From the Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824
Received for publication, November 19, 2002, and in revised form, February 3, 2003
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ABSTRACT |
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The CCR4 family proteins are 3'-5'-deadenylases
that function in the first step of the degradation of poly(A) mRNA.
Here we report the purification to homogeneity of the yeast CCR4
protein and the analysis of its substrate specificities. CCR4
deadenylated a 7N+23A substrate (seven nucleotides followed by 23 A
residues) in a distributive manner. Only small differences in CCR4
activity for different A length substrates were observed until only 1 A residue remained. Correspondingly, the Km for a
25N+2A substrate was found to be at least 20-fold lower than that for a
26N+1A substrate, although their Vmax values
differed by only 2-fold. In addition, the total length of the RNA was
found to contribute to CCR4 activity: up to 17 nucleotides (not
necessarily poly(A)) could be recognized by CCR4. Poly(U), poly(C), and
poly(G) were also found to be 12-30-fold better inhibitors of CCR4
compared with poly(A), supporting the observation that CCR4 contains a non-poly(A)-specific binding site. Surprisingly, even longer substrates ( The regulation of eukaryotic gene expression occurs at multiple
levels, including the control of mRNA stability. The poly(A) tail
at the 3'-end of eukaryotic mRNA plays a major role in controlling mRNA and protein levels (1). In many cases, deadenylation of the
poly(A) tail is the first step required for decapping and 5' to 3'
degradation of the mRNA sequence (2, 3). Not only does rapid
deadenylation lead to degradation of the mRNA and the shutoff of
protein synthesis, but deadenylation itself reduces the efficiency of
translation of mRNA (4). In yeast, mRNAs are deadenylated by
first shortening the poly(A) tail to ~15 nucleotides, after which the
5'-cap is removed. Sequences within the RNA body play a role in
controlling the rate of poly(A) removal as well as
trans-acting proteins, including presumably the deadenylase itself (5, 6).
In yeast, the CCR4 protein, as part of the CCR4-NOT complex, has been
shown to be responsible for cytoplasmic deadenylation (7-10). The CCR4
protein contains three major functional domains: an N-terminal
activation domain, a central leucine-rich repeat (LRR)1 domain that binds
CAF1, and a C-terminal exonuclease III-like domain (9, 11, 12). The
exonuclease III-like domain comprises the apparent CCR4 deadenylase
function based on mutagenic studies (8, 9). We (9) and others (8) have
shown that CCR4 is a 3'-5'-RNase and a single-strand DNA-specific
DNase that acts in a distributive manner with strong preferences for
poly(A) substrates. The CCR4 protein is enzymatically active even in
the absence of the CAF1 protein and other components of the CCR4-NOT
complex. In addition, point mutations of the putative catalytic
residues in the exonuclease domain of CCR4 abrogate its in
vitro and in vivo activities (8, 9). These observations
strongly indicate that the CCR4 protein is the catalytic subunit of the
mRNA deadenylase family of proteins, although CAF1 may display some
deadenylase activity under certain conditions (10).
Model studies have indicated that alterations in deadenylation rates
will have the greatest effects on changes in RNA degradation (13). We
have therefore investigated the substrate preferences and enzymatic
properties of CCR4 to understand the role CCR4 plays in selecting its
mRNA substrates. We found that CCR4 prefers RNA substrates with at
least 2 A residues at their 3'-ends. However, it was most
efficient with substrates that were at least 17 nucleotides in length,
although these sequences need not necessarily be poly(A). Inhibition
studies further support a non-poly(A)-specific binding site for
CCR4. Moreover, longer RNA substrates (at least 45 nucleotides) converted CCR4 to a processive enzyme. In addition, CCR4 by itself displayed no preference for capped substrates. These results suggest that the distributive-to-processive transition for CCR4 and the ability
of CCR4 to contact a certain length or type of sequence may be
sites by which other factors may regulate CCR4 deadenylase activity in vivo.
Plasmids and RNA Substrates--
The full-length CCR4 open
reading frame was cloned into a modified pYES2/CT vector (Invitrogen).
