(Received for publication, September 12, 1995; and in revised form, November 2, 1995)
From the
Reduced tension of O slows the degradation rate of
mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in
catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell
line. The observed increase in half-life (30 h versus 10 h)
correlates with enhanced binding of a 66-kDa protein (hypoxia inducible
protein) to the pyrimidine-rich tract located between bases
1552-1578 in the 3`-untranslated region of TH mRNA
(hypoxia-inducible protein binding site (HIPBS)). The present study
investigates the protein binding site within the 27-base HIPBS, first
by using specific cleavages of HIPBS and its flanking sequences with
antisense oligodeoxynucleotides and RNase H and then by using
mutational analysis of the binding properties. We found that the
27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified
the optimal hypoxia-inducible protein binding site that is represented
by the motif (U/C)(C/U)CCCU, where the core binding site is indicated
by the underlined cytidines. Substitutions of either one of the
cytidines with purine or uridine abolished the protein binding. The
mutations within HIPBS, which partially reduced binding, did not
prevent stimulation of protein binding for extracts from hypoxic cells.
The hypoxia-induced increase in complex formation was proportional to
the strength of binding using proteins from normoxic cells. The HIPBS
element is conserved in TH mRNAs derived from different species.
It is well established that pyrimidine-rich sequences and
specific polypyrimidine (poly(Y))-binding ()proteins
represent important functional elements in RNA metabolism(1) .
Among the best characterized pyrimidine-rich motifs are the poly(Y)
tracts located between the branch site and the 3` splice site of
introns. These tracts are known to regulate the processing of
intervening sequences(2) . Other examples of pyrimidine-rich
motifs include oligopyrimidine sequences in the 5`-untranslated regions
of mRNAs for ribosomal proteins that regulate translation of these
proteins (3, 4, 5) and AUUUA pentamers,
which are well established as destabilizing elements in the
3`-untranslated regions of different short-lived mRNAs(6) .
Recently, the poly(Y) tracts in the 3`-untranslated region of
15-lipoxygenase mRNA were shown to regulate its
translation(7) . A number of pyrimidine-rich tract-binding
proteins have been recently identified, purified, and cloned. Examples
of these include the poly(Y) tract-binding protein(8) , the
essential splicing factor, U2AF
(9) ; the splicing
regulator Sex-lethal, Sxl (10) ; the group of heterogeneous
nuclear ribonucleoprotein particles (hnRNPs) such as the proteins C1
and C2(11) , I(12) , and K and J(13) ; and the
48-kDa protein binding to 15-lipoxygenase 3`-UTR(7) .
Importantly, various pyrimidine-rich tract-binding proteins show
affinities for distinct binding motifs within the pyrimidine-rich
sequences. In this respect the proteins K and J bind to polycytidine
sequences(13) , and proteins C1 and C2 bind to the polyuridine (11) sequences. The distinct consensus binding sequences were
also found to be preferred by the poly(Y) tract-binding, Sxl, and
U2AF
proteins(14) .
We have recently reported
that stability of mRNA for tyrosine hydroxylase (TH) (EC 1.14.16.2),
the regulatory enzyme in the synthesis of catecholamines, a major group
of neurotransmitters, is three times increased during hypoxia in the
pheochromocytoma-derived PC12 clonal cell line(15) . This is
associated with increased TH protein synthesis (16) and
release of the neurotransmitter dopamine during hypoxia(17) .
The effect of hypoxia on TH mRNA stability is specific, because neither
-actin nor GAPDH mRNAs are stabilized, and it represents a novel
and important mechanism by which environmental factors can affect gene
expression. We have since found that this increase in TH mRNA half-life
during hypoxia is accompanied by increased binding of a 66-kDa protein
(hypoxia-inducible protein, HIP) to the pyrimidine-rich tract located
between bases 1552 and 1578 in the 3`-UTR of TH mRNA(18) . This
element has been referred to as the hypoxia-inducible protein binding
site (HIPBS). The present study characterizes the sequence specificity
and maps the HIP binding site within the pyrimidine-rich region of the
TH mRNA 3`-UTR. We report that the motif (U/C)(C/U)CCCU within the
pyrimidine-rich tract represents the optimum HIP-binding site.
Importantly, the middle two cytidines (underlined) are the protein core
binding site.
The experimental procedures were described earlier(18) . The restriction enzymes were purchased from Promega Corp. and Life Technologies, Inc., whereas the chemicals were purchased from Fisher and Sigma.
