From the Department of Biochemistry and Biophysics,
Texas A&M University, College Station, Texas 77843-2128 and the
§ Center for Cancer Biology and Nutrition, Institute of
Biosciences and Technology, Texas A&M University System Health Science
Center, Houston, Texas 77030-3303
Received for publication, July 12, 2000, and in revised form, September 20, 2000
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ABSTRACT |
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Exons IIIb and IIIc of the FGFR2 gene are
alternatively spliced in a mutually exclusive manner in different cell
types. A switch from expression of FGFR2IIIb to FGFR2IIIc accompanies
the transition of nonmalignant rat prostate tumor epithelial cells (DTE) to cells comprising malignant AT3 tumors. Here we used
transfection of minigenes with and without alterations in reading frame
and with and without introns to examine how translation affects
observed FGFR2 splice products. We observed that nonsense mutations in other than the last exon led to a dramatic reduction in mRNA that is abrogated by removal of downstream introns in both DTE and AT3
cells. The mRNA, devoid of both IIIb and IIIc exons (C1-C2), is a
major splice product from minigenes lacking an intron downstream of the
second common exon C2. From these observations, we suggest that
repression of exon IIIc and activation of exon IIIb inclusion in DTE
cells lead to the generation of both C1-IIIb-C2 and C1-C2 products.
However, the C1-C2 product from the native gene is degraded due to a
frameshift and a premature termination codon caused by splicing C1 and
C2 together. Derepression of exon IIIc and repression of exon IIIb lead
to the generation of both C1-IIIc-C2 and C1-C2 products in AT3 cells,
but the C1-C2 product is degraded. The C1-IIIb-IIIc-C2 mRNA
containing a premature termination codon in exon IIIc was present, but
at apparently trace levels in both cell types. The nonsense-mediated
mRNA decay pathway and cell type-dependent rates of
inclusion of exons IIIb and IIIc result in the mutually exclusive
expression of FGFR2IIIb and IIIc.
The FGF1 signal
transduction system is ubiquitous in multicellular organisms and
mediates communication between cell compartments during development and
in adult tissues (1-3). The system comprises activating FGF, heparan
sulfate proteoglycan, and transmembrane receptor tyrosine kinase.
Functional diversity and tissue specificity of the FGF signaling system
results from combinations of at least 22 genetically distinct
homologues of FGF polypeptides, heterogeneity of heparan-sulfate
oligosaccharide chains in FGFR proteoglycans, and alternative splicing
of the FGFR kinase genes. The best characterized example of regulated
alternative pre-mRNA splicing with high biological impact in the
FGFR family is the cell type-specific, mutually exclusive expression of
FGFR2 mRNAs containing either exon IIIb or IIIc from the FGFR2 gene
(4-11). In parenchymal tissues, which exhibit epithelial and stromal
compartments, only FGF receptor 2IIIb (FGFR2IIIb) is expressed in the
epithelial cells and recognizes activating FGF-7 or FGF-10, which are
expressed only in the stromal compartment (1, 3, 11-14). In contrast,
only FGFR2IIIc, which cannot recognize FGF-7 or FGF-10, is expressed in
stromal cell types (4, 14-19). Paracrine signaling from FGF-7/FGF-10
in the stroma to FGFR2IIIb in the epithelium has been implicated in the maintenance of homeostasis between compartments and has an overall effect of limiting cell proliferation and maintaining cell
differentiation (11, 16, 17). The loss of FGFR2IIIb activity severs the epithelial cells from the controlling signals of the stroma. A loss of
expression of FGFR2IIIb in epithelial cells concurrent with a switch to
exclusive expression of FGFR2IIIc has been observed during the
progression of transplantable rat prostate tumors from a nonmalignant
(DT) to a malignant (AT) phenotype (4, 14, 16, 17). The switch from
exclusive expression of exon IIIb- to IIIc-containing mRNA in
epithelial cells appears to be clonal, unidirectional, and irreversible.
Cell type-specific transfection of FGFR2 minigenes and trace analysis
of mRNA products by the PCR has been employed by others to screen
for cis-acting sequences and trans-acting factors
that are involved in cell type-specific alternative splicing of FGFR2 pre-mRNA (5-8, 20). These studies have employed diverse minigene constructs in which partial FGFR2 or viral cDNAs flank the genomic sequence containing exons IIIb and IIIc. The studies noted that transient or permanent transfection and the reading frame of the constructs affected apparent exon inclusion, which complicated interpretation of results (6). In this study, we examined in detail how
the translational reading frame affects the alternative splicing of the
FGFR2 pre-mRNA and the stability of the spliced FGFR2 mRNA.
Experimental minigenes were constructed from FGFR2 sequences and gave
rise to translatable mRNAs containing either an artificial last
exon harboring the termination codon or an upstream exon harboring a
premature termination codon (PTC). Modes of transfection and analysis
of mRNA products by the reverse transcription-polymerase chain
reaction (RT-PCR) and ribonuclease protection assay (RPA) were
compared. Our results confirm those of others that suggested that
inclusion and exclusion of both exons IIIb and IIIc can occur during
pre-mRNA splicing in addition to inclusion of only one or the other
(5). Although results based solely on PCR analysis could be interpreted
as PTC-dependent shifts in exon recognition during FGFR2
pre-mRNA splicing, the collective results after quantitative analysis suggested that the effect of a PTC was simply to depress the
particular transcript containing the PTC. The results suggest that the
mutually exclusive expression of only FGFR2 exon IIIb and IIIc is a
result of cooperation between cell-specific splice-site regulation
(5-8) and nonsense-mediated decay (NMD) of spliced mRNA products
containing a PTC (21-24).