The resultant CCR4 gene under the control of the
GAL1 promoter contained an N-terminal FLAG tag and a
C-terminal His6 tag. Expression of CCR4 was in strain KY803-1a-1 (MAT Protein Extraction and
Purification--
FLAG-CCR4-His6 purification was
conducted by a few modifications as described previously (9). The yeast
culture containing FLAG-CCR4-His6 fusion protein was grown
in a selective medium with 4% glucose to A = 1.0 and
then shifted to 4% galactose/raffinose for growth to A = 1.5. The cells were washed and lysed in Tris acetate buffer (buffer
A: 50 mM Tris HOAc (pH 7.9), 150 mM KOAc, 0.1 mM MgCl2, 10% glycerol, and 0.1% Nonidet
P-40) plus a protease inhibitor mixture. After clarification of the
crude lysate (15,000 × g at 4 °C for 10 min), the
supernatants were ultracentrifuged at 100,000 × g for
45 min at 4 °C. The supernatants were subsequently incubated with
washed Ni2+-agarose beads (with buffer A) for ~4 h at
4 °C. After incubation, the Ni2+-agarose beads were
washed extensively with buffer A containing 50 mM
imidazole, and then the bound proteins were eluted with same buffer
containing 250 mM imidazole. The eluted proteins were dialyzed against buffer A (with 5% glycerol) for 24 h with two changes of buffer. The dialyzed proteins were incubated with
anti-FLAG M2 antibody-agarose (Sigma) at 4 °C for 4 h.
After extensive washes with buffer A, the FLAG beads were washed once
with buffer B (buffer A without Nonidet P-40). To remove the background
proteins, the beads were then incubated for 15 min at 4 °C in buffer
B containing 100 µg/ml nonspecific hemagglutinin competitor
peptide (YPYDVPDYA, Research Genetics). After a pre-elution step, the
bound FLAG fusion proteins were eluted twice with buffer B containing
200 µg/ml FLAG peptide (Sigma). The eluted proteins were dialyzed
against buffer B (with 5% glycerol) for 24 h with two changes of
buffer. The purified proteins were then quantified using the
Bradford assay. The CCR4 protein was further identified by
SDS-PAGE and Western analysis.
In Vitro Deadenylation Assay--
The following assay conditions
were used for in vitro deadenylation: 50 mM Tris
HOAc (pH 7.9), 0.01 mM MgCl2, 20 mM
KOAc, 0.10 units of RNase inhibitor, 10% glycerol, 1-100
µM RNA substrate, and the indicated amount of purified
CCR4. The RNA substrates were radiolabeled as previously indicated (9).
The reaction volume was 30 µl, and the incubations were performed at
37 °C unless otherwise indicated. Five-µl aliquots of the reaction
were removed, and the reaction was terminated by the addition of an equal volume of formamide/EDTA buffer. Reaction products were resolved
by subsequent electrophoresis on 8 M urea and 16%
polyacrylamide (19:1 (v/v) acrylamide/bisacrylamide) sequencing gels.
The products were analyzed and quantified using PhosphorImager software.
Kinetic Assays for Distributive Activity of CCR4--
To
determine the kinetic parameters for the distributive activity of CCR4,
experiments were performed with radiolabeled 26N+1A, 25N+2A, and 22N+5A
RNA oligonucleotides using purified CCR4 protein. The enzyme
concentration was adjusted to obtain linear reaction rates (at least up
to 1 h). Five µl of sample volume were removed at appropriate
times following incubation at 37 °C. The products were fractionated
using a Tris borate/EDTA-polyacrylamide sequencing gel, on which
the poly(A)-excised products were distinguished as discrete bands. The
reaction rate was determined by measuring the quantity of substrate
remaining after 5 min for the 26N+1A and 25N+2A substrates, for which
the predominant products were 26N and 25N+1A, respectively. For the
22N+5A substrate, the distributive rate was determined from the
total amount of deadenylation that occurred in 5 min after quantitating
the abundance of the 22N+5A, 22N+4A, 22N+3A, 22N+2A, and 22N+1A
products. For this analysis at low substrate concentration (see Fig.
3B, lower panel), we assumed that the relative
rate of deadenylation of the 5A substrate to the 1A product was uniform
based on our analysis showing uniform changes in abundance of each of
these products with time (see Fig. 2 and Ref. 9; data not shown). At
substrate concentrations >5 µM, the predominant reaction
was 22N+5A to 22N+4A (see Fig. 3B, lower panel).
