protein prepared from cells transfected with the human TTP expression plasmid CMV.hTTP.tag, only probe ARE-A50 was markedly more degraded (Fig. , compare lanes 3 and 4, lanes 7 and 8, and lanes 11 and 12). The disappearance of probe ARE-A50 in the presence of TTP (lane 12) was accompanied by the appearance of a new band of smaller size (arrow) that migrated to the same position as probe ARE, indicating that the new band represented the accumulation of a deadenylated form of the ARE-A50 probe. Thus, under these experimental conditions, TTP appeared to promote the degradation of only the ARE-A50 probe and not the degradation of a non-ARE-containing poly(A) probe (A50) or an ARE-containing nonpolyadenylated probe (ARE). The migration position of the V probe (MCS sequences alone) is shown in lanes 13 and 14.
FIG. 2. Cell-free deadenylation of polyadenylated, ARE-containing RNA probes. In panels A to C, 293 cell extracts were incubated with 32P-labeled RNA probes on ice (no symbol) or at 37°C (+) for 60 min, and EDTA (final concentration, 20 mM) was (more ...) Effect of nonbinding TTP mutants on deadenylation.
We have shown previously that the increased levels of TNF-α and GM-CSF mRNAs in cells derived from TTP-deficient mice were due to increased stability of those mRNAs (7
). Conversely, TTP promoted the instability of ARE-containing mRNAs in a 293 cell cotransfection system, apparently by first degrading the poly(A) tail of the mRNA (19
). This mRNA destabilizing effect of TTP required its binding to the ARE of these mRNAs through its TZF domain, since TTP lost both ARE-binding and mRNA-destabilizing activities when key amino acids in the TZF domain were mutated (19
). We therefore evaluated the importance of the TZF domain to TTP's ability to promote RNA deadenylation in the cell-free deadenylation assay. As shown in Fig. , an extract containing the TTP zinc finger mutant C124R caused the same minimal degradation of the probe ARE-A50 as seen with extracts from cells transfected with vector alone (Fig. , compare lanes 1 and 2 to lanes 5 and 6). The mutant TTP also did not cause accumulation of the lower-molecular-weight band, as seen with extracts containing wild-type TTP (Fig. , lanes 3 and 4), which again migrated to approximately the same position as the probe ARE (Fig. , lanes 7 and 8; arrow). Previous studies have documented that this mutant TTP protein is expressed at least as well as the wild-type TTP protein under these transfection conditions (see, for example, Fig. in reference 20
). These studies demonstrate that the ability of TTP to bind to the ARE was required to promote the deadenylation of ARE-containing, polyadenylated RNA probes.
FIG. 8. Attempted cross-linking of coexpressed PARN-FLAG and TTP-HA. Extracts were prepared from 293 cells expressing vector alone (BS+) or CMV-hTTP-tag (hTTP), CMV.hPARN.flag (hPARN), or both together. The extracts were then incubated without (−) (more ...) Effect of a mutant ARE on TTP-induced deadenylation.
We have shown previously that a mutant ARE, in which all of the A's of the core AUUUA pentamer were changed to G's, did not bind TTP (21
). We tested whether the deadenylation of a similar mutant probe could be stimulated by wild-type TTP. The probe used was identical to probe ARE-A50 except that the flanking A's of the AUUUA motif in the ARE had been mutated to C's. In this case, TTP did not stimulate the deadenylation of the mutant probe A/C ARE-A50, despite evidence that TTP promoted the deadenylation of the normal ARE-A50 probe (Fig. , compare lanes 3 and 4 to lanes 7 and 8). In a gel shift assay with the same mutant probe, A/C ARE-A50, extracts from cells transfected with vector alone, wild-type TTP, or the C124R mutant formed similar complexes that migrated in similar patterns (Fig. , lanes 2 to 4). When probe ARE-A50 was incubated with extract from cells transfected with CMV.hTTP.tag, there was formation of the usual TTP-probe complex (Fig. , lane 7), which was not seen with extracts of cells transfected with the TTP mutant C124R or vector alone (Fig. , lanes 6, 8). The specificity of this TTP-probe complex has been validated in several previous studies (21
). These results indicated that a probe competent to bind TTP was necessary for deadenylation to occur in this cell-free assay.