Construction of Functional FGFR2 Minigenes with Nonsense and
Frameshift Mutations--
Minigenes were constructed by generating
fragments of rat genomic DNA in the PCR followed by ligation with the
luciferase gene into the pcDNA3.1/Zeo+ vector (Invitrogen,
Carlsbad, CA). FGFR2 cDNA fragments were then inserted 5' and 3' to
the rat genomic DNA such that following pre-mRNA splicing, the
constructs would encode a functional membrane-bound FGFR2 extracellular
domain fused to an intracellular luciferase. These constructs are
preceded by the designation, "M." cDNA constructions identical
to the minigenes, except for the absence of introns, are preceded by
the designation, "C." The pcDNA3.1/Zeo+ vector contains a
cytomegalovirus promoter and an SV40 polyadenylation signal, but codes
for no Kozak consensus sequence for translational initiation. The
natural FGFR2 ATG was utilized as the translational start site.
Minigenes and predicted products are summarized in Fig. 1 (A
and B).
Rat FGFR2 genomic DNA was obtained from the prostate tissue of F344
Fisher rats by standard methods. The PCR was carried out in a final
volume of 100 µl using 1 µg of genomic DNA with reaction conditions
specified in the LA PCR Kit (PanVera, Madison, WI). All products of the
PCR were sequenced.
Cell Culture--
Stock cultures of cloned cell lines from the
Dunning R3327PAP and R3327AT3 tumors were prepared and maintained by
previously described methods (14).
Mammalian Cell Transfections--
Liposomes were prepared
according to manufacturer protocols (Life Technologies, Inc.,
Gaithersburg, MD). Transfection conditions were optimized for: cell
density, liposome reagent, liposome volume, DNA amount, time of
liposome incubation, and time after transfection by varying the
conditions and assaying for luminescence following transfection of the
pGL-3 Control vector (Promega). Cells (5 × 105) were
seeded in 25-cm2 (T25) flasks for the RT-PCR analysis.
Cells (1.5 × 106) were seeded in 75-cm2
(T75) flasks for the ribonuclease protection assays. 5 µg of DNA
vector along with 15 µl of CellFectin (Life Technologies, Inc.) was
used for transfecting DTE cells, whereas LipofectAMINE (Life
Technologies, Inc.) was used for transfecting AT3 cells. DNA (5 µg)
was mixed with 15 µl of liposomes in a total volume of 100 µl of
serum-free RPMI 1640/Dulbecco's modified Eagle's medium for a
total of 45 min for cells in T25 flasks. DTE and AT3 cells were
transfected and analyzed for luminescence 48-h post transfection.
Liposome-DNA complexes were incubated with all cell types for 4 h
at which time the serum-free medium was exchanged for serum-containing
media. Cells were analyzed for luminescence, FGF binding, or by RPA or
RT-PCR analysis 48-h post transfection. Volumes were tripled for cells
in T75 flasks. Following transfection of the pGL-
Stably transfected cell lines were transfected by the same optimized
methods. Following transfection with the Zeocin (Invitrogen) resistant
minigenes for 3 days, cells were detached, subcultured, and reseeded at
a density of 5 × 105 cells/T25 flask and selected in
the presence of 400 µg/ml Zeocin as per manufacturer's instructions
for 2 weeks. Stable cell lines were thereafter maintained in the
presence of 100 µg/ml Zeocin.
Analysis of FGF Binding--
Sources, recombinant production,
iodination, and recovery of FGF-1, FGF-2, and FGF-7, as well as
conditions for measuring specific and nonspecific binding and covalent
affinity cross-linking to FGFR, have been described in detail
previously (25, 26).
RT-PCR and Restriction Enzyme Analysis of Minigene Expression
Product--
Total RNA was isolated from transfected rat prostate
tumor cells using the Ultraspec RNA isolation reagent (Biotecx,
Houston, TX) per the manufacturer's instructions. Total RNA (5 µg)
was used for generation of cDNA template by reverse transcription with a random 6-mer as primer in a total reaction volume of 25 µl. A
5-µl portion was used for subsequent PCR reactions. The PCR was
performed at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for 40 cycles. Along with 20 µl of the PCR mixture, 2 µl of
React 2 buffer (Life Technologies) was added along with the restriction
enzymes AvaI, EcoRV, or SacII for
restriction analysis and were incubated for 1 h. This method
reproducibly digested the products to completion. Each transient
transfection, RT-PCR, and restriction enzyme analysis was carried out
at least three times, and the figures displayed are a single
representative of the three. RT-PCR and restriction analysis of
products from stably transfected cell cultures were repeated at least
three times. The figures displayed are a single representative of three reproductions.
Ribonuclease Protection Analysis of Minigene Products--
Total
RNA was isolated as described above and subjected to ribonuclease
protection using the Hybspeed RPA kit (Ambion, Austin, TX) using the
following probes transcribed with the Maxiscript kit (Ambion) according
to the manufacturer's suggested procedures. All probes were
radiolabeled with [ Nucleotide Sequence Accession Number--
Sequences of minigenes
have been submitted to GenBankTM (accession number AF147757).
Expression of Minigenes Containing Exons in-Frame with the FGFR2
Translational Initiation Site or with PTC--
FGFR2 minigenes and
cDNAs used in the study are summarized in Fig.
1A. Minigenes were designed
using rat genomic DNA, employed the natural FGFR start codon, encoded
the entire extracellular and transmembrane domains of FGFR2, and are
predicted to yield FGF-binding transmembrane receptors following
translation. Minigenes were also fused with the coding sequence for
luciferase at the COOH terminus of full-length expression products.
Initially, four minigenes were constructed. MWt contained no mutations.