To determine Km and Vmax
values, each kinetic experiment contained up to eight different
substrate concentrations. Each substrate was analyzed in duplicate
(sometimes in triplicate), and the experiments were conducted at least
three times. The values obtained were fitted by linear
regression analysis to the Michaelis-Menten equation and used to
determine, from the intercepts, Vmax and Km constants (Table
II).
The relative rates of the distributive deadenylation reaction were
determined for the 2A substrates as described above, for the 5A
substrates by quantitating the total amount of deadenylation that
occurred within 5 min, and for the 20A substrates by determining the
total amount of deadenylation that occurred within 1 min. For the 20A
substrates, within 1 min, little of the completely deadenylated
substrate is found; and a substantial amount of the 20A substrate
remains, with only 19A, 18A, and 17A substrates being predominantly
formed. Therefore, quantitating the total amount of deadenylation
within 1 min provides a reasonable estimate of the 20A reaction rate,
given that very little difference in 20A, 19A, and 18A reaction rates
have been observed (see Fig. 2 and Ref. 9).
Conditions for in Vitro Deadenylation--
We optimized the
conditions for CCR4 deadenylation in vitro using a
radiolabeled 22N+5A RNA substrate. We reported previously (9)
that the exonuclease activity of CCR4 is dependent upon Mg2+. Here we found that CCR4 displayed a very low
Mg2+ optimum (0.01-0.1 mM) (Fig.
1). Other divalent cations such as Mn2+, Zn2+, Ni2+, and
Co2+ were inactive with CCR4 (data not shown). The
monovalent K+ (17 mM), pH (7.9), and
temperature (37 °C) optima were also obtained (Fig. 1). The
Na+ optimum was also found to be low (20 mM)
(data not shown). Moreover, we did not find any difference in these
optimum conditions when we compared the partially purified CCR4
preparation (single-step FLAG purification) (9) with a pure CCR4 enzyme
preparation (see below) (data not shown).
Distributive Action of CCR4 on RNA Substrates--
We initially
analyzed the activity of the CCR4 protein on a 7N+23A RNA substrate. A
time course of CCR4 deadenylation of this substrate (Fig.
2) revealed that CCR4 clearly acted in a
distributive manner. A plot of the relative abundance of each of the
RNA species as a function of time showed that, as CCR4 catalyzed
deadenylation, the abundance of the different species formed a nearly
symmetric bell-shaped curve. This type of profile is consistent with
CCR4 displaying no large differences in its binding and/or catalysis for substrates containing poly(A) tails from 23 A residues to at least
5 A residues (Fig. 2). Our previous results indicated, however, that
CCR4 had major difficulty in deadenylating a substrate with only 1 terminal A residue (9). Careful analysis of the curves in Fig. 2 does
indicate, however, a slight spreading of the abundance of the product
toward shorter poly(A) lengths, suggestive of a slowing of the reaction
rate with shorter substrate lengths (see below).
CCR4 Requires at Least 2 A Residues at the 3'-End to Efficiently
Deadenylate the RNA--
To address whether CCR4 exhibited a binding
or catalytic deficiency with the 1A substrate, we purified CCR4 to
homogeneity prior to analyzing its kinetic parameters. To facilitate
purification of the CCR4 protein from yeast, the CCR4 gene
fused with FLAG and His6 epitopes was overexpressed in a
strain deleted for CCR4. The resultant fusion protein was
purified to near homogeneity by a two-step affinity chromatographic
procedure from yeast strain KY803-1a-1 (ccr4) (Fig.
3A, lane 2).
Using radiolabeled 26N+1A and 25N+2A RNA substrates, we determined the
apparent Km and Vmax for each
substrate with CCR4. Typical reactions as a function of substrate
concentration are displayed in Fig. 3B. With the 25N+2A RNA
substrate, CCR4 displayed a Km of 5.4 µM and a Vmax of 2.1 µM/min. Whereas the Vmax value for
the 26N+1A substrate was comparable to that for the 25N+2A substrate
(2.2 µM/min), its Km was increased by
20-fold. The inability of CCR4 to efficiently deadenylate an RNA
substrate with 1 A residue (9) can be attributed to a decreased apparent binding ability of the 1A substrate. We also determined an
apparent Km of 3.5 µM and a
Vmax of 0.86 µM/min for the 22N+5A
substrate, which are similar to those found for the 25N+2A substrate.