Ability of TTP to promote deadenylation of a GM-CSF ARE probe.
We also tested a probe derived from an mRNA containing a second class II ARE that was contained in the GM-CSF mRNA. This probe was important to test because its ARE ends ca. 54 bases 5′ of the beginning of the poly(A) tail, as occurs in the natural GM-CSF mRNA; this is in contrast to the ARE-A50 probe used for most of these experiments, in which the core ARE from TNF-α mRNA was linked directly to a poly(A) tail, separated only by the XbaI cloning site. When the GM-CSF probe was incubated with 293 cell extracts, TTP caused degradation of the polyadenylated GM-CSF probe (Fig. , compare lanes 5 and 6 with lanes 7 and 8), again accompanied by the appearance of a smaller, deadenylated species (lane 8, arrow); however, TTP had no effect on the nonpolyadenylated probe (lanes 1 to 4). As in the case of the TNF-α probe, the TTP mutant C124R was without effect on degradation of the polyadenylated GM-CSF probe (lanes 9 and 10). As expected, native but not C124R mutant TTP could bind to the GM-CSF ARE probe in a gel shift assay (Fig. ).
FIG. 3. Ability of TTP to promote deadenylation of a GM-CSF ARE probe. (A) Extracts from 293 cells transfected with vector alone (BS+), CMV.hTTP.tag (hTTP), or the TTP zinc finger mutant (C124R) were mixed with the GM-CSF ARE probes g668-775 (lanes 1 (more ...) Effect of TTP-related proteins on probe deadenylation.
Besides TTP, mammals express two additional CCCH TZF proteins, ZFP36L1 and ZFP36L2 (5
). Although the physiological functions of these two proteins are unknown, they have been shown, like TTP, to bind to ARE probes and stimulate the breakdown of ARE-containing mRNAs when coexpressed in cells (21
). To examine whether these proteins caused the deadenylation of the ARE-containing polyadenylated probes in this cell-free system, we performed similar assays with extracts from 293 cells transfected with CMV.cMG1.tag (the rat orthologue of ZFP36L1) or CMV.xC3H-3.tag (the Xenopus
orthologue of ZFP36L2). When probe ARE-A50 was incubated with extracts from cells transfected with TTP, cMG1, or xC3H-3 expression plasmids, the probe was degraded in a characteristic fashion in the presence of all three proteins (Fig. , lanes 4, 6, and 8) compared to extracts from vector alone-transfected cells (Fig. , lanes 1 and 2). The binding of these proteins to this probe could be readily demonstrated by gel shift analysis (Fig. ). Thus, representatives of the two TTP-related proteins behaved like TTP in this cell-free deadenylation assay.
FIG. 4. Effect of TTP-related proteins on probe deadenylation. (A) Extracts from 293 cells transfected with vector alone (BS+), CMV.hTTP.tag (hTTP), CMV.cMG1.tag (cMG1), or CMV.xC3H-3 (xC3H-3) were incubated with probe ARE-A50 (lanes 1 to 8) or ARE (lanes (more ...) Effect of the TZF domain alone on deadenylation.
The members of this CCCH TZF protein family share a highly conserved TZF region that comprises the ARE-binding domain (21
). To determine whether the TZF region alone was sufficient to induce the deadenylating activity in 293 cell extracts, we evaluated extracts from cells transfected with either full-length TTP or the epitope-tagged TZF domain, consisting of amino acids 97 to 173 of human TTP (GenBank RefSeq accession no. NP_003398
). As expected, extracts from cells expressing full-length TTP caused degradation of the probe and accumulation of the deadenylated ARE band (Fig. , lanes 3 and 4, arrow). In contrast, the TZF-containing extract caused minimal degradation of the probe, and no accumulation of the deadenylated probe, after 60 min of incubation (Fig. , lanes 5 and 6), similar to extracts from cells transfected with the vector alone (Fig. , lanes 1 and 2). Despite the lack of activity in the deadenylation assay, the TZF domain polypeptide could readily bind to the ARE-A50 probe, as demonstrated by gel shift analysis (Fig. ). These data indicated that the TZF domain peptide was unable to mimic full-length TTP in this cell-free deadenylation system.
