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INTRODUCTION |
Voltage-gated potassium
(Kv)1 channels play integral
roles in regulating ion balance, membrane potential, secretion, and
cell excitability in many different cells (1). Mammalian Kv channels are encoded by a family of 19 known genes, each with its own unique profile of pharmacological and biophysical properties and tissue distribution (2). Kv channel expression is known to be regulated in
complex ways in both embryonic and adult cells. One member of this
family, Kv1.4, is expressed in brain, heart, and skeletal muscle (3,
4). The functional channel consists of four identical subunits and has
properties resembling a typical rapidly inactivating "A" current
channel (5). Within neurons, Kv1.4 channels are found in nerve
terminals where they are thought to underlie the rapid
after-hyperpolarization during action potentials (6). This channel is
sensitive to external 4-aminopyridine and Ba2+ (7) and is
potently blocked by pandinus toxin (8), as well as by the
dihydroquinoline compound CP339818 (9).
The mKv1.4 gene is known to generate at least three
transcripts, 2.4, 3.5, and 4.5 kb in size (3), that are under the
control of a single GC-rich promoter (4). The 2.4-kb transcript has been found only in skeletal muscle and in the mouse myoblast cell line
C2C12 where it is up-regulated during myotube
formation (3). The 3.5- and 4.5-kb transcripts, which are expressed in
heart and brain, begin at a single transcription initiation site and differ only by their use of alternate polyadenylation sites in their
3'-untranslated regions (UTRs) (4). The 5'-UTRs of typical mammalian
genes are relatively short (average length, ~150 nt) and lack both
AUGs stable secondary structure (10). By contrast, the 5'-UTR of
mKv1.4 (mouse Kv1.4) is long (~1.2 kb), contains 18 upstream AUG codons, and has a predicted complex secondary structure
with numerous stable hairpin structures (Fig.
1). Several of the upstream AUG codons
are contained within a favorable context for initiation (11) and are
followed by open reading frames as long as 40 amino acids, none of
which is contiguous with the authentic coding region.

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Fig. 1.
Sequence and predicted secondary
structure of mKv1.4 5'-UTR. A, sequence of the 5'-UTR
of mouse Kv1.4. Eighteen abortive AUGs are shown in bold and
underlined, and the authentic initiating AUG is shown as
bold only. A polypyrimidine tract near the 3' end is
underlined. The 1.2 kb fragment used in the bicistronic
constructs begins at nt 14 of this sequence, indicated by an
arrow. This sequence can be found in the
GenBankTM data base under accession numbers U03722 and
U03723. B, secondary structure of the 200-nt 3'-proximal
fragment of the mKv1.4 5'-UTR predicted by MFOLD; the
polypyrimidine tract (see Fig. 1A) is shown in
bold. This structure, which has a calculated free energy of
38 kcal/mol, was an element in several of the most stable structures
predicted by MFOLD using both the entire 1.2-kb 5'-UTR and the 200-nt
fragment alone.
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At least three mechanisms are known for eukaryotic translation
initiation, namely 5'
3' scanning, "shunting," and internal ribosome entry. In the scanning model, the 40 S ribosomal subunit (bound to the ternary complex) binds to the 5' cap complex and travels
in a 5'
3' direction until it encounters an initiator AUG codon in
a favorable context; the 60 S ribosomal subunit then joins the complex,
and peptide synthesis begins (11). Translation initiation of most
vertebrate genes is thought to occur by this mechanism. The shunting
mechanism is also 5' cap-dependent but differs from
scanning in that the 40 S subunit bypasses the majority of the 5'-UTR
by shunting or "jumping" to a region at or near the authentic site
of translation initiation, avoiding having to scan through most of the
5'-UTR (12, 13). In contrast, translation initiation by internal
ribosome entry involves the binding of the 40 S ribosomal subunit to an
internal ribosome entry site (IRES) at or near the authentic AUG,
thereby eliminating the requirement both for the presence of a 5' cap
structure and for scanning through the greater part of the 5'-UTR (for
review see Refs. 14-16).
The presence of upstream AUG codons and stable secondary structures are
both known to interfere with ribosome scanning and to inhibit
translation initiation at the authentic start site of typical
vertebrate genes (17-19). The presence of both of these features in
the 5'-UTR of mKv1.4 thus suggests the possibility that translation
initiation for this gene may occur by some mechanism other than 5'
cap-dependent scanning. Here we report that the 5'-UTR of
mKv1.4 contains an IRES that can initiate efficient translation of a reporter protein in a 5' cap-independent fashion. We
propose that translation initiation by internal ribosome entry may
represent a general and important mechanism for regulation of
expression of this gene as well as other members of the extended family
of potassium-selective channels.