Increased Length of Non-adenylated Nucleotides of RNA Molecules
Enhances the Distributive Activity of CCR4--
The substrate
specificity of CCR4 was further investigated using different lengths of
non-adenylated nucleotides bearing constant length poly(A) tails of 2, 5, and 20 A residues. The relative activity of CCR4 was determined
using a concentration of the RNA molecules of 0.31 µM,
which was significantly below the Km for the 25N+2A
and 25N+5A RNA substrates. A typical reaction is displayed in Fig.
4 (A-C) for
RNA substrates containing 2, 5, and 20 A residues at their 3'-ends,
respectively. Table III
summarizes the relative reaction rates for the substrates analyzed.
Several observations can be made from the changes in relative activity for CCR4 distributive deadenylation as a function of the length of the
non-poly(A) region of the RNA substrate. First, for the 2A and 5A
substrates, increasing the N length from 6 to 12 nucleotides substantially enhanced the reaction rate by 2-3-fold. In contrast, increasing the N length further from 12 to 25 nucleotides increased the
relative activity by only 1.4-1.7-fold (Table III). Second, once the
total RNA length became ~17-26 nucleotides, no major differences in
the distributive reaction rate were observed for different N lengths
irrespective of the poly(A) length (Fig.
5). Third, 12N+5A was as active with CCR4
as 12N+20A, indicating that the length of the poly(A) sequences
per se does not alter CCR4 activity. These observations
suggest that CCR4 is able to recognize a certain length of RNA
substrate (at least 17 nucleotides) and that the A content is not
necessarily important for this recognition. These results are also in
agreement with those shown in Fig. 2, where a slight spreading of the
distribution of 7N+23A products indicated a slight slowing of the
reaction rate at shorter substrate lengths.
CCR4 Protein Transitions to a Processive Enzyme with Substrates
Greater than 44 Nucleotides--
It is shown in Fig. 4C
(lanes 2-6) that a significant portion of the 25N+20A
substrate was converted directly to the 25N+1A product. These data
suggest that CCR4 can also act in a processive manner. Because it is
also possible that the 25N+1A product was created by an endonucleolytic
cleavage occurring at the 1A-2A phosphodiester bond, we 3'-end-labeled
the 25N+20A substrate with poly(A) polymerase and reconducted the CCR4
deadenylation reaction. As shown in Fig. 4D, at time 0, an
additional 3-33 A residues were added to the 25N+20A substrate.
Deadenylation of this substrate by CCR4 under conditions in which CCR4
displayed a processive reaction with the 25N+20A substrate (Fig.
4C; data not shown) or a 50N+10A substrate (data not shown)
indicated that the poly(A) tail was being removed without the
concomitant endonucleolytic cleavage at the 1A-2A phosphodiester that
would have resulted in a range of 22-52 poly(A) products (marked with
asterisks in Fig. 4D). Similarly, using an
in vitro synthesized 50N+30A substrate uniformly labeled
with A (14), no 29A endonucleolytic product was observed following CCR4
processive deadenylation that resulted in a 50N+1A product (data not
shown). These data confirm that CCR4 can act in a processive manner.
CCR4 behaved as a processive enzyme to a much greater extent with the
25N+20A substrate than with the 12N+20A substrate (Fig. 4C,
lanes 7-12), indicating that the total length of the RNA
substrate is important for CCR4 processivity and not just the A tail
length. No processivity was observed with the 7N+23A substrate (Fig.
2), although for the 6N+20 substrate displayed in Fig. 4C
(lanes 16-18), a very small amount of 6N+1A substrate was
formed as a result of an apparently processive reaction.
Similarly, CCR4 reacted with the 50N+5A RNA substrate in a processive
manner (Fig. 6, lane 6),
indicating that it is not the length of the poly(A) sequence that
dictates the distributive-to-processive transition for CCR4. In fact,
CCR4 acted primarily in a processive manner with the 50N+5A, 50N+10A,
and 50N+30A substrates (Fig. 6; data not shown), suggesting again that
the total length of the substrate, not its poly(A) length, dictates the
distributive-to-processive transition for CCR4. We conclude that CCR4
can act as a processive enzyme in the presence of RNA substrates with
lengths of at least 45 nucleotides. No difference in CCR4 reaction rate
or ability to act in a processive manner was observed with capped and
uncapped 50N+5A RNA substrates (Fig.