MIIIb-stop contained mutations that caused a PTC in exon IIIb.
MIIIb-frsh contained an insertional mutation that caused a
reading-frame shift within exon IIIb. The shift resulted in a
termination codon in exon C2 of the C1-IIIb-C2 product but also
resulted in stop codons (PTCs) in exon IIIc of any C1-IIIb-IIIc-C2
transcripts. MIIIc-stop contained mutations causing a PTC in exon IIIc.
FGFR2 cDNAs (CIIIb and CIIIc) were constructed and employed as
controls for the expression of minigenes in the absence of pre-mRNA
splicing. Additionally, a construct, MIIIc-1bpstop, containing a
single-base pair mutation that generated a PTC in exon IIIc, was
constructed (Fig. 1A).
The predicted cell type-specific translation products of the minigenes
are illustrated in Fig. 1B. Functionality of minigene constructs from transcription to protein product was first validated prior to use in prostate DTE and AT3 epithelial cells by transient transfection and subsequent measurement of cell surface FGF binding to
COS monkey kidney cells. [125I]FGF-1 binding was used to
assess binding to all FGF receptor products, whereas
[125I]FGF-7 binding was used to assess binding
specifically to FGFR2IIIb (11, 14). COS cells transfected with all
minigenes and cDNAs except those with a PTC in exon IIIc exhibited
a 3- to 5-fold increase in [125I]FGF-1 binding over
untransfected cells (Fig. 2A).
COS cells transfected with all minigenes except those with a stop codon or frameshift in exon IIIb exhibited a 5- to 15-fold increase in the
binding of FGF-7 (Fig. 2B). Covalent affinity cross-linking confirmed that the binding reflected formation of specific radiolabeled FGF-1·FGFR complexes of expected molecular mass of about 133 kDa (Fig. 2C). A separate analysis of luciferase activity from
cells transfected by the minigenes yielded insufficient differences between constructs with and without upstream nonsense mutations to be
of utility in monitoring cell type-specific alternative splicing.
Luciferase activity was subsequently used to monitor transfection
efficiency among minigenes and different cell types. Once minigenes
were validated as capable of producing sufficient levels of translation
product in COS cells, expression was then examined at the mRNA
level in DTE and AT3 cells, which mutually exclusively express
FGFR2IIIb or FGFR2IIIc, respectively (5, 14).
We first screened for the presence of the four possible mRNA
products of the minigenes, C1-IIIb-IIIc-C2, C1-IIIb-C2, C1-IIIc-C2, and
C1-C2, by RT-PCR using paired primers to C1 and C2. Expression was
examined with a 5'-primer specific for the upstream exon common to all
FGFR2 cDNAs and a 3'-primer specific for the luciferase sequence, a
downstream sequence common to minigene-derived cDNAs, to amplify
only minigene products. Restriction enzymes AvaI and EcoRV, which recognize sequences in FGFR2 exon IIIb and
IIIc, respectively, were used to distinguish between exon
IIIb-containing and exon IIIc-containing cDNAs in the PCR. Products
generated in the PCR following treatment with the two restriction
enzymes are summarized in Fig. 3.
Analysis by ribonuclease protection was subsequently employed to verify
that mRNA products indicated by the PCR were present in greater
than trace amounts and to quantify amounts of exon IIIb-C2 or IIIc-C2
products relative to other FGFR2 mRNAs. Amounts of mRNA were
standardized by an internal Expression of Exon IIIc and C1-C2 mRNAs in AT3 Cells and
Reduction in Expression of Exon IIIc-containing mRNA by a PTC in
Exon IIIc--
About 67% of cell lines derived from malignant AT3
tumors express exclusively the FGFR2IIIc mRNA, whereas the other
33% express no FGFR2 products at all (14, 16). An initial analysis by RT-PCR of mRNA from AT3 cells following transient transfection with
minigenes MWt, MIIIb-stop, and MIIIb-frsh revealed only the FGFR2IIIc
cDNA band expected of the cell type (Fig.
4). The nonsense mutation in exon IIIc in
the MIIIc-stop caused a reduction in the C1-IIIc-C2 product concurrent
with an apparent increase in the C1-C2 product devoid of both exons.
Employment of different primers complementary to C2 confirmed the
predominant expression of the C1-IIIc-C2 mRNA from MWt, MIIIb-stop,
and MIIIb-frsh, and the apparent increase in the C1-C2 product from the
MIIIc-stop (results not shown). Minor bands that were resistant to both
enzymes and correlated in size with the C1-C2 transcript were sometimes detected. Dependent on primers and conditions, faint bands appeared from MWt that correlated with the expected size of the C1-IIIb-IIIc-C2 transcript. However, we failed to detect the presence of the C1-IIIb-C2 cDNA by AvaI treatment. At first glance, these results
suggested the predominant recognition of exon IIIc by the splicing
machinery in AT3 cells and that the nonsense mutation in exon IIIc
caused an increase in the C1-C2 splice product.
To confirm results from the PCR, we employed ribonuclease protection
analysis (RPA) with IIIb-C2 (Fig.
5A)- and IIIc-C2 (Fig. 5B)-specific probes. The results revealed that the
C1-IIIc-C2 mRNA comprised 63-80% of FGFR2 mRNAs from AT3
cells. The RPA analysis confirmed that expression of C1-IIIc-C2 from
the MIIIc-stop was dramatically reduced, but yielded no evidence of a
significant increase in either the C1-IIIb-C2 or C1-C2 mRNA
products. In contrast to RT-PCR, the RPA analysis suggested that the
C1-C2 product comprised a constant 20-40% of the FGFR2 products in
AT3 cells. The comparative analysis between RPA and RT-PCR approaches
illustrate limitations in the RT-PCR analysis in estimate of minority
products when common primers across alternative splice sites are
employed. Pitfalls in interpretation of the increase in otherwise
minority products due to a PTC in the majority product in RT-PCR
analysis has been described and reviewed by others (27, 28).