Characterization of the TTP-induced deadenylating activity.
As described more fully below, we found that the activity within the 293 cell extracts that acted with TTP to stimulate deadenylation of ARE-containing polyadenylated probes was sensitive to boiling and could be extracted with phenol-chloroform, both of which are characteristics of proteins. Since the TTP-inducible deadenylating activity was present in a non-detergent-containing, 100,000 × g
supernatant from 293 cells and required magnesium but not ATP (see below), we focused on the human enzyme PARN. Mammalian PARN activity has been shown to depend on Mg2+
but not on ATP (17
). To investigate whether PARN might be involved in the TTP-dependent deadenylation of ARE-containing probes in this cell-free system, we prepared cell extracts in MgCl2
-free buffer and then added back various concentrations of MgCl2
. In the presence of 3 mM MgCl2
at 37°C, probe ARE-A50 was slightly degraded in extracts prepared from cells transfected with vector alone (Fig. , lane 2); however, in the absence of added MgCl2
, the probe was completely stable at 37°C (Fig. , lane 3). When the probe was incubated with extracts from cells transfected with TTP, both the usual deadenylation of the probe and the appearance of the deadenylated probe decreased with decreasing concentrations of MgCl2
(Fig. , lanes 4 to 7). These findings suggested that the ARE-containing, polyadenylated RNA probe was degraded in the presence of TTP by a Mg2+
-dependent activity present in the 293 cell extracts. The presence or absence of ATP had no effect on the TTP-dependent deadenylating activity (data not shown).
FIG. 5. Characterization of the TTP-induced deadenylating activity. Extracts from 293 cells transfected with vector alone (BS+), CMV.hTTP.tag (hTTP), and CMV.hPARN.flag (hPARN) were incubated on ice or at 37°C (+) for 60 min and processed (more ...)
We also examined the effect of Mg2+ on the degradation of probe ARE-A50 in extracts from 293 cells that overexpressed human PARN. In the presence of 3 mM MgCl2, the PARN-containing extracts caused complete disappearance of the polyadenylated probe (Fig. , lanes 9 and 10). This activity decreased with decreasing concentrations of MgCl2 (Fig. , lanes 9 to 13), although there was no accumulation of the deadenylated RNA (arrow) as seen with TTP (lane 5). There was some PARN-induced degradation of the probe in the absence of added MgCl2 (Fig. , lane 13), possibly due to trace amounts of Mg2+ in the cell extracts (see below).
A polyadenylated probe (A50) that did not contain the ARE was minimally degraded by the 293 cell extracts from vector alone-transfected cells, both in the presence or absence of added 3 mM MgCl2 (Fig. , lanes 1 to 3). This probe was also minimally degraded in extracts from TTP-transfected cells, either when various concentrations of Mg2+ were present or in the presence of 1 mM EDTA (Fig. , lanes 4 to 9). However, when extracts from PARN-transfected 293 cells were exposed to the A50 probe, there was dramatic, MgCl2-dependent degradation of the probe that did not occur in the presence of 1 mM EDTA (Fig. , lanes 10 to 15).