6).2 It should be noted that
the apparent faster rate of conversion with uncapped versus
capped substrates shown in Fig. 6 was not borne out by other
experiments. Similar results were obtained with capped and uncapped
50N+30A RNA substrates (data not shown).
CCR4 Activity in the Presence of Various Inhibitors--
To
investigate further the CCR4 deadenylase activity specificity for its
RNA substrate, we analyzed the effect of various competitive inhibitors
on CCR4 poly(A) removal. The above described deadenylation assay using
the 22N+5A RNA substrate was performed in the presence of increasing
amounts of 5'-AMP, 5'-GMP, 5'-UMP, 5'-CMP, poly(A), poly(U), poly(G),
and poly(C). Table IV shows that UMP was
an ~2-fold better inhibitor than AMP, CMP, and GMP. We also observed
that the KI of inhibition for ATP (1.2 pM) was much lower than that for AMP (data not shown).
However, as was previously observed for the poly(A) ribonuclease
(PARN) deadenylase (15), this inhibition was shown to result from the chelating effect of ATP with Mg2+ ions, which thereby
prevented the catalytic potential of the CCR4 enzyme (data not shown).
Interestingly, we observed that poly(U), poly(C), and poly(G) were much
more inhibitory than poly(A), consistent with our above conclusion that
the CCR4 protein may be able to recognize a certain length of the RNA
substrate and that the poly(A) sequence is of little importance to this
recognition.
In this study, we report on the substrate specificities of the
yeast CCR4 deadenylase. Several features of its substrate recognition were identified. First, as previously indicated (9), CCR4 acted in a
distributive fashion to deadenylate a 7N+23A substrate. With other
substrates of comparable or shorter length, CCR4 was also a
distributive enzyme; yet we observed that, with substrates 45 nucleotides or longer, CCR4 could act in both a processive and distributive manner. The ability to be a processive enzyme was also
independent of the length of the poly(A) region of the substrate. One
model that could account for this distributive-to-processive transition
is that CCR4 binds better to longer substrates and therefore remains
attached for a longer period of time. This model is unlikely, however,
in that no difference was observed in the relative distributive
activity with RNA substrates longer than 17 nucleotides (Figs.
4C and 5), implying that CCR4 even with longer substrates
displays no differences in inherent affinity for the substrate. An
alternative model is that CCR4 itself undergoes a transition in the
presence of longer substrates such that a pool of CCR4 can now act in a
processive fashion. The oligomeric structures of the highly processive
Second, we found that, as a distributive enzyme, CCR4 preferred
substrates with lengths of at least 17 nucleotides. The decreases in
reactivity with shorter substrates appeared to be a result of CCR4
decreased binding affinity, as the substrate concentrations used in
these experiments were significantly below the Km of
binding. The length of the poly(A) sequence did not alter these preferences, confirming observations of others2 and
ours3 that CCR4 cannot
specifically bind to poly(A). Whether this binding preference results
from a sequence-specific recognition is not known. However, inhibition
studies with poly(U) and poly(G) indicated that these compounds were
20-40-fold better inhibitors of the CCR4 distributive reaction than
was poly(A). CCR4 might display sequence specificity for non-poly(A)
sequences that might be enriched with U or G. Given that poly(A)
sequences in yeast are up to 70-80 nucleotides in length, one model
suggests that only the extreme 3'-end of the poly(A) tail and the
3'-untranslated region sequences are bound by CCR4, with most of the
poly(A) sequence looped out and not in contact with CCR4.
Third, either as a distributive or processive enzyme, CCR4 was
deficient in removing a single terminal A residue from the mRNA
(9). This requirement for 2 A residues at the 3'-end was shown to be
the result of a much higher (20-fold) Km for the 1A
substrate than for the 2A substrate. CCR4 can remove the terminal A
residue and can also remove C or other nucleotides, albeit at very slow
rates (9). Fourth, CCR4 itself did not display any difference with
capped or uncapped substrates, suggesting that other proteins
interacting with CCR4 aid its recognition of capped mRNA (8).