Mode of Transfection Alters the Pattern of Alternative Splicing in
DTE Cells--
DTE cells, which express exclusively the IIIb isoform
of the FGFR2 receptor, were transfected transiently with minigenes MWt, MIIIb-stop, MIIIc-stop, and MIIIb-frsh. Surprisingly, when the products
were assessed by PCR, the transfection of DTE cells with all four
minigenes yielded no apparent C1-IIIb-C2 cDNA band expected of the
cell type (Fig. 6). Instead, bands
corresponding to C1-IIIc-C2 cDNA and C1-C2 cDNA, in which both
exons IIIb and IIIc were excluded, were the majority products from all
minigenes except MIIIc-stop. MIIIc-stop gave rise to predominately the
589-bp band indicative of the C1-C2 cDNA devoid of both exons.
To determine whether this unexpected pattern was a consequence of
transient transfection rather than the assay method, DTE cells were
stably transfected (see "Experimental Procedures"), and expression
products were again analyzed by RT-PCR. In contrast to the results from
transiently transfected cells, DTE cells stably transfected with the
MWt and MIIIc-stop gave rise to exclusively the expected C1-IIIb-C2
cDNA (Fig. 7). However, minigene
MIIIb-stop still gave rise to bands indicative of the C1-IIIc-C2 and
C1-C2 cDNAs, although the apparent levels of expression were
reduced. As in AT3 cells, these PCR-based results again indicated that a PTC in the respective exon that is characteristic of the cell type
potentially caused a shift to other alternatively spliced products.
The more quantitative RPA analysis was then applied to both transiently
and stably transfected DTE cells. Consistent with the RT-PCR data, the
analysis of mRNA with the R2IIIc-protection probe following
transient transfection of minigenes MWt, MIIIb-stop, and MIIIb-frsh
confirmed the unexpected expression of the C1-IIIc-C2 mRNA at
levels from 40 to 42% (Fig. 8). However,
in contrast to the analysis by PCR, which failed to indicate the
presence of the C1-IIIb-C2 mRNA characteristic of DTE cells,
analysis with the R2IIIb-protection probe revealed that the C1-IIIb-C2
product was significant. Expression of the expected C1-IIIb-C2 mRNA
from minigenes MWt, MIIIc-stop, and MIIIb-frsh, all of which had no nonsense mutation in exon IIIb, was 11, 19, and 21% of total products, respectively (Fig. 9). These results show
that expression from the minigenes in transiently transfected DTE cells
is distributed between the C1-IIIb-C2, C1-IIIc-C2, and C1-C2 products
at about 10-20, 40, and 40-50%, respectively, of total FGFR2
products if one assumes that these are collectively the significant
mRNAs from the minigenes. The apparent 11-19% decrease in the
C1-IIIb-C2 product relative to other products due to the PTC in exon
IIIb could indicate a decrease in selection of the IIIb exon during pre-mRNA splicing. Otherwise, the PTCs in either exon IIIb or IIIc
function only to decrease the respective product to 2 and 5% of the
total without effect on relative amounts of other alternative products
generated by the splicing machinery.
Consistent with the RT-PCR analysis, RPA analysis of RNA from stably
transfected DTE cells revealed that C1-IIIc-C2 mRNA was negligible
from cells transfected by all minigenes, independent of reading frame
(Fig. 10). This property is a hallmark
of the DTE cell type with respect to expression of the native FGFR2
gene and is consistent with the idea that recognition of exon IIIc is
normally strongly repressed by the splicing machinery of DTE cells.
Analysis with the R2IIIb-protection probe indicated that the C1-IIIb-C2
mRNA ranged from 55 to 61% in cells stably transfected with the
MWt, MIIIc-stop, and MIIIb-frsh (Fig.
11). This suggested that, contrary to
results from RT-PCR, DTE cells following stable transfection of
minigenes without a nonsense codon in exon IIIb generated C1-IIIb-C2
and the C1-C2 mRNA variants in a 60/40 ratio (Fig. 11). In sum, the
C1-C2 mRNA appears constant at about 40% of total splice products
independent of the mode of transfection of DTE cells, whereas the mode
of transfection affects the relative amounts of the alternative
C1-IIIb-C2 and C1-IIIc-C2 products generated by the splicing machinery.
The results are consistent with the saturation and neutralization of a
repressor of exon IIIc inclusion in DTE cells by the high levels of
minigene expression per cell in transient transfections relative to the
homogenous and presumably lower expression per cell in stably
transfected cell lines (5).
Finally, in contrast to results by RT-PCR, which suggested that the
C1-IIIb-C2 mRNA was absent from DTE cells stably transfected by
MIIIb-stop, RPA revealed that the exon IIIb-containing mRNA comprised 26% of the total FGFR2 mRNA. The fact that no change in
the level of C1-IIIc-C2 and C1-C2 products was discernible again
suggested that the consequence of the PTC in IIIb exon was destabilization of the C1-IIIb-C2 mRNA rather than alteration of
alternative splicing. This was confirmed by restoration of the
C1-IIIb-C2 mRNA to wild-type levels by mutation of a single base
pair that converted the PTC into a sense codon (MIIIb-resc) (Fig.
11).