Although the experiments described above demonstrate that the TTP-dependent deadenylating activity present in 293 cell extracts on ARE-containing, polyadenylated RNA substrates was dependent on Mg2+, it remained possible that the association of TTP with the ARE was itself Mg2+ dependent. However, neither the gel shift patterns of endogenous 293 cell proteins forming complexes with probe ARE-A50 (Fig. , lanes 1 and 2) nor TTP expressed in 293 cells (Fig. , compare lanes 3, 8, and 9) required added MgCl2. The formation of both TTP-ARE complexes decreased with increasing concentrations of EDTA (Fig. , lanes 3 to 7), perhaps due to chelation of the zinc ions within TTP's zinc fingers. These data indicate that the lack of TTP-induced deadenylation seen in the absence of Mg2+ was not due to inhibited TTP binding to the ARE under these conditions.
Effects of TTP and PARN together to promote deadenylation.
To evaluate the possible synergistic activation of deadenylation caused by TTP and PARN, cells were transfected with cDNAs expressing PARN and TTP, singly and together. Extracts containing TTP alone caused a time-dependent degradation of the ARE-A50 probe and accumulation of the deadenylated probe (Fig. , compare lanes 5 to 8 with lanes 3 and 4). Transfection of PARN alone caused a time-dependent deadenylation of the probe, but no accumulation of the deadenylated band (Fig. , lanes 9 to 12). However, when the effects of PARN and TTP together were evaluated under these conditions, there was complete probe degradation, and marked accumulation of the deadenylated probe (Fig. , lanes 13 and 14). Note that lane 14 (TTP plus PARN) was from only a 15-min incubation and is thus directly comparable to lane 6 (TTP alone) and lane 10 (PARN alone) at this time point. Thus, the two proteins together produced a dramatic and synergistic stimulation of probe ARE-A50 deadenylation under these conditions.
FIG. 6. Effects of TTP and PARN expressed together to promote deadenylation. For panels A and B, extracts from 293 cells transfected with vector alone (BS+), CMV.hTTP.tag (hTTP), CMV.hPARN.flag (hPARN), or either BS+ or CMV.hTTP.tag together with (more ...)
When similar experiments were performed with the A50 probe that lacked an ARE, there was no effect of TTP on probe degradation compared to extracts from cells transfected with vector alone (Fig. , compare lanes 3 and 4 to lanes 1 and 2). In extracts from cells cotransfected with PARN, the time courses of the probe degradation profiles were essentially the same when vector alone or TTP was cotransfected (Fig. , lanes 5 to 12). Thus, TTP had no apparent effect on the ability of PARN to cause deadenylation of a poly(A) probe that lacked an ARE.
We next performed similar experiments on ice, in an attempt to slow the reaction that was essentially complete by 15 min at 37°C in the presence of both PARN and TTP (see lane 14 in Fig. ). At 0°C, extracts from cells transfected with vector alone or various amounts of the TTP expression plasmid did not promote destabilization of the ARE-A50 probe after 60 min (Fig. , lanes 1 to 5). In the extract from cells transfected with PARN alone, there was barely detectable degradation of the ARE-A50 probe on ice, even after 60 min of incubation (Fig. , lanes 19 to 22). However, in extracts from cells expressing both exogenous PARN and TTP, there was a time-dependent degradation of the probe ARE-A50 at 0°C, accompanied by a gradual increase in the accumulation of the deadenylated ARE band (arrow; Fig. , lanes 6 to 18), and the deadenylating activity was dependent on the amount of transfected TTP DNA used. At the lowest concentration of TTP DNA used, 10 ng of CMV.hTTP.tag, there was no apparent degradation of the probe after 60 min on ice (Fig. , lane 18). The TTP zinc finger mutant C124R alone did not induce any endogenous deadenylating activity (Fig. , lane 23); after 60 min of incubation with extract from cells cotransfected with the PARN vector, the probe was found to be degraded to approximately the same extent as occurred with extracts from cells transfected with PARN alone (Fig. , compare lanes 22 and 24).