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MATERIALS AND METHODS |
Constructs--
Bicistronic constructs (20) containing the
reporter genes chloramphenicol acetyltransferase (CAT) and luciferase
(LUC) were gifts from Peter Sarnow. The SV40 promoter and the T7
promoter control transcription of both reporter genes in the SV
construct and the T7 vector, respectively. The intercistronic spacer of both vectors contains a unique SalI site for cloning.
The cDNA for mKv1.4 was isolated from mouse brain
by reverse transcription-PCR using the following primer pair: sense,
5'-AGCTAGAGAGCAAGACTGGG-3', and antisense, 5'-ATAAGGCCATATGGCTGTTGC-3'.
The 5'-UTR was amplified by PCR to generate SalI restriction
sites on both the 3' and 5' ends using the following primer pair:
sense, 5'-AGCTAGAGTCGACGACTGGGATGG-3', and antisense,
5'-ACTCACCATCGCCACGTCGACGGTGG-3'.
The resulting PCR product was inserted into the intercistronic spacers
of both constructs (SV and T7) at the unique SalI site, and
its orientation was determined by restriction digests and DNA
sequencing. This procedure removed 13 nucleotides from the 5' end of
the full-length 5'-UTR.
Constructs containing a hairpin loop were generated by annealing the
complementary oligonucleotides shown below, which are flanked by
SalI extensions, and cloning this synthetic DNA
fragment into the SalI site in the intercistronic
spacer:
The underlined regions form 19-nt RNA stems with a calculated
free energy of
41 kcal/mol, and a SalI site is restored at only one of the two ends (the right-hand end above) after insertion. The orientation of this insert was determined by DNA sequencing, and
clones were chosen that had the restored SalI site at the 3'
end of the insert.
The 200-nt 3'-proximal region of the 5'-UTR was amplified by PCR using
the following primers (which generated SalI sites at their
termini): sense, 5'-AGGCAAGCTGTCGACAGCAACGCC-3', and antisense, 5'-AGCTAGAGTCGACGACTGGGATGG-3'. After digestion with SalI,
the PCR-amplified fragment was cloned into the SalI site of
the intercistronic spacer. The orientation of this fragment was
determined by DNA sequencing.
Cell Culture, Transfection of Constructs, and CAT and LUC
Assays--
NIH-3T3 cells were maintained in minimum essential medium
containing 1 g/liter glucose and 10% fetal calf serum. 24 h prior to transfection cells were plated at a density of 5 × 105 cells/90-mm plate. On the day of transfection, cells
were washed twice with 1× phosphate-buffered saline and then covered
with 8 ml of fresh medium. 20 µg of DNA (isolated either from cesium chloride density gradients or by use of the Qiagen Maxi-prep (Qiagen Inc., Chatsworth, CA)) was combined with 50 µl of 2 M
CaCl2 and 450 µl of sterile water. The DNA solution was
combined with 500 µl of 2× HBS (275 mM NaCl, 10 mM KCl, 42 mM HEPES, 12 mM
dextrose) by vortexing for 15 s. The solution was held at room
temperature for 20 min and gently pipetted onto the cells, and the
cells were washed and resuspended as described above. 64-72 h after
transfection, cells were washed with 1× phosphate-buffered saline,
harvested, and extracted on lysis buffer. Cell lysates were pelleted,
and the supernatants were assayed for LUC activity using a luciferase assay kit (Promega, Madison, WI) with a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). CAT activity was
measured with the Promega CAT assay kit.
In Vitro Transcription of Translation Templates--
DNA
templates for in vitro transcriptions were linearized with
HpaI, and transcriptions using bacteriophage T7 RNA
polymerase were performed as described by Roehl and Semler (21), with
the modification that all transcription reactions were performed in the
presence of 1 mM m7GpppG cap analog. All
transcripts were purified by phenol/chloroform extraction, twice
precipitated with ammonium acetate/ethanol, and desalted using spin
column chromatography. RNAs were quantitated by
spectrophotometry.
In Vitro Translations in the Presence of Extracts from HeLa
Cells--
Translations were performed in 50% (v/v) rabbit
reticulocyte lysate and 40% (v/v) ribosomal salt wash from HeLa cells.