Figure 1: A, sequence of the 162-base pair, KpnI-SphI fragment of TH mRNA template that was used to generate different transcripts (plasmids pSP73-TH162 and pSP73-TH162M). The pyrimidine-rich sequence that encodes HIPBS is boxed. The naturally occurring restriction sites are indicated. The newly generated restriction sites, NcoI and BamHI, are marked by asterisks. B, gel retardation assays using S-100 protein extract and transcripts that were obtained after linearization of templates with HindIII (lane 1), BamHI (lane 2), and NcoI (lane 3). The formation of the complex was the same when HindIII or BamHI linearized templates were used, but the complex was not formed when the template was linearized with NcoI.
In the second series of experiments, synthetic RNA
oligonucleotides were used. RNA oligonucleotides were synthesized on a
nucleic acid synthesizer (Applied Biosystems model 380B) using
phosphoramidites, support columns, and a deprotection protocol from
Glen Research. All RNA oligonucleotides used were run on 10% acrylamide
gel to determine the presence of a single band. For RNA gel shift
assays, RNA oligonucleotides were labeled at the 5` end using 50
µCi of [-
P]ATP (DuPont NEN) and
polynucleotide kinase (Promega Corp.). Labeled oligonucleotides were
purified using RNase-free G-25 columns (Boehringer Mannheim).
Figure 2: Effects of specific deletions within the HIPBS using antisense oligodeoxynucleotides on TH mRNAprotein complex formation. A, sequence of the HIPBS (boxed) and its 5`- and 3`-flanking regions (gray letters). The arrows (a-i) indicate the fragments of RNA that were specifically cleaved during hybridization with complementary oligodeoxynucleotides. B, gel shift assays with the cleaved RNA fragments (a-i) and S-100 protein fraction (lanes 1-11). The thick arrow indicates the specific complex. C indicates a control experiment where the RNA molecule is intact. Cleavage of the RNA sequence outside HIPBS does not affect the binding (lanes 2 and 7). Cleavage within the HIPBS decreases the binding (lanes 3, 4, 8, and 9). Cleavage within the 11-pyrimidine tract abolishes formation of the typical complex (lanes 5, 10, and 11).
Fig. 1A shows the DNA sequence corresponding to the KpnI-SphI fragment of the 3`-UTR of TH mRNA that was used to generate transcripts for gel retardation assays. The location and sequence modification created by generation of BamHI and NcoI restriction sites are also shown. Fig. 1B shows the binding pattern of the protein to this fragment of TH mRNA 3`-UTR when the wild type DNA template (pSP73-TH162) was linearized with HindIII (full-length transcript, lane 1) and when the pSP73-TH162M template was digested with either BamHI (transcript ends immediately 3` from HIPBS, lane 2) or NcoI (transcript ends immediately 5` from HIPBS, lane 3). It is clear that the RNA fragment that contains HIPBS but no additional 3` sequence binds HIP in the same manner and to the same extent as the full-length wild type transcript (compare lanes 2 and 1). However, RNA that did not include HIPBS did not bind protein (lane 3).
In order to map the protein binding site within HIPBS, we have performed selective cleavages of this sequence from either the 5` or 3` end using antisense oligodeoxyribonucleotides and RNase H endogenously present in the protein extracts. Fig. 2A shows the HIPBS including some of the 5`- and 3`-flanking sequence. The HIPBS contains a stretch of 11 uninterrupted pyrimidines (Fig. 2A, i), and our initial experiments were oriented to determine if this element is directly involved in the protein binding. The arrows (Fig. 2A, a-i) indicate the fragments of RNA that were selectively cleaved by hybridization with individual complementary antisense oligodeoxynucleotides. Fig. 2B shows gel retardation assays using RNA transcripts that have progressive deletions of HIPBS from the 5` end (Fig. 2B, left) and from the 3` end (Fig. 2B, right). It is clear that cleavage of the flanking sequence 5` (Fig. 2B, lane 2) or 3` (Fig. 2B, lane 7) outside of HIPBS did not affect the protein binding. The cleavage within HIPBS from the 5` end somewhat reduced the binding (Fig. 2B, lanes 3 and 4), but only cleavages within the stretch of 11 pyrimidines prevented formation of the normally observed complex (Fig. 2B, lane 5). Similarly, cleavage from the 3` end revealed that although binding was decreased by consequent shortening of HIPBS, it was not abolished until the deletion occurred within the stretch of 11 pyrimidines (Fig. 2B, compare lanes 8-10). In addition, the antisense oligodeoxynucleotide that was complementary to this stretch of 11 pyrimidines was able to abolish the binding (lane 11). Moreover, in all cases where the formation of RNA-protein complexes was only reduced but not abolished, the binding was enhanced when protein extracts were obtained from hypoxic cells (not shown).