Restoration of Open Reading Frame Restores the Expression Pattern
of PTC-containing Minigenes to That of MWt, Which Contains Normal
Exons--
A 4-bp mutation was initially employed to introduce
nonsense mutations into the normal FGFR2 reading frame (Fig. 1). A
single-base pair substitution at the site of the 4-bp mutation in
MIIIb-stop and MIIIc-stop was used to restore the reading frame in the
MIIIb-resc and MIIIc-resc constructs. Analysis by RT-PCR indicated that
both the MIIIb-resc and MIIIc-resc minigenes (Fig.
12) exhibited the cell-specific
expression pattern characteristic of the MWt minigene (Figs. 4 and 7).
RPA analyses confirmed that the 1-bp substitutional mutation in most
cases restored levels of mRNA to that of the MWt minigene with
unmutated exons (Figs. 8, 9, and 11). These results strongly suggested
that the 4-bp mutation in the respective minigene acted by generating
PTCs, which elicit degradation of the mRNA product rather than
alteration of exon recognition and disruption of a splicing
enhancer.
To eliminate the possibility that the results of the 4-bp mutation were
due to creation of a splicing silencer (the TTAATTAA sequence), two
additional minigenes were constructed (Fig. 1). One construct,
MIIIc1bp-stop, was examined, which contained a single 1-bp substitution
in exon IIIc that converted a TCA to TGA. In the second construct,
MIIIc1bp-resc, the TGA was mutated to TTA to restore the open reading
frame. Following transient transfection of the minigenes into DTE cells
and analysis by RPA with the IIIc-protection probe, the relative levels
of C1-IIIc-C2 and the C1-C2 mRNAs were examined (Fig.
13). Similar to MIIIc-stop, MIIIc1bp-stop caused a significant reduction in relative amount of the
C1-IIIc-C2 mRNA compared with the 41% level of C1-IIIc-C2 mRNA
exhibited by the MWt. Restoration of the reading frame in MIIIc1bp-resc
restored the relative abundance of the C1-IIIc-C2 product to that of
MWt. The fact that the single-base pair substitution generating a PTC
caused a reduction in the level of C1-IIIc-C2 mRNA, whereas another
single-base pair mutation had no effect, strongly suggests that
nonsense-mediated destabilization of the mRNA occurred rather than
creation of a spicing silencer by the TTAATTAA sequence.
Nonsense Mutations Do Not Affect Levels of Nonspliced FGFR2
mRNA Expressed from an Intronless cDNA--
To determine
whether the reduction in levels of exon IIIb or IIIc mRNA
containing a PTC was dependent on splicing of pre-mRNA, we examined
the levels of the C1-IIIc-C2 mRNA in cells transfected with an
FGFR2 cDNA (CIIIc-stop) containing the same PTC-generating 4-bp
mutation employed in the minigene MIIIc-stop. By RPA analysis, no
difference in stability of the C1-IIIc-C2 mRNA could be detected between AT3 or DTE cells transfected with either the CIIIc-stop or
CIIIc cDNAs (Figs. 5, 8, and 9). This indicated that pre-mRNA splicing is required for the destabilizing effect of the PTC-generating mutations.
Inclusion of Both IIIb and IIIc Exons Can Be Detected by PCR Using
Paired Primers Specific for Exons IIIb and IIIc--
To date,
strategies using PCR primers common to alternative splice products of
the native FGFR2 gene have failed to report either the C1-IIIb-IIIc-C2
or the C1-C2 products. Using the same strategy, we clearly demonstrated
conditions under which the C1-C2 mRNA can be detected in both DTE
and AT3 cells at up to 40% of products by use of our artificial
minigenes where the termination codon in FGFR2 exon 10 (C2) is in the
last exon. In contrast, neither the PCR nor the RPA analyses that were
employed suggested the presence of the C1-IIIb-IIIc-C2 from the
minigenes under the conditions and cell types tested. Yet the product
has been detected by others using minigenes designed without regard to
reading frame or use of intact FGFR2 C1 and C2 cDNAs in the
constructions (5, 9). To specifically test for the C1-IIIb-IIIc-C2
product, which is presumably absent or expressed at very low levels
from our minigenes, we employed 5'- and 3'-primers complementary to
specifically exons IIIb and IIIc, respectively. Surprisingly, the
paired exon IIIb and IIIc primers revealed the presence of the product
in not only cells transfected by the MWt minigene, but also in
untransfected cells as well (Fig. 14).
This confirmed that the C1-IIIb-IIIc-C2 mRNA was present in at
least trace amounts and further confirmed the underestimation or
failure in detection of minor splice products when common primers
across the alternative sequences are employed in the PCR (27, 28).
Cell Type-dependent Expression of Exons IIIb and IIIc
Assessed by Translatable Products from FGFR2 Minigenes--
The aims
of this study were to assess cell type-dependent expression
of translatable mRNAs from minigenes comprised of FGFR2 cDNA
flanking genomic sequences containing alternative exons IIIb and IIIc,
and to examine the potential impact of reading frame on pre-mRNA
splicing or mRNA stability. Consequently, we first confirmed that
our minigenes could not only be transcribed and that the pre-mRNA
was spliced into predicted products, but also that the mRNA was
translated. This distinguished our minigene expression system from
those described previously.