Ribosomal salt wash was prepared from uninfected and
poliovirus-infected HeLa cells as described by Brown and Ehrenfeld
(22). All translation reactions (total volume, 20 µl) were programmed
with 500 ng of in vitro transcribed RNA and incubated for
2 h at 30 °C. LUC and CAT activities were determined as
described above.
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RESULTS |
The 5'-UTR of mKv1.4 Mediates IRES-dependent
Translation Initiation in Mammalian Cells--
To test for the
presence of an IRES in the 5'-UTR of mKv1.4, we introduced
this sequence into the intercistronic spacer of a bicistronic vector
(20) containing two reporter proteins, CAT and LUC, present on a single
transcript under the control of the SV40 promoter. Translation of CAT
occurs by the 5' cap-dependent ribosome scanning mechanism,
whereas efficient translation of LUC, on the other hand, occurs only if
the intercistronic spacer contains a sequence with IRES activity that
allows internal binding of ribosomes. These constructs were transfected
into NIH-3T3 or COS-1 cells, and CAT and LUC activities were measured
64-72 h post-transfection. CAT activity served as an indicator of
transfection efficiency, and results (determined in triplicate) for
each experimental condition are displayed as the ratio of LUC to CAT
activity.
Representative results of such transfections are shown in Fig.
2. The control vector lacking the
mKv1.4 5'-UTR sequence (Fig. 2, line 1) shows
some LUC activity in NIH-3T3 cells, possibly because of leaky
translation termination at the CAT stop codon and ribosomal
read-through (11). To reduce this background activity, we inserted a
stable hairpin structure downstream from the CAT stop codon, as
indicated in line 2 of Fig. 2. This structure has a
predicted free energy of
41 kcal/mol and reduces translational read-through to negligible values (Fig. 2, line 2). All
subsequent studies of the IRES were carried out using constructs
containing this stem-loop. In the presence of the 1.2-kb 5'-UTR of
mKv1.4 (Fig. 2, line 3), LUC/CAT activity is
increased to nearly 400, indicating that this sequence possesses IRES
activity. A construct containing only the 3'-most 200 nt of the 5'-UTR
(Fig. 2, line 4) also shows activity comparable with that of
the 1.2-kb fragment, suggesting that IRES activity is largely contained
within this region. Neither the 1.2-kb nor the 200-nt fragments exhibit
substantial IRES activity when cloned into the SVsl vector in the
antisense direction (Fig. 2, lines 5 and
6), confirming the sequence specificity of this activity.
Similar results were obtained when transfections were carried out in
COS-1 cells (data not shown).

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Fig. 2.
IRES activity of the 5'-UTR of mKv1.4 in
NIH-3T3 cells. Results are shown as the ratio of LUC/CAT activity
following transfection of bicistronic SV40 promoter-containing
constructs into NIH-3T3 cells. Line 1, vector with no
insert. Line 2, vector with stable stem-loop insert alone
(Vsl); all subsequent constructs contain this stem-loop. Line
3, Vsl with 1.2 kb mKv1.4 5'-UTR, forward (sense)
orientation. Line 4, Vsl with 200-nt 3'-proximal fragment of
mKv1.4 5'-UTR, sense orientation. Lines 5 and 6,
same as lines 3 and 4 but with inserts in reverse
(antisense) orientation.
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IRES Activity in mKv1.4 Is 5'
Cap-independent--
IRES-dependent translation initiation
is expected to be 5' cap-independent, and extracts from
poliovirus-infected cells have been used to suppress
cap-dependent translation while not affecting IRES activity
in a bicistronic expression vector (23). In poliovirus-infected cells,
viral mRNA is translated efficiently, whereas translation of host
mRNA is severely inhibited due to the proteolytic cleavage of the
eIF-4G (formerly called p220 or eIF-4
) subunit of the cap binding
complex eIF-4F (24) by viral proteinase 2A. The only cellular mRNAs
translated in poliovirus-infected cells are those that use a 5'
cap-independent internal ribosome entry mechanism. If the
mKv1.4 5'-UTR does indeed encode an IRES, its translation should be 5' cap-independent, and translation initiation should be
unaffected by the presence of extracts from poliovirus-infected cells.