The possible effects of PARN on the association of various concentrations of expressed TTP with the ARE-A50 probe were evaluated in a gel shift assay (Fig. ). The presence of PARN in the extract did not increase the binding of TTP to the RNA probe, nor did it result in the formation of a “supershifted” complex, as might be expected if there were direct physical association between TTP and PARN (Fig. ). PARN alone did not shift this probe into the gel (Fig. , compare lanes 1 and 10). At the concentration of TTP DNA, 10 ng, at which no detectable synergistic activation of deadenylation with PARN occurred at 0°C (Fig. , lane 18), there was barely detectable formation of a TTP-probe complex (Fig. , lanes 5 and 9).
Effects of affinity-purified TTP and PARN on deadenylation.
To begin to address the question of whether PARN plus TTP alone could promote the ARE-dependent deadenylation of poly(A) probes, the fusion proteins hTTP-FLAG or hPARN-FLAG were isolated by affinity chromatography from 293 cells transfected with the expression plasmids (Fig. ). The fusion proteins were eluted from the affinity matrix with FLAG epitope peptide in an attempt to decrease nonspecific elution of contaminating proteins. When the probe ARE-A50 was incubated at 37°C with either the TTP eluate alone (Fig. , lane 1) or the PARN eluate alone (P) (Fig. , lane 2), there was minimal degradation of the intact probe compared to the effects of extract from cells transfected with vector alone (Fig. , lane 5). However, the probe was almost completely degraded, along with formation of the characteristic lower band, when both FLAG eluates were combined (Fig. , lane 3). When the FLAG-TTP eluate was added to the vector-transfected extract, the ARE-A50 probe degradation profile was virtually identical to that seen when extract from TTP-transfected cells was used (Fig. , compare lanes 6 and 10). The addition of PARN-FLAG in the absence of TTP caused more degradation of the probe than the presence of the endogenous deadenylating activity (Fig. , compare lanes 4 and 7). When both eluates were added, the probe was almost completely degraded (Fig. , lane 8), with formation of the lower band (Fig. , lane 8) that migrated to the same position as probe ARE (arrow; Fig. , lanes 11 to 14). Thus, under these conditions, the individual TTP and PARN eluates each had minimal deadenylating activity; however, the combination of the two had marked deadenylating activity toward the ARE-A50 probe. Similar results were seen when the TTP and PARN eluates were incubated together with probe ARE-A50 on ice (results not shown).
FIG. 7. Effects of affinity-purified TTP and PARN on deadenylation. Deadenylation assays were performed with the fusion proteins hTTP-FLAG or hPARN-FLAG that had been isolated by affinity chromatography from 293 cells transfected with the appropriate expression (more ...)
To characterize further the endogenous factor(s) in 293 cells whose deadenylating activity was stimulated by TTP in the degradation of the ARE-containing poly(A) probes, we treated 293 cell extracts by phenol-chloroform extraction or by boiling. When the phenol-chloroform extracted or boiled 293 extracts were incubated with the ARE-A50 probe at 37°C, there was little degradation of probe compared to untreated extract (Fig. , lanes 1 to 4). Likewise, the ARE-A50 probe stability in the FLAG peptide eluate added to untreated, or extracted, or boiled extracts at 37°C was comparable to that seen in the absence of the eluate (Fig. , lanes 5 to 8). When the eluted FLAG-TTP was incubated with the probe, there was a slight degradation of the probe but no change in the size of the probe was seen (Fig. , lane 9). However, in the presence of the untreated 293 extract, almost all of the probe was degraded, with the formation of the smaller band (Fig. , lane 10) that migrated to the same position as that seen when extracts from 293 cells transfected with TTP were used (Fig. , lane 14). FLAG-TTP added to phenol-chloroform or heat-treated 293 extracts did not cause the probe to degrade (Fig. , lanes 11 and 12), suggesting that the deadenylation factor(s) effectively activated by TTP was a protein and was heat labile. Probe ARE incubated with the TTP-containing extract was also shown in this experiment (Fig. , lanes 15 and 16).
Attempted cross-linking of coexpressed FLAG-PARN and hemagglutinin (HA)-TTP.
