Interactions of CCCH Zinc Finger Proteins with mRNA
BINDING OF TRISTETRAPROLIN-RELATED ZINC FINGER PROTEINS TO
AU-RICH ELEMENTS AND DESTABILIZATION OF mRNA*
Wi S.
Lai
,
Ester
Carballo,
Judith M.
Thorn,
Elizabeth A.
Kennington, and
Perry J.
Blackshear§¶
From the Office of Clinical Research and Laboratory
of Signal Transduction, NIEHS, National Institutes of Health, Research
Triangle Park, North Carolina 27709 and the § Departments of
Medicine and Biochemistry, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, February 25, 2000
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ABSTRACT |
Macrophages derived from tristetraprolin
(TTP)-deficient mice exhibited increased tumor necrosis factor 
(TNF) release as a consequence of increased stability of TNF
mRNA. TTP was then shown to destabilize TNF mRNA after
binding directly to the AU-rich region (ARE) of the 3'-untranslated
region of the TNF mRNA. In mammals and in Xenopus,
TTP is the prototype of a small family of three known zinc finger
proteins containing two CCCH zinc fingers spaced 18 amino acids apart;
a fourth more distantly related family member has been identified in
Xenopus and fish. We show here that representatives of all
four family members were able to bind to the TNF ARE in a cell-free
system and, in most cases, promote the breakdown of TNF mRNA in
intact cells. Because the primary sequences of these CCCH proteins are
most closely related in their tandem zinc finger domains, we tested
whether various fragments of TTP that contained both zinc fingers
resembled the intact protein in these assays. We found that amino- and
carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid
fragment that contained both zinc fingers, could bind to the TNF ARE
in cell-free cross-linking and gel shift assays. In addition, these
truncated forms of TTP could also stimulate the apparent deadenylation
and/or breakdown of TNF mRNA in intact cells. Alignments of the
tandem zinc finger domains from all four groups of homologous proteins
have identified invariant residues as well as group-specific
signature amino acids that presumably contribute to ARE
binding and protein-specific activities, respectively.
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INTRODUCTION |
Zinc finger domains within proteins can mediate interactions with
DNA, RNA, other proteins, and small molecules such as diacylglycerols. One relatively uncommon class of zinc finger proteins contains fingers
of the CCCH type, in which three cysteines and one histidine are
thought to coordinate a single atom of zinc. Members of a subclass of
the larger family of CCCH zinc finger proteins contain two tandem zinc
fingers consisting of
CX8CX5CX3H,
where X refers to variable amino acids, spaced exactly 18 amino acids apart. The prototype of proteins of this CCCH double zinc
finger subclass is tristetraprolin
(TTP),1 also known as TIS11
and Nup475. It was first identified as the product of an immediate
early response gene in fibroblasts and other cells stimulated with
insulin, serum, or phorbol esters (1-4). The same stimuli that
increase transcription of this gene also stimulate its rapid serine
phosphorylation (5) and rapid nuclear to cytosol translocation (6) in fibroblasts.
TTP-deficient mice appear normal at birth but rapidly develop a wasting
syndrome accompanied by erosive arthritis, dermatitis, alopecia,
autoantibodies, and myeloid hyperplasia. Essentially all of these
abnormalities were prevented by the injection of monoclonal antibodies
specific for mouse tumor necrosis factor (TNF) (7). Macrophages
derived from these mice exhibited enhanced TNF release, accompanied
by increases in TNF mRNA levels (8). This was due to an increase
in stability of TNF mRNA in macrophages derived from
TTP-deficient mice (9). These findings implicated TTP as an
intracellular regulator of TNF mRNA stability and thus of TNF
biosynthesis. More recently, we have shown that TTP deficiency has a
similar effect on the stability of another mRNA containing a
so-called class II AU-rich element (ARE), that encoding
granulocyte-macrophage colony-stimulating factor (GM-CSF) (10). In this
case, there was a marked stabilization of GM-CSF mRNA in bone
marrow stromal cells derived from TTP-deficient mice compared with
control cells, indicating that TTP is also a physiological regulator of
GM-CSF mRNA stability and thus of GM-CSF secretion (10).
TTP can bind directly to the ARE of TNF mRNA (9, 11). The
integrity of both zinc fingers was required for this direct protein-RNA
interaction, because a single C 
R mutation within the CCCH motif
from either finger abolished the ARE binding activity of TTP (11). The
same mutations abrogated the ability of TTP to destabilize the TNF
mRNA in intact cells (11). These studies indicated that TTP could
bind directly to the TNF ARE and destabilize the TNF mRNA in
a zinc finger-dependent manner, apparently by initially
stimulating its deadenylation (11).
Aside from TTP, two other members of this subclass have been identified
to date in mammals: cMG1 (TIS11b, ERF1, and Berg-36) (12-15) and
TIS11d (ERF2) (15, 16). All three of these proteins contain the two
typical CCCH fingers, spaced 18 amino acids apart, with the sequence
RYKTEL or a variant leading into each finger. Proteins with nearly
identical double zinc fingers spaced 18 amino acids apart have been
identified in Drosophila and yeast (17-19). In addition to
the Xenopus homologues of the three mammalian proteins described above, which all contain two tandem zinc fingers, we recently
cloned a fourth cDNA encoding a Xenopus protein (XC3H-4) that contained two CCCH zinc fingers that were 18 amino acids apart and
were preceded by the R(K)YKTEL sequence, as well as an additional more
carboxyl-terminal pair of CCCH zinc fingers that were more closely
spaced and lacked the lead-in R(K)YKTEL sequence (20). This mRNA
was expressed only in the oocyte, eggs, and early embryo; its mammalian
homologue has not been identified, to our knowledge.
Although TTP appears to be a normal physiological regulator of TNF
and GM-CSF mRNA levels, secondary to its ability to bind to and
promote the destabilization of their mRNAs, the physiological functions of the other vertebrate CCCH double zinc finger proteins are
unknown. In the present study, we assessed the ability of these family
members to bind to the ARE of the TNF mRNA, and their ability to
influence the stability of this mRNA in intact cells. Our data
indicate that representatives of all four known members of this
subclass of CCCH proteins exhibit TNF ARE-binding activity, as
assessed by cell-free mRNA cross-linking and gel shift assays.
Members of at least three of the families can also exhibit TNF
mRNA-destabilizing capabilities in intact cell transfection assays.
Somewhat surprisingly, both cell-free and intact cell activities were
exhibited by both amino- and carboxyl-terminal truncated forms of TTP
and by a 77-amino acid internal domain of TTP that contains both zinc
fingers. Alignments of this tandem zinc finger (TZF) domain from all
vertebrate members of this subclass revealed many invariant residues
within this domain, as well as group-specific signature amino acids
that may be important for the specific cellular functions of these proteins.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction
Parent Plasmids--
The human (21) and mouse (2) TTP cDNAs
were obtained as described. The cDNAs encoding the
Xenopus CCCH proteins XC3H-1, XC3H-3, and XC3H-4 were
obtained as described (20).
Expression Constructs--
Human TTP expression constructs
H6E·HGH3' and CMV·hTTP·tag were made as described (11).
CMV·mTTP·tag, which contained the entire protein coding region of
mouse TTP, was made using the same methods. CMV·hTTP (97-173)·tag,
CMV·hTTP (1-173)·tag, and CMV·hTTP (97-326)·tag, which all
contained the double zinc finger domain (amino acids 104-166 from Ref.
21) and part of the protein sequence of human TTP, were made as
described (11). CMV·hTTP (97-173)·tag contained essentially only
the zinc fingers and seven flanking amino acids on both ends.