These substrate specificity analyses suggest the following model for
CCR4 recognition of its RNA substrate. CCR4 binds to at least 2 terminal A residues, presumably in its active site. It also displays
recognition for at least another 15 nucleotides; whether they are
completely contiguous to these 2 A residues is not known. Finally, CCR4
may change its conformation or alter its oligomeric structure with
substrates of even greater length ( These features of CCR4 recognition of its substrates may, of course, be
targets for control of the deadenylation process. Regulating the
ability of CCR4 to bind specific mRNA or to transition to its
processive state would greatly influence its enzymatic activity. The
CAF1 protein and other components of the CCR4-NOT complex do not appear
to be likely regulators of these features. For example, partially
purified preparations of CCR4 that contain the complete CCR4-NOT
complex (9) do not display significant differences in deadenylation
compared with the purified protein in terms of requiring at least 17 nucleotides or in the transition to processivity. Other
trans-acting proteins such as PUF3 and PAB1 could control
these processes (7, 8, 23). The role of the other components of the
CCR4-NOT complex may be to confer additional regulatory aspects to the
deadenylation process. These could include binding to the mRNA cap
structure or recognition of additional specific RNA sequences not
present in our model RNA substrates used in this study.
Previous studies on the deadenylation of several mRNAs in
vivo (5, 7, 8) clearly indicate that the rate of deadenylation can
vary considerably for different mRNAs. Careful analysis of these
in vivo deadenylation studies indicates that
PGK1, which deadenylates slowly, does so in a distributive
manner (5). In contrast, faster deadenylated mRNAs such as
MFA2, STE3, COX17, and
GAL10 deadenylate with a combination of both processive and distributive processes (5, 7, 23). This is an expected result given
that processivity would allow the more rapid accumulation of product
compared with the comparatively slow distributive reaction. Because
CCR4 can display both distributive and processive activities, future
studies will need to address how regulating these processes relates to
the observed differences in mRNA deadenylation. In addition, the
previously observed initially slow deadenylation of all mRNAs up to
loss of the first 10 A residues (5) followed by more rapid
deadenylation of the bulk of the poly(A) tail suggests an additional
site at which CCR4 deadenylation may be controlled.
45 nucleotides) stimulated CCR4 to become a processive enzyme, suggesting that CCR4 undergoes an additional transition in the presence
of such substrates. CCR4 also displayed no difference in its activity
with capped or uncapped RNA substrates. These results indicate
that CCR4 recognition of its RNA substrates involves several features
of the RNA that could be sites in vivo for controlling the
rate of specific mRNA deadenylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
leu2-1 PET56 trp1-1 ura3-52 gal2
gcn4-1 ccr4::ura3). Commercially synthesized
RNA oligonucleotides are described in Table
I. The RNA substrates were generally
labeled with T4 polynucleotide kinase and [
-32P]ATP
and further purified by gel filtration on Sephadex G-25 spin columns.
The capped and uncapped 50N+5A and 50N+30A substrates (pT3-L3(A5) and pT3-L3(A30)) were synthesized
in vitro as previously described (14). Poly(A) polymerase
was used to attach radioactive 3'-AMP to the RNA substrates as
described (7), and the resultant labeled RNAs were gel-purified.
RNA substrates
Kinetic values of the CCR4 distributive reaction
1, respectively.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Optimum reaction conditions for CCR4
deadenylase activity. Left panels, the deadenylase
activity of CCR4 was assayed under various conditions using the 22N+5A
RNA substrate. Upper panel, temperature dependence;
second panel, pH dependence; third panel,
Mg2+ concentration dependence; lower panel,
K+ concentration dependence. Right panels, shown
are graphical representations of the results, where the percent of the
22N+1A product formed in 5 min is plotted versus the
relevant parameter. B lanes, no protein.
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Fig. 2.
Distributive action of CCR4. Left
panel, radiolabeled 7N+23A RNA substrate (0.48 µM)
was incubated with 30 ng of partially purified CCR4 under the optimum
assay conditions, and aliquots were taken at the times indicated.
Samples were analyzed on a denaturing 8% urea and 14% polyacrylamide
gel. Right panel, shown is a graphical representation of
deadenylation products at the indicated times as quantified using
PhosphorImager software.
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Fig. 3.
A, SDS-PAGE analysis of the CCR4
protein. FLAG-CCR4-His6 fusion protein was analyzed
on a denaturing 8% polyacrylamide gel and stained with Coomassie Blue.