The mode of transfection and the RT-PCR analysis of minigenes initially
complicated the interpretation of results in DTE cells in which the
exon IIIb-containing mRNA is exclusively expressed from the native
gene. Using PCR analysis and a different transfection host, Breathnach
and colleagues (6, 9) reported that FGFR2 minigenes lacking an open
reading frame had to be stably transfected to detect exon
IIIb-containing mRNA. The dependence of IIIb-containing mRNA
expression on transfection mode was also observed by Carstens et
al. (5) using the DTE cell host and a minigene with artificial exons flanking the FGFR2 genomic sequence. It has been suggested that
this phenomenon is due to the high rate of minigene transcription in
some cells during transient transfection, which titrates the activity
of an exon IIIc repressor (5). Consequently, the inclusion of exon IIIb
decreases as the inclusion of exon IIIc increases during pre-mRNA
splicing. A potential difference in accessibility of the pre-mRNA
to cis-acting splice factors in transiently transfected cells cannot be ruled out. In contrast, the more uniform lower expression across all cells in permanently transfected clones when the
gene is stably integrated in the genome gives rise to the expected
inclusion and exclusion of exons IIIb and IIIc, respectively, in
mRNA from the DTE cell type. However, the C1-C2 product devoid of
both exons appeared to be the majority product in DTE cells as well as
a significant product in AT3 cells even after correction for the
shortcomings of the PCR analysis and variations in mode of
transfection. In toto, our results are consistent with the presence of a strong exon IIIb activator (or derepressor) and exon IIIc
repressor in normal and premalignant epithelial cells (5). The DTE
cells are a prototype of nonmalignant epithelial cells from prostate
tumors in which the putative trans-acting factor is lost
during and may contribute to progression to the malignant AT3 tumor
phenotype. Our results raise the possibility that the C1-C2 transcript
in which both exons are excluded may be a constitutive product that
comprises 35-50% of products created following splicing of the FGFR2
pre-mRNA. The counterpart of the C1-C2 product is not significantly
expressed in untransfected cells from the native FGFR2 gene. Our
minigene differs from the native gene in that the termination codon
generated by exclusion of both exons IIIb and IIIc is in the last exon
of the resultant mRNA. This is in contrast to the premature
termination codon (PTC) in exon 10 (out of 19) that arises from the
native gene. Therefore, a mechanism must exist to reduce the
translatable native C1-C2 as well as other aberrantly spliced FGFR
transcripts that would result in an unproductive or potential
dominant-negative fragment of an FGFR.
Impact of Nonsense Mutations Causing a PTC on Observed FGFR2
mRNA Products--
According to RT-PCR analysis, under all
conditions where they were present, both exon IIIb-containing and exon
IIIc-containing mRNA products were quenched when nonsense mutations
were introduced into a coding sequence for the respective exons in the
minigenes. It was not apparent whether alterations in pre-mRNA
splicing or stability of spliced mRNA was affected, because
alternative transcripts that were otherwise very low or undetectable
appeared in the PCR mixtures when the major product was reduced by
mutation. However, the RNase protection analysis revealed that the
major effect of the PTC-generating mutations was to depress the
respective product containing the PTC rather than to shift exon
recognition at the level of pre-mRNA splicing. Sequential
alteration of a single base at the same site that disrupted or
maintained the reading frame was sufficient to decrease or have no
effect on the expression of specific transcripts, respectively. This
further confirmed that PTCs impact mRNA stability rather than exon
recognition. However, pre-mRNA splicing was necessary for the
PTC-dependent reduction in mRNA products. An intronless
minigene, CIIIc-stop, containing an identical stop codon to MIIIc-stop,
gave rise to mRNA that was of equal abundance to that from the
unmutated intronless cDNA (CIIIc).
The NMD Pathway Cooperates with Cell Type-specific Pre-mRNA
Splicing to Ensure Cell-specific Expression of mRNAs Containing
Only Exon IIIb or IIIc--
Taken together, our observations on
expression of FGFR2 products are consistent with proposed mechanisms
for quality control of mRNAs by the nonsense-mediated decay (NMD)
pathway in metazoans. This mechanism is thought to act through the
tagging of exon-exon junctions during nuclear pre-mRNA splicing
possibly by hnRNP-like proteins. These proteins are then recognized,
either at the nuclear-cytosolic boundary or in the cytosol, by the
translation apparatus. A part of the translational machinery then scans
the mRNA for intact reading frames in relation to the exon-exon
junctions and elicits degradation of mRNAs that exhibit a PTC
upstream of an exon-exon junction (29). NMD is thought to have evolved
to select mRNAs with an intact reading frame from a pool of splice
combinations generated by the splicing machinery (30, 31). This
protects against expression of aberrant protein products due to
nonsense mutations or mistakes by the splicing machinery, particularly those giving rise to truncated products that would perturb activity of
multimeric complexes such as FGFR (32-34).
The NMD mechanism necessarily must distinguish between a PTC and a stop
codon in the last exon with respect to eliciting mRNA decay. This
exception explains why the C1-C2 mRNA from which both exons are
excluded is a significant product from our minigenes, but not from the
native FGFR2 gene. Similarly, we showed that the mRNA containing a
frameshift mutation in exon IIIb that also resulted in a stop codon in
the last exon of our minigene was not subject to decay. The counterpart
of the C1-C2 product from the endogenous FGFR2 gene results in a stop
codon in the third codon following the C1-C2 junction. Because native
exon C2, which is exon 10 out of 19 within the native FGFR2 gene, is
not the last exon, the stop codon in exon 10 is premature and subjects the mRNA to NMD. To date, the C1-C2 mRNA excluding both exons has not been reported from the four FGFR genes in nature. Likewise, a
significant presence of the C1-IIIb-IIIc-C2 mRNA has not been reported from the native FGFR2 gene. The mRNA that includes both exons IIIb and IIIc was undetectable by RPA and generally undetectable by RT-PCR except with specifically designed primers and conditions. This mRNA is an obvious product of FGFR2 minigenes devoid of a translational initiation site and open reading frame (5,
9).2 Like the native gene,
exons IIIb and IIIc are in-frame with the native translational start
site in our minigenes. Therefore, the C1-IIIb-IIIc-C2 product exhibits
a PTC in the fifth codon in exon IIIc following the IIIb-IIIc exon
junction, which would subject the mRNA to NMD. Our results suggest
that the C1-IIIb-IIIc-C2 product is generated at the level of
transcription and splicing. However, its decay via the NMD pathway,
possibly combined with a low level of product generated by the splicing
machinery relative to the other three possibilities, results in a
functionally insignificant level of the C1-IIIb-IIIc-C2 mRNA.