CMV·hTTP (1-173)·tag contained amino acids 1-173, and CMV·hTTP
(97-326)·tag contained amino acids 97-326 (the last amino acid) of
human TTP.
CMV·CMG1·tag was made by inserting a PCR fragment containing the
entire protein coding region of rat cMG1 (Ref. 13; bp 108-1190 of
GenBankTM accession number X52590), into the vector
CMV·BGH3'/pBS+. The template cMG1 for the PCR reaction was generously
provided by Dr. K. D. Brown (AFRC Institute of Physiology and
Genetics Research, Babraham, Cambridge, UK). The epitope tag derived
from the influenza virus hemagglutinin protein (22) was attached to the
last amino acid of the cMG1 protein as described (23).
CMV·XC3H-1·tag and CMV·XC3H-3·tag were made by inserting PCR
fragments containing the entire protein coding region of the corresponding Xenopus cDNA clones (20), as well as the
epitope tag fused to the last amino acid of each protein, into the
vector CMV·BGH3'/pBS+.
CMV·U2AF35 was made by inserting a PCR fragment
containing the entire protein coding region of the splicing factor
U2AF35 (24) into the vector CMV·BGH3'/pBS+. Plasmid pRSET
B, which contained the entire coding region of U2AF35, was
provided by Drs. B. R. Graveley and T. Maniatis (Harvard University, Cambridge, MA), and was used as a template in the PCR reaction.
CMV·mTNF containing a NarI-XbaI fragment
spanning bp 127-1325 of a mouse TNF cDNA sequence
(GenBankTM accession number X02611) was made as described
(11). The mTNF cDNA clone, provided by Dr. B. Beutler
(University of Texas Southwestern Medical Center, Dallas, TX),
contained an incomplete 3'-untranslated region that ended at bp 1325 (GenBankTM accession number X02611), with 33 adenylate
residues attached to the last T. This sequence is shown in Fig. 1 of
Ref. 11.
Transfection of HEK 293 Cells, Northern Analysis, and Cytosolic
Extract Preparation
HEK 293 cells were maintained in minimal essential medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transient transfection of 1.5 × 106 cells with CMV·hTTP·tag
or with expression constructs containing the protein coding regions of
the other CCCH zinc finger proteins or with vector pBS+ alone in
calcium-phosphate precipitates was performed as described previously
(23, 25), except that the transfection mixture was allowed to stay on
the cells for 16-20 h, and the glycerol shock step was omitted. When
cells were co-transfected with CMV·mTNF and CCCH protein
expression constructs, human growth hormone expression plasmid pXGH5
(Nichols Institute Diagnostics, San Juan Capistrano, CA) was also
co-transfected to monitor transfection efficiency.
24 h after the removal of the transfection mixture, samples were taken
from the cell culture medium, and the human growth hormone released was
assayed according to the manufacturer's protocol. Total cellular RNA
was then harvested from the HEK 293 cells using the RNeasy system
(Qiagen, Valencia, CA). Northern blots were prepared as described (2).
Blots were hybridized to random-primed, -32P-labeled
cDNA probes coding for various CCCH zinc finger proteins, including
mouse TTP (2), Xenopus XC3H-1 and XC3H-3 (20), rat cMG1
(13), or splicing factor U2AF35 (24). Blots were also
hybridized with a ~1-kb NarI-BglII fragment of
a mTNF cDNA (11) and a ~ 0.3-kb fragment of mouse
cyclophilin cDNA (bp 166-480; GenBankTM accession
number X52803).
Cytosolic extracts were prepared from HEK 293 cells 24 h after the
removal of the transfection mixture. The cells were incubated on ice
for 20 min in a buffer consisting of 10 mM HEPES, pH 7.6, 3 mM MgCl2, 40 mM KCl, 5% (v/v)
glycerol, 0.5% (v/v) Nonidet-P40, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 8 µg/ml leupeptin
(lysis buffer). Lysis of the cells and maintenance of intact nuclei
were carefully monitored by microscopy. The nuclei and cell membrane
debris were removed by centrifugation at 16,000 × g at
4 °C for 15 min. Glycerol was added to the supernatant (cytosolic
extract) to 20% (v/v), and the resulting extract was stored at
70 °C.
Analysis of RNA-Protein Complexes by SDS-PAGE, Electrophoretic
Mobility Shift Assay, and Western Blotting
Preparation of RNA Probes--
Plasmid pTNF 1281-1350 (bp
1281-1350; GenBankTM accession number X02611) contained
seven AUUUA motifs, five of them being overlapping UUAUUUAUU nonamers.
This was constructed as described (11).
Plasmid pTNF 1309-1332 (bp 1309-1332; GenBankTM
accession number X02611), containing four overlapping UUAUUUAUU
nonamers, was constructed by inserting double-stranded oligonucleotides spanning bp 1309-1332 into the EcoRV-XbaI
cloning sites of pSK
.
Plasmid pTNF 1309-1332 (A/G) contained five Gs (italics) replacing
the five flanking As of bp 1309-1332 of GenBankTM
accession number X02611
(UUGUUUGUUUGUUGUUUGUUUUUU)
and was constructed as described for pTNF 1309-1332.
Correct sequences of all plasmid inserts were confirmed by dRhodamine
Terminator Cycle Sequencing (Perkin-Elmer, Foster City, CA). To label
RNA transcripts with [-32P]UTP (800 Ci/mmol), the
above plasmids linearized with XbaI were used as templates,
and the Promega Riboprobe in vitro Transcription Systems
protocol was employed. The resulting products were precipitated with
ammonium acetate and ethanol.
Cross-linking of Proteins to RNA--
Cytosolic extracts
prepared from HEK 293 cells transfected with CMV·hTTP·tag or other
zinc finger protein expression constructs or vector (5 or 20 µg of
protein) were incubated with 1.5 × 106 cpm of RNA
probe in a 96-well plate at room temperature for 20 min in 20 µl of
lysis buffer (without protease inhibitors). Heparin and yeast tRNA were
added to final concentrations of 2.5 µg/µl and 50 ng/µl,
respectively, for an additional 10 min. The 96-well plate was then
placed on ice and irradiated at 254 nm UV light in a Stratalinker
(Stratagene, La Jolla, CA) for 30 min at a distance of 5 cm from the
light source. RNA not associated with protein was digested with 100 units of RNase T1 (Life Technologies, Inc) for 20 min at room
temperature and further digested with 25 µg of RNase A (Amersham
Pharmacia Biotech) at 37 °C for 15 min. The remaining RNA-protein
complexes were analyzed by SDS-PAGE (12 or 16% acrylamide gel)
followed by autoradiography.
Western Blotting--
Cell extracts (5-50 µg of protein) were
mixed with 
volume of 5× SDS sample buffer (26), boiled for 5 min, and then loaded onto 12 or 16% SDS-PAGE gels. Western blotting was performed by standard techniques. Membranes were incubated in
Tris-buffered saline/0.3% Tween 20 (TBS/T) with either polyclonal antiserum HA.11 (1:2, 500) or an antiserum to U2AF35 (27).
Incubation of the membranes with second antibody and development were
performed as described (8). For some blots, 125I-protein A
(0.2 µCi/ml in TBS/T; Amersham Pharmacia Biotech) was used in place
of second antibody.
RNA Electrophoretic Mobility Shift Assay--
Cytosolic extracts
prepared from HEK 293 cells transfected with either vector alone or
expression constructs driven by the CMV promoter (5 or 20 µg of
protein) were incubated with 2 × 105 cpm of RNA probe
at room temperature for 20 min in 20 µl of lysis buffer (without
protease inhibitors). Heparin and yeast tRNA were added to final
concentrations of 2.5 µg/µl and 50 ng/µl, respectively, for an
additional 10 min. RNA not associated with protein was digested with
100 units of RNase T1 (Life Technologies, Inc.) for 20 min at room
temperature; the reaction mixture was then loaded onto a 6%
nondenaturing acrylamide gel and subjected to electrophoresis at 250 V
for 90 min, in 0.4× Tris borate/EDTA buffer.