We inserted the 1.2-kb fragment of the 5'-UTR of mKv1.4 into
the intercistronic spacer of a bicistronic construct similar to the one
used in the transfection experiments, but with transcription directed
by the bacteriophage T7 promoter. The stem-loop sequence described
above was inserted downstream of the CAT stop codon to reduce leaky
translation termination and ribosomal read-through. Bicistronic
mRNAs were synthesized in vitro using T7 RNA polymerase in the presence of the m7GpppG 5' cap analog. RNAs were
then added to a rabbit reticulocyte lysate containing 40% ribosomal
salt wash from either poliovirus-infected or uninfected HeLa cells. In
the presence of extracts from uninfected HeLa cells, the control vector
RNA lacking the 1.2-kb mKv1.4 sequence showed detectable CAT
activity but little or no LUC activity, leading to a low LUC/CAT
ratio (Fig. 3A). Insertion of
the 1.2-kb mKv1.4 5'-UTR resulted in a significant
increase in the LUC/CAT ratio, suggesting that the mKv1.4 5'-UTR was
able to efficiently direct translation of the downstream cistron via an
internal ribosome entry mechanism. Insertion of the mKv1.4 5'-UTR into
the intercistronic spacer in the antisense orientation displayed
considerably reduced levels of IRES-like activity (Fig. 3A)
compared with those detected in the sense orientation.

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Fig. 3.
IRES activity in rabbit reticulocyte
lysates. T7 promoter-containing bicistronic vector contains
stem-loop (see Fig. 2). A, reaction mixtures supplemented
with extracts of control HeLa cells. The results are expressed as the
ratio of LUC/CAT activity. Bars (from left): no
added RNA; vector alone; 1.2-kb 5'-UTR of mKv1.4 in sense
(1.2-kb-s) or antisense (1.2-kb-as) orientation.
B, reaction mixtures supplemented with extracts of
poliovirus-infected HeLa cells. The results are expressed as the
percentage of maximum activity seen in the assay (1.9 × 103 units for CAT, and 1.8 × 106 for LUC;
see text). The bars are labeled as in A.
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When extracts from poliovirus-infected HeLa cells were added to the
in vitro translation reactions, dramatic effects were seen
on the translation of the upstream cistron. Because CAT activity was
reduced to nearly background levels in the presence of extracts from
poliovirus-infected HeLa cells, the ratio of LUC/CAT is not meaningful
in evaluating IRES activity. Thus, the data shown in Fig. 3B
were expressed as CAT or LUC activity in extracts from infected cells
relative to the maximum values obtained when translations were carried
out in the presence of extracts from uninfected cells (1.9 × 103 units for CAT, and 1.8 × 106 for
LUC). The data reveal a dramatic suppression of
cap-dependent translation (CAT activity) compared with
cap-independent translation (LUC activity) when translations were
carried out in the presence of extracts from poliovirus-infected cells.
Although ~20% of maximum LUC expression is retained, CAT activity is
reduced to ~0.3% of its maximum. Fig. 3B also shows that
this remaining LUC activity is dependent on the presence of the
mKv1.4 5'-UTR and that the LUC signal with the insert in the
antisense direction is very low. These data show that the IRES activity
in the mKv1.4 5'-UTR is 5' cap-independent.
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DISCUSSION |
Translation initiation by internal ribosome entry was first
demonstrated for picornaviruses (23, 25) and has since been shown for
other RNA viruses (26, 27). In addition, although the precise mechanism
for internal ribosome loading is not yet understood, at least one
picornavirus IRES has been shown to initiate translation in a
completely 5' end-independent manner (28). However, only a few cellular
genes are known to be translated in this manner, including
Antennapedia in Drosophila (29), and Bip (30),
fibroblast growth factor 2 (31), and c-myc (32) in mammalian
cells. Thus, mKv1.4 represents one of a limited number of
mammalian genes known to contain a functional IRES. The 200-nt stretch
in mKv1.4 containing IRES activity shares two important characteristics with known IRES elements from RNA viruses, namely a
polypyrimidine tract (Fig. 1A) and extensive
computer-predicted RNA secondary structures (Fig. 1B) that
might be targeted by trans-acting cellular factors necessary
for the formation of the 40 S pre-initiation complex. Indeed,
trans-acting cellular factors corresponding to known
cellular RNA binding proteins have been shown to have functional interactions with IRES elements of picornavirus and hepatitis C virus
(33-40). Known IRES elements have also been reported to have complex
secondary structures (41), and stem-loop elements within these regions
are thought to contain motifs necessary for IRES activity (42, 43).