In summary, our results show that the partnership between NMD and cell
type-specific regulation of inclusion of exons IIIb and IIIc at
splicing underlies the cell-specific mutually exclusive expression of
FGFR2IIIb and FGFR2IIIc mRNAs. To date, cis-acting sequence elements have been proposed, which appear to promote, derepress, or repress inclusion of exon IIIb, dependent upon cell type
(6-8). In addition, a sequence element has been characterized that
concurrently mediates both repression of exon IIIc and activation or
derepression of exon IIIb in FGFR2IIIb-expressing cells (5). The C1-C2
mRNA devoid of both exons that is subject to NMD appears to be a
constitutive default product of FGFR2 transcription. This is consistent
with the presence of constitutive repressors of inclusion of both exons
IIIb and IIIc that are overcome by cell-specific derepression
(activation). Results of preliminary experiments to rescue the
C1-IIIb-IIIc-C2 transcript by mutations, which eliminated the PTC in
exon IIIc by shifting the termination codon into C2, failed to
significantly increase the product. This suggests that generation of
the C1-IIIb-IIIc-C2 mRNA may be severely limited at splicing prior
to further degradation by NMD. The absence of repressors of both exons
in a single cell type would be a requirement for generation of
significant levels of the C1-IIIb-IIIc-C2 variant at splicing. To date,
there is no evidence for this condition that would give rise to
concurrent expression of both the C1-IIIb-C2 and C1-IIIc-C2 mRNAs
in the same cell type. However, we have observed that up to 30% of
malignant prostate tumor AT3 cell clones from cultures expressing
exclusively the FGFR2IIIc mRNA express no detectable FGFR2
transcripts (16, 17). Whether this is due to loss of transcription of
the FGFR2 gene or due to the dominance of repressors, e.g.
the loss of derepressors, for both exons IIIb and IIIc in the tumor
cells is under investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gal vector
(Promega) and staining for
-galactosidase activity following the
manufacturer's protocol, the transfection efficiency of DTE cells was
estimated to be ~15%, whereas AT3 cell transfection efficiency was
found to be ~1%.
-32P]UTP during transcription by
either T3 or T7 RNA polymerase. The FGFR2IIIb probe was constructed by
excising a 224-bp HaeIII-SacII fragment from the
CIIIb construction and ligation into pBluescript SK that was digested
with SmaI and SacII. Following digestion by
HindIII, the probe was transcribed by T3 RNA polymerase. The FGFR2IIIc probe construction was made by excising a 218-bp
EcoRV-SacII fragment from the CIIIc construct,
and following Klenow treatment, ligating it into pBluescript SK
digested by SmaI. Following linearization by
EcoRI, the construct was transcribed by T3 RNA polymerase. Rat
-actin probe was made by ligation of a 117-bp AluI
fragment from a rat
-actin cDNA into pBluescript SK digested by
EcoRV. Following linearization with EcoRI, the
construct was transcribed by T7 RNA polymerase. The predicted products
of the two probes and their location within FGFR2 minigenes are
illustrated schematically in Fig. 3. Each probe was hybridized with the
corresponding RNA sample for 10 min and incubated in the presence of
Ambion RNase A1/T1 mixture for 30 min. Protected fragments were
separated on 6% polyacrylamide-sequencing gels and subjected to
autoradiography, and the relative intensities of products was
quantitated by phosphorimaging analysis using the ImageQuaNT program.
Unless otherwise noted, RPA experiments were reproduced at least three times.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FGFR2 minigenes and predicted translation
products. A, minigenes were constructed with four exons
and three introns as indicated. Constant exon C1 was comprised of
coding sequence for the NH2-terminal signal sequence,
immunoglobulin module II and the invariant exon coding for the first
half of immunoglobulin module III (native FGFR2 exons 2, 4-7). Variant
exons IIIb (native exon 8) and IIIc (native exon 9) are denoted.
Minigene constant exon C2 was comprised of coding sequence for the
FGFR2 extracellular juxtamembrane, transmembrane domain
(TM), and part of the intracellular juxtamembrane domain of
FGFR2 (native exon 10), which was fused to the full coding sequence of
luciferase (Luc). The sequences within exons IIIb and IIIc
in which mutations were made to generate stop codons (boxed
TAA) in-frame with the normal translational start site of FGFR2
(ATG) are indicated with the changes in boldface.
The single mutated cytosine that was utilized to restore reading frame
is also indicated. Minigenes with introns are designated
"M" followed by the altered exon and consequence of the
alteration (Wt = wild-type with no alteration;
frsh = frameshift causing a downstream stop codon;
stop = stop codon at the altered site;
resc = mutational rescue of open reading frame).
Intron-less minigenes (cDNAs) were designated "C"
followed by the alternative exon IIIb or IIIc. MIIIc-1bp stop contains
a 1-bp C to G substitutional mutation changing a TCA codon to a TGA
codon (boxed). MIIIc-1bp resc contains a 1-bp G
to T substitutional mutation changing the TGA codon to a TTA codon.
B, scheme for functional analysis of cell-specific
expression of FGFR2. Top panel: minigenes MIIIb-stop and
MIIIb-frsh were designed to give rise to a functional product in AT3
cells only. Product can be measured by the following methods: FGF
binding, immunochemical reactivity, and luciferase activity.