We next examined the effects of mutations within the pyrimidine tract on binding of the HIP using RNA gel shift assays with the S-100 fraction of cytoplasmic proteins from PC12 cells. Table 1shows sequences of HIPBS mutations that were introduced using the NcoI-BamHI restriction frame. Fig. 3shows the binding pattern of HIP to the mutated HIPBS. The quantification of average data from several such experiments is presented in Table 1.
Figure 3:
Effects of mutations within HIPBS on the
formation of TH mRNA-protein complex. A, gel retardation
assays using indicated mutated RNAs (lanes 2, 3, and 5-8) as compared with wild type RNA (lanes 1 and 4). Mutation A prevents formation
of the complex (lane 6). Mutation C
increases the binding (lane 2). Other mutations have
intermediate effects on the binding.
First we tested whether increasing the
pyrimidine content of HIPBS sequence would affect the binding.
Interestingly, substitution of cytidines for three naturally occurring
adenines, mutation C, that created additional
stretches of oligo(C) in HIPBS, increased the binding by approximately
80% (Fig. 3, lane 2). Substitution of adenines by the
other pyrimidine nucleotide, uridine, mutation U
, was
less effective than the above mutation and only increased the binding
by approximately 20% as compared with the wild type control (Fig. 3, lane 3).
We have also tested whether
decreasing the pyrimidine content of HIPBS and in particular whether
interrupting the stretch of 11 pyrimidines affects the HIP binding
properties. Mutation A, which disrupted the stretch
of 11 pyrimidines and the oligo(C) sequence, completely abolished the
binding (Fig. 3, lane 6). In contrast, the mutation
A
located immediately 3` to A
,
which also disrupted the stretch of 11 pyrimidines but without
affecting the UCCCCU element, did not abolish the binding, but only
reduced it (Fig. 3, compare lanes 6 and 7).
The other mutations, A
and A
, in which
adenines were substituted for pyrimidines outside the 11-pyrimidine
stretch and which did not disrupt the UCCCCU element, reduced the HIP
binding to about 30-40%. The affinity of the mutated HIP-binding
sequences for HIP was further tested using competition experiments in
which the binding of HIP to the wild type transcript was competed with
the indicated doses of unlabeled, mutated RNAs in the RNA gel shift
assays. Fig. 4shows the results of these experiments. The
binding was reduced by approximately equal doses of cold wild type and
mutant U
RNAs. The dose of the cold RNAs that
completely abolished the binding was somewhat above 100 ng, because at
100 ng of cold competitor some binding was still visible in both (Fig. 3, lanes 2-4 and 8-10). The
binding was competed more strongly with the C
RNA
that also showed stronger binding affinity in the gel shift assays. The
competing dose was below 100 ng (Fig. 3, lanes
5-7). The mutant A
RNA that did not show
any binding activity in RNA gel shift assays did not compete for
binding at doses of cold probe up to 500 ng (Fig. 3, lanes
14-16). The three other mutant RNAs, A
,
A
, and A
, showed intermediate
strength in the competition experiments that paralleled their HIP
binding affinities.
Figure 4:
Competition of TH mRNAprotein binding with
wild type and mutated (mutated HIPBS) cold RNAs at the indicated
concentrations. Mutation A did not compete for the
binding (lanes 14-16), and mutation C
was the strongest competitor (lanes 5-7). The
mutations that moderately increased (lanes 8-10) or
partially decreased (lanes 11-13 and 17-22) the binding reduced the complex formation
parallel to their binding affinities.
Finally, we tested whether the HIPBS-mutated
RNAs maintained the ability to bind cytoplasmic protein in the
hypoxia-inducible manner. Fig. 5shows that except for mutant
A, which does not bind protein (Fig. 5, lanes 9 and 10), all other mutant RNAs were capable
of forming increased RNA-protein complexes when S-100 protein extracts
were obtained from PC12 exposed to 5% O
(hypoxia) for 3 h
as compared with the extracts obtained from cells grown in normoxia
(21% O
). However, the hypoxia-induced increase in the
complex formation was proportional to the binding affinity with
normoxic protein extracts. The results of the above experiments
indicate that the core binding site for the hypoxia-inducible protein
is within the UCCCCU element and is represented by the stretch of four
cytidines.