Computer-predicted models of the 200-nt mKv1.4 IRES-containing region
predict a similarly complex structure with multiple hairpin loops (Fig.
1B). Because the 5'-UTR of mKv1.4 is
significantly longer (~1.2 kb) than that of the average mammalian gene, and contains multiple (eighteen) abortive upstream AUGs, the IRES
present in the 200-nt stretch might bind cellular factors that
ultimately enable the ribosome to efficiently translate the channel
protein from the authentic translation initiation site. In support of
this possibility, we have obtained preliminary data from
electrophoretic mobility shift experiments showing specific binding of
cellular proteins to the mKv1.4
IRES.2
IRES Control of Translation May Be a Common Mechanism Utilized by
Kv Channel Genes--
Our demonstration of IRES activity in the 5'-UTR
of mKv1.4 may account for its normal translation despite the
presence of several unusual features (length, abortive AUGs, and stable
secondary structure) that are known to inhibit 5'
cap-dependent ribosome scanning. We have also previously
shown (4) that AUUUA motifs present in the long 3'-UTR of Kv1.4 can
significantly inhibit translation, presumably by affecting translation
initiation, and that alternate use of polyadenylation sites plays a
role in regulating this process.
The UTRs of many other mammalian Kv channel transcripts also show
strikingly different structures from the typical vertebrate pattern.
Inspection of Fig. 4 shows that there is
no Kv transcript known to have either of its UTRs as short
as the typical mean values, and most are considerably longer. It should
be noted that lengths of these UTRs represent only lower limits,
because the ends of many of these transcripts have not yet been mapped
(including, for example, both of the long UTRs of Kv1.1), and many
longer transcripts have yet to be characterized at all (e.g.
>9-kb transcripts of Kv1.1 and Kv1.3). Fig. 4 also illustrates the
presence of multiple upstream AUGs in the 5'-UTRs and of AUUUA motifs
in the 3'-UTRs of many Kv transcripts. The UTRs of Kv genes are not
only strikingly unusual with respect to their lengths and their
containing these unusual motifs, but as has been pointed out by us and
others (see Ref. 2), their sequences are considerably more highly
conserved than in most other mammalian genes, lending further support
to the notion that they have been conserved over evolutionary time for
some important function(s).

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Fig. 4.
Lengths of 5'- and 3'-UTRs of mammalian
voltage-gated channel transcripts, showing the presence of 5' AUGs and
3' AUUUAs. The ruler at the top indicates
the lengths of the UTRs represented below, and the two downward
arrowheads on this ruler mark the mean length of
vertebrate UTRs (see text), ~150 nt for the 5' and ~500 nt for the
3'-UTRs. Short vertical bars on each UTR mark the location
of AUG (in the 5'-UTRs) or AUUUA (in the 3'-UTRs), and upward
arrowheads indicated alternate end points due to alternate
transcription start sites (5' ends) or use of alternate polyadenylation
signals (3' end). The termini of those transcripts for which the ends
of the mRNA have been experimentally determined are indicated by
solid circles; UTRs lacking this symbol represent minimum
estimates of their true length. The dotted line in the
5'-UTR of Kv1.5 represents a sequence that was not available for
analysis. The dotted line in the 5'-UTR of Kv3.1 represents
a region that is removed by alternate splicing. CDS
represents the coding region for these mRNAs (not drawn to
scale).
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Thus, translational control by internal ribosome entry may be important
not only for Kv1.4 but may represent a mechanism more generally
utilized for regulation of gene expression by the extended family of Kv
genes. In fact, despite the more limited data available for the UTRs of
other families of potassium selective channels, it is not difficult to
find examples of transcripts whose UTRs share the unusual features we
have described in Kv1.4. The mouse calcium-activated K+
channel slowpoke (44), for example, has at least three
abortive AUGs in its >820-nt 5'-UTR and three AUUUA motifs in its
>627-nt 3'-UTR. Likewise, the recently described transcript of the
human inward rectifier hGIRK1 (45) has no fewer than 12 AUGs
in its >1360-nt 5'-UTR and five AUUUA motifs in its 314-nt 3'-UTR.
Translation initiation through the use of internal ribosome entry and
its regulation by 3' elements may therefore be a significant factor in
the regulation of expression not only of Kv1.4 and its voltage-gated cousins but also of other members of the extended family of
K+-selective ion channels.
We thank Peter Sarnow for kindly providing the
SV40 and T7 promoter-containing bicistronic vectors.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U03722 and U03723.