Expression of XC3H-4 Protein and Its Fragments in Xenopus
Oocytes
Preparation of XC3H-4 RNA in Vitro--
cDNAs encoding
various regions of the XC3H-4 protein (20), full length (amino acids
1-276); amino acids 1-120 (containing the tandem CCCH zinc fingers of
the TTP-type); and amino acids 121-276 (containing the second pair of
CCCH zinc fingers), were inserted into the BglII cloning
site of plasmid pSP64TEN (a gift from Dr. Douglas Melton, Harvard
University, Cambridge, MA). The epitope tag derived from the influenza
hemagglutinin protein (22) was attached to the last amino acid of each
of the peptides as described (23). Correct sequence of the inserts was
confirmed by dRhodamine Terminator Cycle Sequencing (Perkin-Elmer). The plasmids were linearized by XbaI digestion and were used as
templates to synthesize RNA in vitro. The RNAs were prepared
with the use of the mMESSAGEmMACHINE SP6 Kit (Ambion, Inc., Austin, TX)
following the manufacturer's protocol.
Microinjection of Oocytes--
Ovary was removed from adult
Xenopus females (Xenopus I, Ann Arbor, MI), and
stage VI oocytes were separated from the ovary and manually
defolliculated. Oocytes were allowed to recover for 16 h at
18 °C in buffer OR-2 (5 mM HEPES, pH 7.8, 82.5 mM NaCl, 2.5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 3.8 mM NaOH). Oocytes were injected with 30-50 ng of mRNA
and incubated for 24 h at 18 °C. Oocyte cytosolic extracts were
prepared as described above for 293 cell extracts.
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RESULTS |
The other two human members of the mammalian CCCH double zinc
finger protein family, ERF1 (cMG1 and TIS11b) and ERF2 (TIS11d), share
strikingly similar amino acid sequences in the TZF region with TTP
(Fig. 1, underlined). Although
the carboxyl termini exhibit some sequence similarities, there are
major differences between TTP and the other two proteins, whereas the
amino acid sequences of ERF1 and ERF2 are more closely related to each
other (Fig. 1).
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Fig. 1.
Alignments of human CCCH zinc finger
proteins. The three known human CCCH proteins hTTP (Ref. 21;
GenBankTM accession number M63625), hsERF1 (11B) (Ref. 14;
accession number X71901), and hsERF2 (11D) (Ref. 16; accession number
X78992) sequences were aligned using ClustalW Alignments (MacVector
6.5, Oxford Molecular) with an open gap penalty of 10.0 and an extended
gap penalty of 0.05. The shaded areas indicate amino acid
identity. The closely related double zinc finger domains are
underlined, with the key cysteine and histidine residues
indicated by dots under the sequence.
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To determine whether these other two family members shared the ability
of TTP to bind to the ARE region of the TNF mRNA and destabilize
it, ARE binding studies were performed using proteins expressed in HEK
293 cells, and co-transfection assays with the TNF mRNA
expression construct were performed in the same cell type. Besides the
TTP expression constructs, the new expression constructs used were made
from rat cMG1 (13), which is the rat homologue of mouse TIS11b, human
ERF1, and Xenopus XC3H-2, and Xenopus XC3H-3
(20), which is the Xenopus homologue of mouse TIS11d and
human ERF2.
Effects of TTP-related Proteins on TNF mRNA--
In the
expression studies described below, we used a previously described (11)
TNF expression construct, CMV·mTNF. This construct does not
contain the full 3'-untranslated region of mTNF; instead, it ends at
base 1325 (of GenBankTM accession number X02611) followed
by 33 adenylate residues encoded by the cDNA. The expression of
TNF mRNA from this construct allowed the detection of both the
adenylated and deadenylated forms of this mRNA in the presence of
TTP (11). TTP has similar effects on the full-length mouse TNF
mRNA (data not shown), but for technical reasons involving size
overlap with the 18 S ribosomal RNA, these are more difficult to quantitate.
The HEK 293 cells used in the transfection experiments do not express
endogenous TNF or TTP mRNA (11). In the following paragraph
referring to Fig. 2, all quantitation of
Northern blot mRNA expression was determined by PhosphorImager and
was corrected for transfection efficiency by human growth hormone
secretion and for gel loading by quantitating endogenous cyclophilin
mRNA levels. When these cells were co-transfected with
CMV·mTNF and a range of concentrations of the human TTP expression
construct CMV·hTTP·tag, the mTNF mRNA exhibited a
characteristic expression pattern. At a low concentration of TTP DNA
(0.005 µg/dish of cells; Fig. 2A, lane 2), the
total amount of mTNF mRNA was reduced to ~40% of control in
this experiment (Fig. 2A, lane 1). When the amount of co-transfected TTP DNA was increased to 0.01 µg of DNA, the
total amount of hybridizable TNF mRNA was further reduced to
23% of control (Fig. 2A, lane 3). When 0.1 and 1 µg of TTP were co-transfected, a smaller species of mTNF mRNA
was increased in intensity, whereas the upper band decreased markedly
(Fig. 2A, lanes 4 and 5). We have
previously shown by RNase H experiments that the lower band is a
deadenylated species of TNF mRNA (11). At these higher
concentrations of TTP DNA, the total accumulated hybridizable mTNF
mRNA was actually greater than that seen in the cells
co-transfected with vector alone (Fig. 2A, lane
1). After correcting for transfection efficiency, the amounts of
hybridizable mTNF mRNA from cells co-transfected with 0.1 or 1 µg of CMV·hTTP·tag were 254 and 481% of control, respectively.
The apparent decrease in hybridizable mTNF mRNA seen at the
highest concentration of TTP DNA (1 µg; Fig. 2A,
lane 5) is likely to be due to the global inhibition of
transcription seen at this level of TTP expression, as noted previously
(11); this was reflected in a marked decrease in human growth hormone
expression from these cells, accounting for the elevated
"corrected" value for the sample shown in lane 5. These
results are similar to those described previously (11), in which
PhosphorImager values from four independent experiments were normalized
for both transfection efficiency and gel loading and then averaged. In
that study, an average decrease of TNF mRNA to 17% of control
was seen at 10 ng of CMV·hTTP·tag DNA; this value increased to 173 and 300% of control at 50 and 100 ng of DNA, respectively. The
mechanism of the increased accumulation of the deadenylated species of
TNF mRNA seen at higher TTP expression plasmid concentrations is
not known but is a consistent and highly reproducible finding (11).
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Fig. 2.
Effect of TTP-related proteins on
TNF mRNA stability. CMV·mTNF (1 µg/plate) was co-transfected into 293 cells with TTP or with the
other CCCH zinc finger protein expression constructs or with vector
alone. Total cellular RNA was harvested as described under
"Experimental Procedures." Each gel lane was loaded with 10 µg of
total cellular RNA. Electrophoresis and Northern hybridization were
performed as described under "Experimental Procedures."