Figure 5:
Effects of hypoxia on the formation of
mutated TH mRNA-protein complex. In all cases, except when mutation
abolished the binding, the formation of the complex was increased when
protein extracts were obtained from PC12 cells exposed to 5% O for 3 h. C, extracts from PC12 cells exposed to
normoxia; H, extracts from PC12 exposed to
hypoxia.
We next mapped the protein binding site within the UCCCCU
element using mutated synthetic RNA oligoribonucleotides. Fig. 6shows the binding features of the hypoxia-inducible
protein to the synthetic 27-base RNA oligonucleotide that has the HIPBS
sequence. It is clear that the binding complex between protein and the
HIPBS RNA oligonucleotide (Fig. 6A, lanes 3 and 4) migrates at the same level as the complex formed
between the same amount of protein extract and the wild type (162 TH)
RNA transcript (Fig. 6A, lane 1). RNA
oligonucleotide that has a random sequence does not form a complex with
the proteins (Fig. 6A, lanes 6 and 7). In addition, the formation of the complex between the
27-base HIPBS oligoribonucleotide and the protein is increased when
protein extracts are obtained from PC12 cells exposed to hypoxia (Fig. 6A, H, lane 4) as compared with
extracts of cells exposed to 21% O (Fig. 6A, C, lane 3). Moreover,
we showed that the HIPBS RNA oligonucleotide is capable of competing
for the HIP with the full-length 162-base RNA transcript, because the
addition of 100 ng of HIPBS abolishes the formation of the RNA-HIP
complex (Fig. 6B, lanes 2-4). In
contrast, the random 27-base RNA oligonucleotide that does not bind
protein (Fig. 6A, lanes 5-7) does not
abolish the binding (Fig. 6B, lanes
5-7).
Figure 6: The 27-base HIPBS oligoribonucleotide is sufficient to bind cytoplasmic protein in the hypoxia-inducible manner. A, protein binds in the same manner to the 162 TH mRNA transcript (lanes 1) and to the 27-base HIPBS RNA oligonucleotide (lanes 3 and 4) but not to the RNA oligonucleotide that has random sequence (lanes 5 and 6). Proteins were obtained from PC12 cells grown in normoxia (C) or hypoxia (H) for 3 h. B, the formation of the complex between 162 TH mRNA transcript and HIP (lane 1) was reduced and abolished with the cold 27-base HIPBS RNA oligonucleotide (lanes 2-4) but not with the random RNA oligonucleotide (lanes 5-7).
Table 2lists sequences of the synthetic RNA oligonucleotides that were tested. Fig. 7A shows RNA gel shift assays with these RNA oligonucleotides. Fig. 7B shows competition experiments in which the wild type and each mutated synthetic oligoribonucleotide (100 ng) were tested for their ability to reduce or abolish formation of the complex between HIP and the wild type 162-base fragment of TH mRNA (162tr). Quantification of the average data is included in Table 2.
Figure 7:
Effects of mutations within the HIPBS
oligoribonucleotide on the formation of the complex with HIP. The wild
type sequence is indicated on top. A, gel shift assays with
indicated mutants of HIPBS oligoribonucleotide. B, competition
of 162tr TH mRNA-HIP binding with 100 ng each of mutated
oligoribonucleotides. W, competition with the wild type HIPBS
oligoribonucleotide. Mutations A, A
,
U
, and U
most substantially reduced the
binding (A, lanes 4, 5, 10, and 11). They were also the weakest competitors for the binding (B, lanes 5, 6, 12, and 13).
Point substitutions
of adenines for each of the bases within the UCCCCU element were
detrimental to binding below 50% of the binding with the 162
transcript. The binding reduction was extreme (to 10%) when either
of the two middle cytidines was substituted by an adenine (A
or A
; Fig. 7A, lanes 4 and 5). These two mutants did not compete with the wild transcript
for protein binding (Fig. 7B, lanes 5 and 6). Interestingly, substitution with the other pyrimidine,
uridine, for C
or C
also strongly reduced the
binding to approximately 20% (U
and U
; Fig. 7A, lanes 10 and 11). These
mutant HIPBS were also weak competitors, although stronger than A
and A
(Fig. 7B, lanes 12 and 13). The binding was partially reduced (30-40%)
when U
, C
, C
, or U
were substituted by an adenine (A
, A
,
A
, or A
; Fig. 7A, lanes
2, 3, 5, and 6). These mutants also had
the ability to compete with 162tr for protein binding in proportion to
the complex formation (Fig. 7B, lanes 3, 4, 7, and 8). Substitution of both external
cytidines by adenines, creating the sequence UACCAU, completely
abolished the binding (not shown).