Lane 1, CCR4 protein following Ni2+-agarose
chromatography; lane 2, purified CCR4 following both
Ni2+-agarose chromatography and anti-FLAG M2
antibody-agarose chromatography. The molecular mass of proteins (in
kilodaltons) is indicated on the left. B, sample reactions
determining the effects of poly(A) tail length on the kinetic
parameters of CCR4. The indicated concentrations of radiolabeled
26N+1A, 25N+2A, and 22N+5A RNA substrates were incubated for 5 min
under standard assay conditions for in vitro deadenylation
in the presence of 300 pg of pure CCR4, except 26N+1A was incubated
with 10 times the amount of enzyme. For the 22N+5A substrate, please
note that for the 16, 12.8, 9.6, and 6.8 µM
concentrations, the 22N+1A band represents a contaminating band present
in the original material (B lanes) (9).
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Fig. 4.
Assays analyzing effects of non-poly(A) RNA
length on CCR4 deadenylase activity. A, 2A RNA
substrates. Radiolabeled 22N+2A, 12N+2A, and 6N+2A RNA substrates (0.31 µM) were incubated with 300 pg of purified CCR4 protein,
and aliquots were removed at the times indicated. The lengths of the
RNA substrates are indicated on the left. B, 5A RNA
substrates. Reactions were conducted as described for A. C, CCR4 becomes a processive enzyme with long RNA
substrates. Radiolabeled 25N+20A, 12N+20A, and 6N+20A RNA substrates
were incubated with 150 ng of purified CCR4. Reactions were conducted
as described for A. The length of each of the RNA substrates
is indicated on the left. The reaction product labeled 1A
for each substrate denotes the locations of the completely deadenylated
products. D, CCR4 does not act endonucleolytically. The
25N+20A substrate was radiolabeled at its 3'-end with poly(A)
polymerase to which ~3-33 A residues were added. The
asterisks denote the range in positions from the 22A to 52A
products that would have formed if endonucleolytic cleavage had
occurred at the 1A-2A phosphodiester of the original 25N+23A to
25N+53A substrates.
Relative rates of CCR4 distributive activity with RNA
oligonucleotides of different lengths
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Fig. 5.
Total length of the RNA influences the
distributive activity of CCR4. The relative reaction rates for
different RNA substrates with CCR4 are plotted versus the
lengths of the RNA substrates (see Table III).
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Fig. 6.
Comparison of deadenylation rates with capped
and uncapped substrates. Capped and uncapped uniformly labeled
50N+5A RNA substrates, as indicated, were incubated with 30 ng of
partially purified CCR4 under standard conditions for the indicated
times. The products were fractionated by electrophoresis on a
denaturing 7% polyacrylamide gel. The resultant products are indicated
on the left.
In vitro deadenylation assay conducted in the presence of inhibitors
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-exonuclease and poly(A) ribonuclease (PARN) deadenylase (16, 17)
suggest that one such transition may be for CCR4 to become oligomeric.
Perhaps longer RNA substrates provide the structure that accelerates
this process by, for instance, providing significantly long enough sequences to which multiple CCR4 proteins can attach.
45 nucleotides) to become a
processive enzyme. Preliminary modeling of CCR4 binding to a 17-mer RNA
based on the structure of the exonuclease III family protein HAP1
(18-20) indicates that the C-terminal catalytic domain of CCR4 would
not easily accommodate binding to RNA of such length. However, other
regions of CCR4, especially its LRR, could be involved in RNA binding.
Given that two other LRR-containing proteins require their LRR domain
for contacting their cognate RNA substrates (21, 22), it is quite possible that the LRR of CCR4 directly or indirectly facilitates CCR4
recognition of its substrate.
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ACKNOWLEDGEMENT |
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The technical assistance of G. Quigley is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM41215 and HATCH Project H291. This is Scientific Contribution 2155 from the New Hampshire Agriculture Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of New Hampshire, Rudman Hall, Rm.
387, 46 College Rd., Durham, NH 03824. Tel.: 603-862-2427; Fax:
603-862-4013; E-mail: cldenis@cisunix.unh.edu.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M211794200
2 R. Parker, personal communication.
3 P. Viswanathan, J. Chen, Y.-C. Chiang, and C. L. Denis, unpublished data.
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ABBREVIATIONS |
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The abbreviation used is: LRR, leucine-rich repeat.
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