A, lane 1, vector alone (BS+, 5 µg/plate); lanes 2-5, CMV·hTTP·tag (0.005, 0.01, 0.1, and 1 µg of DNA/plate, respectively); lanes 6-9,
CMV·CMG1·tag (0.005, 0.01, 0.1, and 1 µg/plate, respectively);
lanes 10-13, CMV·XC3H-3·tag (0.005, 0.01, 0.1, and 1 µg/plate, respectively). B, lane 1, vector
(BS+, 5 µg/plate); lanes 2-5, CMV·mTTP·tag (0.005, 0.01, 0.1, and 1 µg/plate, respectively); lanes 6-9,
CMV·XC3H-1·tag (0.005, 0.01, 0.1, and 1 µg/plate, respectively);
lanes 10-13, CMV· U2AF35 (0.005, 0.01, 0.1, and 1 µg/plate, respectively). In both A and B, for
all samples represented in lanes 2-13, vector DNA was also
added to make the total amount of co-transfected DNA 5 µg/plate. The
Northern blots shown in the upper panels were probed with
32P-labeled mTNF and mouse cyclophilin cDNA probes.
The two arrows labeled TNF indicate the two
species of TNF mRNA discussed in the text. The arrow
labeled Cyclo indicates endogenous cyclophilin mRNA.
Identical RNA samples as in the upper panels were blotted
and probed with 32P-labeled mouse TTP, rat cMG1, and
Xenopus XC3H-3 cDNA probes (indicated as CCCH
in A), or mouse TTP, Xenopus XC3H-1 (indicated as
TTP), and human U2AF35 cDNA probes (in B),
and are shown in the lower panels. The position of the 18 S
ribosomal RNA is indicated.
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In the same co-transfection experiment, we tested the ability of the
two TTP-related proteins to destabilize TNF mRNA and to promote
the formation of the deadenylated species. When either CMV·CMG1·tag
(a rat cMG1 expression plasmid, representing the cMG1/TIS11b/ERF1
proteins) or CMV·XC3H-3·tag (a Xenopus XC3H-3 expression
construct, representing the TIS11d/ERF2 proteins) was co-transfected
with CMV·mTNF into 293 cells, each exhibited a similar pattern to
TTP in influencing the accumulation of TNF mRNA. With very low
amounts of co-transfected CMV·CMG1·tag DNA, 0.005 and 0.01 µg,
the total hybridizable amounts of TNF mRNA were decreased to 40 and 27% of control, respectively (Fig. 2A, lanes
6 and 7). The accumulation of the smaller species of
TNF mRNA was obvious at 0.1 and 1 µg of co-transfected
CMV·CMG1·tag DNA; total hybridizable TNF mRNA was 106 and
663% of control, respectively (Fig. 2A, lanes 8 and 9). Expression of the Xenopus protein,
XC3H-3, resulted in a similar pattern. Transfection of low
concentrations of CMV·XC3H-3·tag DNA (0.005 and 0.01 µg) caused a
decrease in total TNF mRNA to 57 and 49% of control,
respectively (Fig. 2A, lanes 10 and
11). At 0.1 µg of CMV·XC3H-3·tag DNA co-transfection, the characteristic two sizes of TNF mRNA were detected (Fig. 2A, lane 12; 78% of control), whereas at 1 µg
of CMV·XC3H-3·tag DNA, the deadenylated species of the mRNA
accumulated to 461% of control (Fig. 2A, lane
13). The bottom panel of Fig. 2A
demonstrates the levels of expressed TTP, cMG1, and XC3H-3 mRNA in
the same samples.
As a control, we used the human RNA splicing factor U2AF35
(24). This protein contains two putative zinc fingers of the CCCH class, which, instead of being 18 amino acids apart, are widely separated by 116 amino acids and are also not preceded by the YKTEL
lead-in sequence. The U2AF35 protein is the smaller subunit
of the essential splicing factor U2AF (24). Its heterodimeric complex
with U2AF65 is thought to be required for recognition of
the 3' splice acceptor site in pre-mRNA splicing (for reviews see
Refs. 28 and 29). U2AF35 has also been shown to interact
with other proteins involved in splicing, such as SC35,
SF2/ASF, tra, and tra2, and was originally not thought to directly bind
to RNA (27, 30); however, this conclusion has been revised recently
(31-33). Although endogenous U2AF35 mRNA in 293 cells
was readily detectable (Fig. 2B, lower panel), the transfection markedly increased the expression of this mRNA (Fig. 2B, bottom panel, lanes 12 and
13) and protein (data not shown). In this co-transfection
experiment, the TNF mRNA was not affected by the expression of
increasing amounts of U2AF35 (Fig. 2B, top
panel, lanes 10-13). This is in contrast to the effects of two other TTPs, mouse (2) and Xenopus (XC3H-1
(20)) (Fig. 2B, top panel, lanes
1-9), both of which behaved like their human counterpart in this assay.
Binding of CCCH Zinc Finger Proteins to the ARE of TNF
mRNA--
Using the 70 base 32P-labeled TNF ARE
probe (bases 1281-1350; GenBankTM accession number
X02611), we performed UV cross-linking experiments with extracts from
293 cells that had been transfected with constructs expressing these
CCCH zinc finger proteins driven by the CMV promoter. In addition to
human TTP (Fig. 3A, lane
3), both rat cMG1 (lane 4) and Xenopus
XC3H-3 (lane 5) were cross-linked by the TNF ARE probe
(Fig. 3A). Two major cross-linked proteins bands were formed with XC3H-3; one with an Mr of ~48,000,
presumably representing the intact protein, and one with an
Mr of ~32,000, presumably representing a
proteolytic fragment. Human U2AF35 protein, whose widely
spaced zinc fingers lack the YKTEL lead-in sequence, did not form
detectable complexes with the TNF ARE probe (not shown). The
identities of the endogenous 293 cell proteins that cross-linked to
this probe, with Mr values of ~85,000,
~70,000, ~46,000, and ~35,000 (see lane 6), are
unknown; their possible relationship to other ARE binding proteins
described in the literature has been discussed previously (11).
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Fig. 3.
Binding of CCCH zinc finger proteins to the
mTNF-ARE. Cytosolic extracts of 293 cells
transfected with either vector alone or constructs expressing the CCCH
zinc finger proteins were prepared as described under "Experimental
Procedures." A, UV cross-linking assay. Incubation of
extracts (each sample in lanes 2 and 3 contained
5 µg of protein; lanes 4-6 contained 20 µg of protein)
with 1.5 × 106 cpm of 32P-labeled mTNF
ARE probe (bases 1281-1350; GenBankTM accession number
X02611), UV cross-linking, and RNase digestion were performed as
described under "Experimental Procedures." Lane 1 (P'), probe alone (1.5 × 106 cpm) after
RNase digestion. Lanes 2 and 6 (BS+),
extracts from 293 cells transfected with 5 µg of vector plasmid.
Lanes 3-5, extracts from 293 cells transfected with 1 µg/plate of plasmid CMV·hTTP·tag, CMV·CMG1·tag, or
CMV·XC3H-3·tag, respectively; vector was added to make the total
transfected DNA 5 µg/plate. The RNA-protein complexes were resolved
by SDS-PAGE (12% gel) followed by autoradiography. The positions of
molecular mass standards are indicated. B, electrophoretic
mobility shift assay. The same cell extracts prepared from 293 cells
and described in A were incubated with 2 × 105 cpm of a TNF ARE probe (1281-1350). RNA mobility
shift assays and RNase T1 digestion were performed as described under
"Experimental Procedures." Lane 1 (P'), probe
alone (RNase T1 digested). Lane 6, extract (20 µg) from
293 cells transfected with 1 µg/plate of plasmid CMV·U2AF35. The
RNA-protein complexes I, II, and III formed with endogenous 293 cell
proteins and the migration position of the free probe are indicated.
C, protein expression analysis. Cell extracts prepared from
293 cells as described in A were analyzed by Western
blotting using a polyclonal antibody to the epitope tag of the fusion
proteins. The antibody was recognized by 125I-protein A in
this case. The positions of molecular mass standards are indicated to
the left of the gel.