Interestingly, U C
substitutions in position 8 (U
) (UUCCCU) substantially
increased the binding to 163 ± 12.7% (Fig. 7A, lane 9). This mutant was also a strong competitor of the wild
type 162tr for the binding of protein; however, it was not a stronger
competitor than the wild HIPBS oligo (Fig. 7B, lane
11). In contrast, U
C substitutions in position 11
(U
) (UCCCUU) decreased the binding to 55 ± 6% (Fig. 7A, lane 12). Substitution of both
external cytidines (C
and C
) by uridine, which
created the sequence UUCCUU, completely abolished binding (not shown),
similar to corresponding substitutions with adenines. In addition,
insertions of an individual adenine between the cytidines in the UCCCCU
motif, creating the sequence UCCACCU or UCCCCAU, reduced the binding
below 20% (not shown). Interestingly, the substitutions C
U
(C
, lane 14) and C
U
(C
, lane 15) had the opposite effects on
the formation of RNA-protein complexes. The C
mutation
caused nearly a 2-fold increase in binding, whereas the C
mutation decreased binding by 30%. Both substitutions together
(C
) had an additive effect such that the total increase
in binding was less pronounced than in the case of the C
mutation alone (lane 16). In spite of the fact that the
C
mutant increased binding, its ability to compete with the
162tr for protein binding was slightly decreased compared with that of
the wild type HIPBS RNA oligonucleotide (Fig. 7B, lane 17).
None of the point mutations in the HIPBS sequence outside the UCCCCU element substantially affected binding. On the other hand, mutation of the the pyrimidine-rich region into the random sequence on either or both sides of the UCCCCU element completely abolished binding (not shown).
We have previously shown that the pyrimidine-rich region (HIPBS) in the 3`-untranslated region of tyrosine hydroxylase mRNA binds a 66-kDa protein in a hypoxia-inducible manner(18) . Those results suggested potential involvement of HIPBS in the regulation of TH mRNA stability during hypoxia(15) . In this study we have shown that under in vitro conditions, the 27-base-long fragment of HIPBS RNA is sufficient not only for the protein binding to occur but also to preserve the hypoxia-inducible feature. We have also characterized the HIP core binding site within the pyrimidine-rich sequence of TH mRNA. This is important because it is clear that different poly(Y) tract-binding proteins not only show binding specificity for distinct pyrimidine motifs, but mutations within the poly(Y) tracts, at least in introns, are able to affect the function of the poly(Y) sequences and the efficiency of splicing(21, 22) .
Our results demonstrate that the
major binding site for the HIP is the UCCCCU motif, as point mutations
only within this element could either substantially reduce or almost
abolish the complex formation, and multiple mutations within this
region completely abolished the binding. In view of the facts that 1)
this element has an oligo(C) stretch, 2) the protein binding is
strongly competed by polycytidine RNA(18) , and 3) creation of
specifically oligo(C) but not simply an increase in pyrimidine content
(uridine) of HIPBS increased the binding, we have hypothesized that the
binding site may be represented by the oligo(C) sequence. As expected,
any purine substitutions or insertions within this motif are
detrimental to the binding. Importantly, however, some pyrimidine
substitutions are also detrimental. Substitution of any one of the
three cytidines (C, C
, and C
) by
uridine had an effect similar to the corresponding substitution by an
adenine, indicating a strong requirement specifically for the presence
of cytidine in these positions. This effect was particularly strong in
the case of C
and C
, indicating that these
nucleotides may represent the core binding site. A simple elongation of
the cytidine (C
-C
) stretch, however, did
not improve protein binding. Elongation of the oligo(C) stretch by C
U
substitution decreased binding and attenuated the
increase in binding caused by C
U
substitution,
indicating a preference for uridine rather than cytidine in the
U
position. Although the mutation C
U
elongated the oligo(C) tract and increased the formation of
RNA-protein complexes, the mutation did not result in an increased
ability to compete with the wild type RNA transcript for the protein
binding. There is, therefore, preference for the UCCCCU sequence rather
than the oligo(C) stretch as the optimal binding site for HIP.