These studies do not establish the mechanism by which TTP effectively increases PARN activity toward ARE-containing, polyadenylated RNA substrates. One simple model is that TTP acts as a tether or adaptor molecule, physically linking PARN to the RNA by a direct physical interaction between TTP and PARN. To test this possibility, we performed protein cross-linking experiments in cell extracts by using the bifunctional cross-linker DSS. These studies used 293 cell extracts in which one or both proteins were overexpressed as fusions with different epitope tags, followed by cross-linking in the presence or absence of magnesium. Figure shows the results of the present study; each panel of Fig. is a similar Western blot of the various 293 cells extracts, in each case probed with a different antibody.
Figure shows a Western blot of extracts probed with the FLAG antibody to recognize the FLAG epitope on PARN but not on TTP. This antibody identified immunoreactive PARN as an Mr
~80,000 protein (lanes 9 to 16); this band was not seen in extracts from cells transfected with vector alone (lanes 1 to 4) or with HA-tagged TTP alone (lanes 5 to 8). Cross-linking with DSS revealed a new complex of Mr
~190,000 that strongly reacted with the FLAG antibody (bracket in Fig. ). This complex was only seen after cross-linking; it was not affected by the presence of cotransfected TTP (lanes 13 to 16) or by the presence or absence of magnesium. The identity of the components of this complex is not known, except that the immunoreactivity identifies one component as PARN-FLAG; this complex may represent the oligomeric form of the activated human enzyme, as shown previously (26
). However, the presence of this larger complex confirms that the cross-linking was effective. An identical blot was then probed with a PARN antibody (Fig. ). This showed the expected reactivity with transfected PARN at Mr
~80,000 (lanes 9 to 16) and confirmed that the higher-Mr
complex (bracket) also contained immunoreactive PARN. A much smaller amount of immunoreactive PARN was noted in the cells transfected with vector alone (lanes 1 to 4) or with TTP-HA alone (lanes 1 to 8). This endogenous PARN immunoreactivity decreased in amount after cross-linking, and longer exposures of this blot showed that the large complex was also formed between endogenous PARN and unknown partners after cross-linking (not shown).
An identical blot was then probed with the HA antibody to identify TTP-HA (Fig. ). TTP was not detected in the vector alone transfected cells (lanes 1 to 4) or the cells transfected with PARN alone (lanes 9 to 12); however, a broad band of immunoreactivity of the appropriate size was present when TTP was transfected, alone (lanes 5 to 8) or with PARN (lanes 13 to 16). A faint band at Mr
~110,000 is probably the TTP dimer noted in previous studies (20
). Neither the amount of TTP nor its larger complex was affected by the presence or absence of the cross-linker, PARN, or magnesium.
If there had been direct complex formation between TTP and PARN, we would have expected formation of a novel Mr ~120,000 complex that reacted to all three of the antibodies used: FLAG, PARN, and HA. However, no such complex was observed with any of the three antibodies used, either in the presence or in the absence of magnesium (Fig. , lanes 13 to 16). Therefore, despite abundant expression of both TTP and PARN and despite evidence that cross-linking had occurred, we found no evidence for a direct physical association between the two proteins under these conditions.
Effect of inactive PARN mutants.
To determine whether the TTP effect could be mediated or inhibited by inactive PARN mutants, we relied on previous data demonstrating that mutation of any one of four key amino acids within the primary sequence of human PARN completely inactivated the enzyme (30
). We therefore made similar mutations in our PARN expression vector and examined both their enzymatic activity and their ability to influence TTP activity in 293 cell extracts.