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The ARE binding activity of the two TTP-related proteins was also
tested in RNA mobility shift assays. As we have shown (11), the TNF
ARE probe formed three RNA-protein complexes (I, II, and III) with a
control extract prepared from 293 cells transfected with vector alone
(Fig. 3B, lanes 2 and 7). When an
extract from cells transfected with CMV·hTTP·tag was used, new
complexes were formed, whereas complexes I, II, and III seen in the
extract from vector-transfected cells decreased or disappeared (Fig.
3B, lane 3). Formation of new RNA-protein
complexes was likewise observed when extracts from 293 cells
transfected with either CMV·CMG1·tag or CMV·XC3H-3·tag were
used in the assay (Fig. 3B, lanes 4 and 5). Considerable radioactivity also remained in the gel
wells when extracts containing these two proteins were used (Fig.
3B, lanes 4 and 5). Human
U2AF35 RNA splicing factor did not form detectable
complexes with the TNF ARE probe under these conditions (Fig.
3B, lane 6). Expression of these proteins from
the expression constructs was readily detectable using antibodies to
the epitope-tagged proteins (Fig. 3C, lanes 2,
4, and 5).
Binding of TTP and Related Proteins to Mutant TNF ARE
Probes--
We previously determined that the flanking A nucleotides
of the AUUUA motifs in the TNF ARE were essential for binding of TTP
to the TNF ARE (11). We next evaluated the ability of the TTP-related proteins to bind to mutant ARE probes.
We first compared a longer TNF ARE probe (bases 1281 to 1350;
GenBankTM accession number X02611) that contained seven
AUUUA motifs to a probe (bases 1309-1332) that contained only four of
the AUUUA sequences (see bottom of Fig.
4). Using probe 1281-1350, cytosolic complexes of Mr ~85,000, ~ 70,000, ~46,000, and ~35,000 were seen when vector-transfected cell
extracts were used; these were less apparent when the shorter probe
1309-1332 was used (Fig. 4, compare lane 2 with lane
7 or lane 6 with lane 11). Extracts prepared from 293 cells transfected with the hTTP, cMG1, and XC3H-3 expression constructs all formed complexes with both long and short probes (Fig.
4, lanes 3-5 and 8-10).
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Fig. 4.
UV cross-linking assays of TTP and related
proteins with mutant mTNF-ARE probes.
Cytosolic extracts of 293 cells transfected with either vector alone or
constructs expressing the CCCH zinc finger proteins were prepared as
described under "Experimental Procedures." Assays used
32P-labeled mTNF ARE probes 1281-1350, 1309-1332, or a
mutant of probe 1309-1332 (A/G), as indicated. Incubation of extracts
(each sample in lanes 2, 3, 7,
8, 13, and 14 contained 5 µg of
protein; lanes 4-6, 9-11, and 15-17
contained 20 µg of protein) with 1.5 × 106 cpm of
probe, UV cross-linking, and RNase digestion were performed as
described under "Experimental Procedures." Lanes 1, 12, and 18 (P', 1.5 × 106
cpm/sample), probe alone after digestion with RNase. Lanes
2, 6, 7, 11, 13, and
17 (BS+), extracts from 293 cells transfected
with 5 µg of vector plasmid. Lanes 3-5, Extracts from 293 cells transfected with 1 µg/plate of plasmid CMV·hTTP·tag,
CMV·CMG1·tag, or CMV·XC3H-3·tag, respectively; vector DNA was
added to make the total transfected DNA 5 µg/plate. Extracts
described in lanes 3-5 were also used for lanes
8-10 and 14-16. The RNA-protein complexes were
resolved by SDS-PAGE (12% gel) followed by autoradiography. The
exposure time for the gel using probes 1281-1350 and 1309-1332 was
4 h at 70 °C, and it was 8 h for the gel using
1309-1332 (A/G). The positions of molecular mass standards are
indicated to the left of the gel. The sequences of the
probes used are shown at the bottom; the adenosine residues
mutated to guanosine residues in probe 1309-1332 (A/G) are indicated
by the triangles.
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We next studied the binding specificity of TTP and its related proteins
to a mutant of the short probe 1309-1332. None of the proteins was
able to form detectable complexes with probe 1309-1332 (A/G)
(bottom of Fig. 4, mutation sites indicated by triangles), a mutated ARE probe in which the flanking A
residues in the AUUUA motif were substituted with Gs (Fig. 4,
lanes 13-17).
Characteristics of a Fourth CCCH Protein--
We previously
identified a fourth prospective family member of the CCCH TZF protein
family in Xenopus (accession number AAD24210 (20)). This
protein, XC3H-4, contained two zinc fingers spaced 18 amino acids apart
that contained all of the hallmarks of the TZF domains from the three
proteins discussed above; in addition, it contained two additional,
more carboxyl-terminal CCCH zinc fingers, spaced more closely together
and containing more degenerate lead-in sequences (20). Data base
searches revealed sequence similarity to the amino-terminal portions of
two CCCH proteins from zebrafish (34) and carp (35). Subsequent
correction of the fish DNA sequences (CAA71245.2 for carp CTH1 and
CAB55775.1 for zebrafish CTH1) showed apparent homology with the
Xenopus sequence over the entire lengths of the proteins
(Fig. 5). To our knowledge, mammalian
homologues of these proteins have not been cloned to date.
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Fig. 5.
Alignments of XC3H-4-like CCCH zinc finger
proteins. The three known proteins that contain four CCCH zinc
fingers were aligned using ClustalW Alignments (MacVector 6.5, Oxford
Molecular) with an open gap penalty of 10.0 and an extended gap penalty
of 0.05. The shaded areas indicate amino acid identity, the
boxed areas indicate similarities. The closely related
tandem zinc finger domains are underlined, with the key
cysteine and histidine residues indicated by dots under the
sequence. The sequences are: Carp CTH1 (Ref. 35; GenBankTM
accession number CAA71245.2); Zebrafish CTH1 (Ref. 34;
GenBankTM accession number CAB55775.1); and
Xenopus XC3H-4 (Ref. 20; GenBankTM accession
number AAD24210).
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Attempts to express the Xenopus member of this group,
XC3H-4, or various subdomains, in 293 cells failed to yield significant levels of mRNA or
protein.2 However,
significant expression of protein was readily achieved by injecting
mRNA into Xenopus oocytes; extracts from these oocytes were then used in ARE cross-linking studies.
Using the 32P-labeled TNF ARE probe (bases 1281-1350;
GenBankTM accession number X02611), we performed UV
cross-linking experiments with extracts from Xenopus oocytes
that had been injected with in vitro transcribed RNAs
encoding the full length, the first half (amino acids 1-120 of
accession number AAD24210), or the second half (amino acids 121-276)
of the XC3H-4 protein. Similar to human, mouse, or Xenopus
(XC3H-1) TTP expressed in 293 cells (Fig.
6A, lanes 3,
4, and 10), extracts prepared from oocytes injected with Xenopus XC3H-4 RNA that encoded the
full-length protein were cross-linked by the TNF ARE probe (Fig.
6A, lane 6), whereas no probe-protein complex was
detectable when extracts of buffer (mock) injected oocytes were used
(Fig. 6A, lane 5). When an extract from oocytes
injected with RNA encoding the first 120 amino acids of XC3H-4 protein
was used in the UV cross-linking assay, a probe-protein complex with an
apparent Mr of 15,000 was observed (Fig.