It is
important to note that not all oligo(C) stretches are capable of
binding HIP protein. For example a 21-base-long sequence within the
3`-UTR of TH mRNA that is relatively rich (although less so then HIPBS)
in pyrimidines and has an ACCCCCA sequence at the beginning of the
poly(Y) tract (Table 3) does not bind HIP(18) . Thus, the
optimal HIP binding element is represented by the sequence UCCCCU with
the similar binding properties for sequence UUCCCU and CUCCCU and may
be summarized by the consensus (U/C)(C/U)CCCU. In addition,
the (U/C)(C/U)CCC
U consensus element must be surrounded by
a pyrimidine-rich sequence.
It is also important to note that in spite of decreased binding affinity, when binding was not abolished, in all cases the formation of the RNA-HIP complex was augmented by exposure of cells to hypoxia. This finding indicates that a number of similar but not optimally binding sequences should be capable of binding protein in the hypoxia-inducible manner.
We have analyzed the presence of a HIPBS-like element in the 3`-UTRs of TH mRNA from different species. Table 3lists the HIPBS-like elements in these mRNAs. It is clear that a homologous pyrimidine-rich sequence is present in all of them, and importantly, each case starts within 50 bases of the translation stop codon. In addition, each of these mRNAs has an additional stretch of pyrimidines further downstream from HIPBS. This additional sequence is usually shorter and in the case of rat TH mRNA has been shown not to bind HIP. Based on the analysis of rat HIPBS, we predict that the HIPBS element will be strong in rat, in human, and particularly in quail TH mRNAs and less so in bovine and mouse TH mRNAs, because in the last two cases, one or two uridines around the oligo(C) stretch are replaced by purines. It is still possible, however, that these elements will bind HIP in a weaker but hypoxia-inducible manner. Also, both bovine and mouse TH mRNA have additional UCCCCCU elements within the downstream pyrimidine-rich tracts that may be able to compensate for the reduced binding in the upstream tract.
A number of different poly(Y) tract-binding
regulatory proteins have been recently characterized. Attempts have
also been made to define the RNA consensus elements that preferentially
bind these proteins using in vitro genetic selection (14) as well as mutational analysis of their binding
properties(20, 21) . In this respect two of these
proteins, the poly(Y) tract-binding protein (8) and the Sxl
protein(10) , show preferential affinity for the polyuridine
containing sequences, and their consensus binding sites are different
from the HIP binding site(14) . There is some degree of
similarity between the HIP binding site and the consensus element that
binds the U2AF splicing factor(9) . Results from
the genetic selection showed that the U2AF
binds
preferentially to the sequences rich in uridines that are frequently
interrupted by two to three cytidines(14) . HIP, however, seems
to bind preferentially to the sequences enriched in cytidines that are
interrupted by one to two uridines. In view of the high affinity of HIP
for oligo(C) sequences, the polycytidine-binding protein K, which is
similar in molecular mass to HIP (68 kDa)(13) , needs to be
considered as the potential HIPBS-binding protein. The HIP consensus
sequence is also homologous with the polypyrimidine motif
(C
PuC
UCUUUC
AAG) located in the
3`-UTR of 15-lipoxygenase(23) . However, the 15-lipoxygenase
mRNA-binding protein has a molecular mass of 48 kDa, which is
substantially smaller than the 66-kDa HIP.
We have also analyzed for
the presence of the HIPBS-like sequences a number of 3`-UTRs from other
mRNAs that are likely to be regulated by hypoxia (Table 4). We
were able to identify a HIPBS-like element in the 3`-UTR of
erythropoietin, inducible nitric oxide synthase, tumor necrosis factor
, myoglobin, and tryptophan hydroxylase. In contrast, we have not
found a HIPBS-like element, for example, in the GAPDH, endothelin, and
phenylalanine hydroxylase mRNAs. Currently, the post-transcriptional
regulation of the genes listed in Table 4is not well known. It
has been studied best in the case of erythropoietin mRNA, to which
binding of cytoplasmic protein to the HIPBS-like region was
reported(24) . In other cases there is only indirect evidence
of this regulation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M10244[GenBank], M69200[GenBank], X05290[GenBank], M36705[GenBank], M24778[GenBank], X02157[GenBank], D14051[GenBank], X57321[GenBank], D00409[GenBank], and X5301e.