First, we examined the activity of the PARN mutants on a poly(A) substrate (Fig. ); in all panels of Fig. , the reactions were conducted at 30°C in an attempt to slow them somewhat. As noted earlier, cell extracts enriched in transfected TTP were essentially identical to extracts from cells transfected with vector alone in their inability to promote deadenylation of the poly(A) substrate (Fig. , compare lanes 2 and 3). Cell extracts enriched in native (i.e., nonmutant) PARN exhibited the usual ability to cause shortening of this substrate (Fig. , lane 4). However, each of three PARN mutants—D28A, E30A, and D382A—when expressed in 293 cells exhibited essentially no deadenylating activity under these conditions (Fig. , lanes 5 to 7). The expression of TTP plus native PARN had no effect on PARN′s ability to promote deadenylation of this non-ARE-containing probe (Fig. , lane 8). Similarly, the coexpression of TTP with the three mutant PARN proteins had no effect on their inability to promote probe deadenylation (Fig. , lanes 9 to 11). Each of the three mutant proteins was expressed at levels comparable to that observed with the native protein, as determined by Western blotting of the same extracts with the HA epitope antibody (Fig. ). The expression of the mutant PARN proteins was modestly increased by the coexpression of TTP in this experiment, whereas the expression of FLAG-tagged TTP was not affected by the coexpression of native or mutant PARN (Fig. ).
FIG. 9. Effect of inactive PARN on probe deadenylation in the presence or absence of TTP. Extracts (5 μg of protein) from 293 cells transfected with vector alone (BS+), CMV.hTTP.tag (hTTP), CMV.hPARN.flag (hPARN), or its mutants (D28A, E30A, and (more ...)
We then examined the effect of TTP on the ability of cotransfected native and mutant PARN to deadenylate the ARE-containing, polyadenylated substrate. The concentrations of expressed TTP and native PARN were adjusted so that each would have a relatively minor effect alone, making synergy between the two readily detectable. The deadenylation reactions were performed at 30°C for the same reason. As shown in Fig. , there was essentially no probe degradation after the incubation of extracts from cells transfected with vector alone for 60 min at 30°C (compare lane 19 to lane 18). The expression of native PARN led to a modest shortening of the probe under these conditions (Fig. , compare lane 20 to lane 19). As noted with the poly(A) substrate, there was no difference in deadenylating activity between extracts from cells transfected with vector alone (Fig. , lane 19) and those from cells transfected with the plasmids encoding the PARN mutants D28A (lane 21), E30A (lane 22), or D382A (lane 23). These data indicate that these PARN mutants had no effect on the ARE-containing, polyadenylated substrate, as noted for the poly(A) substrate.
We next performed time courses of probe degradation with extracts containing TTP alone and then with extracts containing TTP plus either native or mutant PARN proteins. Under these conditions of relatively low protein concentration and 30°C incubation, TTP alone had a modest, time-dependent effect on probe degradation and accumulation of the deadenylated substrate (Fig. , lanes 2 to 5). Coexpression of native PARN plus TTP resulted in the expected marked increase in time-dependent probe degradation accompanied by accumulation of the deadenylated probe (Fig. , lanes 6 to 8). The 60-min time point for TTP plus PARN (lane 8) exhibited markedly increased disappearance of the polyadenylated probe and appearance of the deadenylated probe compared to the control extract (lane 19), native PARN alone (lane 20), or TTP alone (lane 5) at the same time point. When TTP and the mutant PARN were cotransfected, there was no apparent increase in the net effect on deadenylation of this probe compared to TTP alone (compare lanes 3 to 5 with lanes 9 to 11, lanes 12 to 14, and lanes 15 to 17), demonstrating that TTP could not “effectively activate” the mutant enzyme. Importantly, the effects of TTP to promote probe deadenylation and the accumulation of the deadenylated probe were not inhibited by the coexpression of the mutant PARN, suggesting that the endogenous deadenylating activity that was increased in the presence of TTP was not inhibited by the presence of the mutant PARN. This apparent lack of an inhibitory effect of the mutant PARN proteins was more evident at either 37°C or when higher concentrations of TTP were used (data not shown). Figure also demonstrates the complete lack of effect of TTP alone (lane 26), PARN alone (lane 27), and TTP plus PARN (lane 28) on the stability of the nonpolyadenylated, ARE-containing probe.