6A, lane 7). This complex formation is presumably due to the TZF domain that is related to the one in TTP (see Fig. 8B below). When an extract from oocytes injected with RNA
encoding the second half of the XC3H-4 protein (amino acids 121-276)
was used in the UV cross-linking assay, no probe-protein complex could be detected (Fig. 6A, lane 8). The second half of
the protein contains a pair of CCCH zinc fingers that each have the
internal spacing of the TTP-type zinc fingers, but the two fingers are separated by only 7 amino acids, and they lack the R(K)YKTEL lead-in sequence. Human U2AF35 protein also did not form detectable
complexes with the TNF ARE probe (Fig. 6A, lane
9). Expression of the XC3H-4 protein and its fragments from the
RNA-injected oocytes was readily detectable using antibodies to the
epitope-tagged proteins (Fig. 6B, lanes 4-6).
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Fig. 6.
Binding of XC3H-4 protein to the
mTNF-ARE. Cytosolic extracts of 293 cells
transfected with either vector alone or constructs expressing the CCCH
zinc finger proteins and extracts prepared from Xenopus
oocytes injected with in vitro transcribed RNAs encoding the
XC3H-4 protein or its fragments were prepared as described under
"Experimental Procedures." A, UV cross-linking assay.
Incubation of 293 extracts (lanes 2-4 and 9-11)
or oocyte extracts (lanes 5-8) with 1.5 × 106 cpm of 32P-labeled mTNF ARE probe (bases
1281-1350; GenBankTM accession number X02611), UV
cross-linking, and RNase digestion were performed as described under
"Experimental Procedures." Each sample in lanes 2-4
contained 5 µg of protein; lanes 5-11 contained 20 µg
of protein. Lane 1 (P'), probe alone (1.5 × 106 cpm) after RNase digestion. Lanes 2 and
11 (BS+), extracts from 293 cells transfected
with 5 µg of vector plasmid. Lanes 3, 4,
9, and 10, extracts from 293 cells transfected
with 1 µg/plate of plasmid CMV·hTTP·tag, CMV·mTTP·tag,
CMV·U2AF35, or CMV·XC3H-1·tag, respectively; vector was added to
make the total transfected DNA 5 µg/plate. Lane 5, extract
from Xenopus oocytes injected with buffer. Lanes
6-8, extract from Xenopus oocytes injected with XC3H-4
RNAs encoding the full-length protein (lane 6), the
amino-terminal fragment 1-120 (lane 7), or the
carboxyl-terminal fragment 121-276 (lane 8), respectively.
The RNA-protein complexes were resolved by SDS-PAGE (16% gel) followed
by autoradiography. The positions of molecular mass standards are
indicated. B, cell extracts prepared from 293 cells or
Xenopus oocytes as described in A were analyzed
by Western blotting using a polyclonal antibody to the epitope tag of
the fusion proteins. The positions of molecular mass standards are
indicated to the left of the gel.
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Interaction of TTP Fragments with TNF mRNA--
As shown in
Fig. 1, the human proteins ERF1 and ERF2 exhibit much greater
similarity in amino acid sequence with TTP within the TZF domain than
in other regions of the proteins, suggesting that the common TZF domain
may be the key component of these proteins that binds to and regulates
the stability of ARE-containing mRNAs. We have already shown that
the integrity of both zinc fingers is necessary for TTP
binding to the TNF ARE in cell-free assays and for destabilizing
TNF mRNA in cell transfection experiments (11).
We asked next whether the TZF domain alone was sufficient
for TTP to interact with the TNF mRNA. Three expression
constructs were prepared that all contained the TZF domain; this spans
amino acids 104-166 of human TTP ( (21) GenBankTM
accession number M63625). CMV·hTTP (1-173)·tag contained amino acids 1-173; CMV·hTTP (97-326)·tag contained amino acids 97-326 (the carboxyl terminus of the protein); and CMV·hTTP (97-173)·tag contained the double zinc finger domain flanked by seven amino acids at
each end.
When cell extracts from 293 cells expressing these protein fragments
were used in UV cross-linking experiments using TNF ARE probes, TTP
and all of its fragments could be cross-linked to a longer probe
containing the full ARE (bases 1281-1350) as well as a shorter probe
containing only four AUUUA motifs (bases 1309-1332) (Fig.
7A, lanes 1-10).
Neither the full-length TTP protein nor any of its fragments was able
to form a detectable complex with the mutant probe 1309-1332 (A/G), in
which the flanking As of its four AUUUA motifs were replaced by Gs
(Fig. 7A, lanes 11-25).
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Fig. 7.
Interaction of TTP protein fragments with
TNF mRNA. A, UV
cross-linking assays with wild-type and mutant TNF ARE probes.
Cytosolic extracts of 293 cells transfected with either vector alone or
constructs expressing the truncated human TTP proteins were prepared as
described under "Experimental Procedures." 32P-Labeled
mTNF ARE probes (1.5 × 106 cpm/sample) 1281-1350,
1309-1332, or mutant probe 1309-1332 (A/G), as indicated at the top,
were incubated with extracts (20 µg of protein). UV cross-linking and
RNase digestion were performed as described under "Experimental
Procedures." Lanes 1, 6, and 11 (BS+), extracts from 293 cells transfected with 5 µg/plate
of vector plasmid. Lanes 2, 7, and 12,
extracts from 293 cells transfected with 1 µg/plate of plasmid
CMV·hTTP·tag (full). Lanes 3, 8, and
13, extracts from 293 cells transfected with 1 µg/plate of
plasmid CMV·hTTP(1-173)·tag. Lanes 4, 9, and
14, extracts from 293 cells transfected with 1 µg/plate of
plasmid CMV·hTTP(97-326)·tag. Lanes 5, 10,
and 15, extracts from 293 cells transfected with 1 µg/plate of plasmid CMV·hTTP(97-173)·tag. Vector DNA was added
to make the total transfected DNA 5 µg/plate. The RNA-protein
complexes were resolved by SDS-PAGE (16% gel) followed by
autoradiography. The positions of molecular mass standards are
indicated. Sequences of the probes used are shown at the
bottom of Fig. 4. B, RNA electrophoretic mobility
shift assays using TNF ARE probe 1309-1332. Extracts (10 µg of
protein) from 293 cells transfected with full-length or truncated human
TTP as described above were incubated with 2 × 105
cpm of probe in the absence () or presence (HA.11) of a polyclonal
anti-epitope tag antibody. RNA mobility shift assays and RNase T1
digestion were performed as described under "Experimental
Procedures." Lane 1 (P'), probe alone (RNase T1
digested). Lanes 2 and 7 (BS+),
extracts from 293 cells transfected with 5 µg/plate of vector
plasmid. Lanes 3 and 8, extracts from 293 cells
transfected with 1 µg/plate of plasmid CMV·hTTP·tag (full).
Lanes 4 and 9, extracts from 293 cells
transfected with 1 µg/plate of plasmid CMV·hTTP(1-173)·tag.
Lanes 5 and 10, extracts from 293 cells
transfected with 1 µg/plate of plasmid CMV·hTTP(97-326)·tag.
Lanes 6 and 11, extracts from 293 cells
transfected with 1 µg/plate of plasmid CMV·hTTP(97-173)·tag.
Vector DNA was added to make the total transfected DNA 5 µg/plate.
The super-shifted (SS) RNA-protein complexes are indicated.
The RNA-protein complexes I, II, and III formed with endogenous 293 cell proteins and the migration position of free probe (FP)
are indicated. C, Western analysis of full-length and
truncated TTP protein expression. Cell extracts (5 µg of protein)
prepared from 293 cells transfected with 1 µg of DNA from the
constructs described above were separated on a 16% SDS-PAGE gel and
immunoblotted using a polyclonal antibody to the epitope tag of the
fusion proteins. Antibody was visualized with 125I-protein
A, and an autoradiograph of this blot is shown. Molecular mass
standards are indicated to the left of the blot. D, effect
of truncated TTP fragments on TNF mRNA stability. CMV·mTNF
(1 µg/plate) was co-transfected into 293 cells with either
full-length or truncated human TTP expression constructs or vector
alone. Total cellular RNA was harvested as described under
"Experimental Procedures." Each lane was loaded with 10 µg of
total RNA. Electrophoresis and Northern hybridization were performed as
described under "Experimental Procedures." Lane 1,
vector (BS+, 5 µg/plate); lanes 2-4,
CMV·hTTP·tag (full) (0.1, 1, and 5 µg/plate, respectively);
lanes 5-7, CMV·hTTP(1-173)·tag (0.1, 1 and 5 µg/plate, respectively); lanes 8-10,
CMV·hTTP(97-326)·tag (0.1, 1 and 5 µg/plate, respectively);
lanes 11-13, CMV·hTTP(97-173)·tag (0.1, 1 and 5 µg/plate, respectively). Vector was added to each transfection
mixture to make the total amount of co-transfected plasmids 5 µg/plate. The Northern blot was probed with a 32P-labeled
mTNF cDNA (upper panel). The two arrows to
the right of upper panel indicate the two species
of TNF mRNA discussed in the text. Identical RNA samples as in
the upper panel were blotted and probed with a full-length
32P-labeled mouse TTP cDNA, as shown in the lower
panel. The position of the 18 S ribosomal RNA is indicated.
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In RNA mobility shift assays using the TNF ARE 1309-1332 probe and
293 cell extracts prepared from cells transfected with these TTP
expression constructs, each of these TTP fragments, like the
full-length protein, was able to form a probe-protein complex (Fig.
7B, lanes 1-6). When an epitope tag antibody was included in the mobility shift assay, all of these TTP fragments formed
super-shifted complexes (Fig. 7B, lanes 8-11),
whereas there was no super-shifted complex formation with extracts from vector-transfected 293 cells (Fig. 7B, lane 7).
Although the amount of mRNA and protein expression from these TTP
fragment expression constructs was somewhat decreased relative to
full-length TTP, readily detectable amounts of these fragments were
seen by probing a Western blot with an antibody to the epitope tag
(Fig. 7C).
We then tested the ability of these truncated forms of TTP to cause
deadenylation and/or degradation of the TNF mRNA in intact cells. When 293 cells were co-transfected with CMV·mTNF and either CMV·hTTP (1-173)·tag (Fig. 7D, lanes 1 and
5-7) or CMV·hTTP (97-326)·tag (lanes 1, 8-10), the
TNF mRNA exhibited the shortening to the deadenylated form seen
with full-length TTP (Fig. 7D, lanes 1-4). When
construct CMV·hTTP (97-173)·tag was used, the characteristic two
bands of TNF mRNA were seen at both 1 and 5 µg of transfected DNA (Fig. 7D, lanes 11-13). These data indicated
that the 77-amino acid peptide containing the TZF domain alone was
capable of promoting the decrease in size of the TNF mRNA, which
we have attributed to its deadenylation (11). The apparent differences
in potency of these constructs to promote deadenylation of the TNF
mRNA appear to be due, at least in part, to differences in
expression (Fig. 7, D, lower panel, and
C); this conclusion is supported by more extensive
concentration-response experiments (not shown).
Alignment of TZF Domains of Known CCCH Proteins--
To begin to
identify critical sequence requirements for ARE binding, we have begun
an analysis of the TZF domain of the vertebrate CCCH proteins. We
aligned the TZF domains from the four vertebrate proteins discussed
here, making the assumption (borne out in every case in which it has
been tested directly) that the domains from homologues from other
animal species would bind to the ARE probe similarly to the prototype
protein. The TZF domains, with one exception, are from published
sequences already in GenBankTM, as listed in the legend to
Fig. 8. The single exception is from an
unpublished Xenopus expressed sequence tag, in which a
single open reading frame predicted a Xenopus allelic
variant of XC3H-3.3 This is
now listed as XC3H-3.2, with the original allele (20) listed as
XC3H-3.1. The 64-amino acid TZF domains from all of these proteins were
aligned using the program Pileup from GCG. In Fig. 8A is
illustrated the dendrogram produced by these alignments; despite the
facts that these proteins are much more disparate outside of the TZF
domains than within them, the alignment program still aligned them into
homologous groups (Fig. 8A). The alignment itself is
pictured in Fig. 8B. To simplify the discussion of the alignment, the amino acids within the TZF domains are numbered from 1 to 64. Although similar TZF domains have been identified in
invertebrate animals and in plants, we excluded these sequences from
the alignment because we have no direct evidence at present that they
are capable of ARE binding or mRNA destabilization.
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Fig. 8.
Alignment of tandem zinc finger domains of
known CCCH proteins. The 64-amino acid TZF domains from the
proteins described in the text were aligned with the Pileup function
from GCG. In A, a dendrogram is shown in which only the
64-amino acid TZF domains shown in B were used to calculate
sequence similarities. The four major groupings are indicated. In
B is shown the alignment of the TZF domains. Identical amino
acids are shaded in black; related amino acids are shaded in
gray. The accession numbers for the proteins listed are as
follows: for the cMG1 group: rat cMG1, X52590; human cMG1, X71901;
mouse cMG1, P23950; and Xenopus XC3H-2, AAD24208. For the
tis11d group: human TIS11d, X78992; mouse tis11d, P23949;
Xenopus XC3H-3.1, AAD24209; and Xenopus
XC3H-3.2.3 For the TTP group: human TTP, P26651; bovine
TTP, P53781; mouse TTP, P22893; rat TTP, P47973; and Xenopus
XC3H-1, AAD24207. For the XC3H-4 group: carp CTH1, CAA71245.2;
zebrafish CTH1, CAA76889; and Xenopus XC3H-4, AAD
24210.
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Examination of Fig. 8B reveals that 34 of the 64 amino acids
in the TZF domains (53%) have been conserved among all four proteins from species as diverse as humans, Xenopus, and zebrafish.
These include the RYKTEL lead-in sequence for the first zinc finger, the lead-in sequence KYKTEL in the second zinc finger, and several other amino acids in the inter finger 18-amino acid spacer, including a
Gly residue at position 27, an acidic residue at 30, a Leu at 31, a His
at 37, and a Pro at 38. Within both zinc fingers, the canonical CCCH
residues were conserved. Within the first finger, a Glu residue was
conserved at position 12, a Gly at 14, a Tyr at 18, a basic residue at
21, and Gln-Phe-Ala at 23-25. Within the second finger, a Gly residue
was conserved at position 52, a Tyr at 56, an Arg at 59, a Phe at 62, and a branched chain amino acid at 63.
The TZF domains also appeared to contain protein-specific
"signatures," which allowed the domains from a given protein
(e.g. TTP) to be grouped appropriately with its homologues
(Fig. 8A), despite the great evolutionary distance between
the animal species examined. For example, TTPs from human, cow, rat,
mouse, and Xenopus all contain Thr residues at position 9, Ser residues at position 11, Ala residues at position 20, Asn residues
at position 35, His-Lys at 46 and 47, Tyr-Leu at 49 and 50, and Ser
residues at position 58, differentiating the TTP homologues from all
other proteins examined. Similarly, the cMG1 homologues all contain Asp
residues at position 20 and Ile residues at position 28, distinguishing them from the others. The TIS11D proteins had as their only signature a
Phe residue at position 28. The more distantly related XC3H-4 proteins
also contained signature amino acids, including Ser-Arg at 8 and 9, Ala
at 11, and Leu at 61.
These comparisons also identified some positions within the TZF domains
that can tolerate significant amino acid diversity. For example, Pro,
Thr, and Arg were present at position 9, Glu and Ser at position 11, Asn, Ser, and Thr at 13, Ala, Ser, Thr, Arg, and Phe at 15, Asp, Glu,
Ala, and Asn at 20, Ile, Phe, Leu, Pro, and Lys at 28, His, Gly, Ile,
and Ser at 29, etc. Less diversity is evident at other positions; for
example, only hydrophobic residues were present at positions 10, 48, and 49, and only basic residues were present at position 21.
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DISCUSSION |
We recently showed that TTP, the prototype of a small family of
vertebrate proteins with tandem CCCH zinc fingers, exhibited several
measurable activities in both cell-free and intact cell experiments. In
cell-free experiments, TTP bound and could be cross-linked to the ARE
of the TNF mRNA, as determined by both gel shift and UV light
cross-linking experiments; this interaction could be prevented by
mutating a single cysteine to arginine in either zinc finger (11). In
cell transfection studies, TTP promoted the apparent deadenylation and
breakdown of a 3'-truncated TNF mRNA that contained most of the
native ARE (11). That these effects of TTP are likely to be
physiologically relevant has been demonstrated by experiments in TTP
knockout mice, which are characterized by a chronic inflammatory state
that appears to be largely due to excess circulating TNF (7-9).
This excess TNF was derived, at least in part, from macrophages,
because macrophages from the TTP knockout mice overproduced TNF.
This in turn was due to an increased stability of the TNF mRNA
in the TTP-deficient cells (7-9).
More recently, we found that the GM-CSF mRNA, which contains a
similar ARE in its 3'-untranslated region, was also greatly stabilized
in bone marrow stromal cells derived from the TTP knockout mice (10).
Importantly, these studies also identified an apparently deadenylated
intermediate breakdown form of the GM-CSF mRNA in the cells from
normal mice, whose formation was greatly inhibited in the cells derived
from the TTP-deficient mice (10). Taken together with earlier data,
these studies suggest that TTP plays an important physiological role in
the destabilization of two important, ARE-containing
mRNAs, those encoding TNF and GM-CSF, and that this TTP effect
appears to involve mRNA deadenylation.
It appears likely that the first step in the effect of TTP to
destabilize these mRNAs is its binding to the ARE, because
nonbinding zinc finger mutants do not stimulate TNF mRNA
breakdown in intact cells (11). The experiments described here begin to
explore the structure-function requirements for this first step in this protein-RNA interaction. First, we showed that the two other known mammalian members of this class of tandem CCCH zinc finger proteins exhibited similar activities in our assays. Specifically, the rat cMG1
protein, whose homologues include mouse TIS11b, human ERF1, and
Xenopus XC3H-2, and the Xenopus XC3H-3 protein,
whose homologues include mouse TIS11d and human ERF2, stimulated the apparent deadenylation and destruction of the TNF mRNA in intact cell transfection experiments. Second, these proteins, as well as a TZF
domain from the fourth known vertebrate member of this protein class,
Xenopus XC3H-4, could bind to the ARE of the TNF mRNA
in cell-free experiments, as demonstrated by gel shift and UV light
cross-linking experiments. Finally, we found that a 77-amino acid TTP
fragment containing the TZF domain of TTP was sufficient to mediate the
TTP effect on TNF mRNA stability in cell transfection experiments, as well as in cell-free gel shift and cross-linking experiments.
The three mammalian and one Xenopus/fish CCCH proteins
studied here comprise the four known members of a subclass of
vertebrate CCCH proteins in which the following features characterize
the TZF domains: 1) Both fingers within the TZF domain in the four proteins are preceded by a conserved 6-amino acid lead-in sequence, R(K)YKTEL; 2) Both fingers in all proteins contain the following conserved residues and spacing,
CXX(F/Y)X3GXCXYXX(K/R)CXFXH,
where X represents variable amino acids; 3) Both fingers in
all proteins are separated by exactly 18 amino acids, i.e.
between the terminal H of the first finger and the first C of the
second finger. These characteristics are identical in the protein
homologues despite their species of origin, ranging from human to
Xenopus laevis; 4) The three mammalian proteins and their
homologues are basic, with overall pIs ranging from 8.75 to 9.91. In
contrast, the Xenopus protein XC3H-4 has an overall pI of
5.9; however, the TZF domain itself is basic, with a pI of 9.1, similar
to the pIs of the TZF domains from the other three proteins (20).
In contrast, the hU2AF35 protein exhibited no activity in
our assays. This smaller subunit of the essential mRNA splicing
factor hU2AF was not thought to bind to RNA directly (24, 30); however, three recent reports suggest that direct binding to RNA can occur (31-33). This protein contains two related CCCH zinc fingers that are
present in the protein homologues from man to yeast but that are
separated by 116 amino acids in the human protein. Thus, the mere
presence of two zinc fingers of this type does not appear to confer
TNF mRNA destabilizing and ARE binding activity, in the absence
of the other characteristics of the more closely spaced TZFs of TTP and
its relatives. However, it will be of interest to determine whether the
CCCH zinc fingers are involved in direct contacts with the pre-mRNA
in this case.
The fact that TTP and its two related proteins all exhibited similar
mRNA binding and destabilizing activities in our assays suggested
that the domain they all hold in common, the TZF domain, is critical
for these activities. That the TZF domain is necessary for these
activities was demonstrated by our findings that mutation of a single
cysteine in either zinc finger to an arginine (11) completely abrogated
ARE binding and mRNA destabilizing activities of TTP. The present
experiments demonstrate that a 77-amino acid domain of TTP that
contains the TZF domain was also sufficient for both ARE binding and
TNF mRNA destabilizing activities. Therefore, at present, this
77-amino acid domain is the minimum known TTP sequence required for
these activities. The corresponding minimum known ARE sequence required
for this binding is 24 bases (bases 1309-1332; GenBankTM
accession number X02611).
These findings raise the possibility that these related proteins may
have TTP-like activities in normal physiology, perhaps to regulate
TNF mRNA stability or perhaps to regulate the stability of other
mRNAs containing class II or related AREs. The physiological importance of these findings will probably require the generation of
knockout mice for each gene.
Many questions remain to be answered about these proposed interactions,
particularly concerning the mechanisms of the effect of the CCCH
proteins on mRNA stability as well as their specificity and
physiological relevance. Among the most important is what confers the
specificity of CCCH protein binding to Class II AREs, because all three
CCCH proteins can be expressed in the same cell types, such as
macrophages? There are major differences in the patterns of expression
of the three proteins in the mouse, particularly in their biosynthetic
responses to external stimuli and their tissue- and cell-specific
expression.4 These
differences, as well as the markedly different primary sequences among
the three proteins outside of the TZF domains, may help to explain why
the endogenous mouse equivalents of cMG1 and XC3H-3 do not prevent the
development of the TTP deficiency syndrome in mice and, in fact, were
present in normal amounts in tissues of these animals (7). There are
many previous examples in the literature in which disruption of the
gene encoding a single member of a gene family is not compensated by
the remaining members (36, 37).
Another important question is that because the TZF domain alone seems
to be adequate for at least the TTP activities measured here, what are
the functions of the other domains in the three proteins? There are
rapid, mitogen-induced changes in the biosynthesis, subcellular
localization, and serine phosphorylation of TTP. What role do these
modifications play in the behavior of TTP, both in cells expressing
TNF and in cells not expressing this cytokine? Do similar
modifications of the oth
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ABSTRACT
INTRODUCTION