Bottom panel: MIIIc-stop was designed to give rise to
functional product in only DTE cells.
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Fig. 2.
The binding of FGF-1 and FGF-7 to
transmembrane products of minigenes expressed in COS cells.
Plasmids bearing the indicated minigene constructs (Fig. 1) (1.5 µg
of DNA) were transfected into 150,000 COS cells per well of 24-well
plates as described under "Experimental Procedures." On day 3, 1 ng
of radiolabeled FGF-1 (A) or FGF-7 (B) was added
in 0.25 ml of serum-free RD at 37 °C for 1 h, and the
radiolabeled FGFR complexes were extracted with 1% Triton X-100 and
counted. The data presented are the mean (±S.D.) of four wells from
one experiment and are representative of three independent experiments.
C, covalent affinity cross-linking assay of FGF-1 and FGF-7
to minigene expression products. After removal of free radiolabeled
FGF, 4 µl of 100 mM DSS was added to the cells described
in A and B for 15 min at room temperature. Cells
were then extracted with 2% SDS and analyzed electrophoretically on
7.5% polyacrylamide gels under reducing conditions. Bands were
visualized by autoradiography. Mean of the indicated bands was the
expected 133 kDa.
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Fig. 3.
Analysis of mRNA products from FGFR2
minigenes by RT-PCR/restriction enzyme analysis and ribonuclease
protection. PCR analysis: Minigene products were specifically
amplified with primers R2UPCM (AAGGTTTATAGTGATGCCCA) and VL4Luc1
(GCAACTCCGATAAATAACGCGCCCAAC) or VL4Luc2 (TTCCATCTTCCAGCGGATAGA). The
latter two primers were complementary to different coding sequences for
luciferase. PCR mixtures using either primer pair were subjected to the
indicated restriction enzymes, and the products were analyzed by
agarose gel electrophoresis. The numbers 1 and 2 in the table refer to 3'-primers VL4Luc1 and VL4Luc2, respectively.
Unless otherwise noted, the data presented in the text employed
VL4Luc1, whereas VL4Luc2 was utilized for confirmation. Ribonuclease
protection: The labeled probes specific for exon IIIb and IIIc employed
in the text and the size of products resulting from hybridization with
different splice combinations are indicated. Only the FGFR2 portion of
the IIIb and IIIc probes is indicated. Both the IIIb and IIIc probes
contained flanking sequences from the pBluescript plasmid resulting in
full-length probes of 256 and 290 nt, respectively. The IIIb probe can
distinguish between exon IIIb-containing mRNAs and all other FGFR2
mRNA products. The IIIc probe can distinguish between the sum of
the exon IIIb-IIIc- and exon IIIc-containing mRNA and the sum of
the C1-C2 and exon IIIb-containing mRNA.
-actin control. Transfection efficiency,
measured as intensity of minigene bands relative to
-actin bands,
varied slightly among transfections but exon inclusion ratios did not
(data not shown). All RPAs were reproduced at least three times, unless
otherwise noted, and inclusion ratio data varied less than 5% from
transfection to transfection. Additionally, because the exon inclusion
analysis involves quantitative comparison of bands within the lanes and
not from different lanes, slight variability in transfection efficiency
will not affect the analysis. The RPA probes and products that resulted
from the four potential variants of the FGFR2 minigenes are summarized
in Fig. 3. RPA probes were validated and conditions standardized with
mRNA from cells transfected with intronless cDNAs, CIIIb and
CIIIc, which were comprised of the identical coding sequences
corresponding to the C1-IIIb-C2 and C1-IIIc-C2 products of the
minigenes. The IIIb and IIIc cDNAs yielded the expected
probe-specific bands at 224 and 218 nucleotides (nt), respectively, and
bands that were 13 and 5 nt shorter. This was presumably due to
"breathing" of the RNA duplex at the 3'-end of the constant exon C2
in both probes and sensitivity to RNase at that site. The three bands were included in the quantitative analyses.
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Fig. 4.
RT-PCR analysis of FGFR2 minigene expression
in transfected malignant prostate tumor cells (AT3). AT3 cells
were transfected with expression plasmids bearing the indicated
minigene followed by extraction of total RNA, which was
reverse-transcribed into cDNA and then introduced into PCRs using
paired primers R2UPCM and VL4 Luc1 (Fig. 3, upper panel). A
20-µl portion of the reaction mixture was treated with
AvaI or EcoRV as indicated and compared with
untreated product (N) by analysis on 1.5% agarose gels
containing ethidium bromide. Bands were visualized by fluorescence
under UV light. Excision, cloning, and sequencing confirmed that the
735-bp band encoded the C1-IIIc-C2 product and the 589-bp band
resistant to AvaI and EcoRV encoded the C1-C2
product.
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Fig. 5.
Ribonuclease protection analysis of
expression of FGFR2 minigenes in AT3 cells. Total RNA was analyzed
with the IIIb (A) or IIIc (B) probe as described
in Fig. 3 (lower panel). Total RNA was extracted from AT3
cells transfected with the indicated constructs. After incubation with
105 cpm of gel-purified IIIb or IIIc probe, the RNA was
precipitated, resuspended and then treated with ribonuclease. Protected
fragments of the probe were then separated, subjected to
autoradiography, and quantitated by phosphorimaging analysis using the
ImageQuaNT program from Molecular Dynamics. Products indicated are
described in Fig. 3, lower panel. The amount of IIIb or IIIc
product is expressed as a percentage of the sum of the respective
product and the C2 bands. Results of phosphorimaging from single
experiments are displayed to avoid breaking out single lanes. The
%IIIb-C2 or IIIc-C2 values are the mean of two experiments. The
shorter bands are due to RNase cleavage at the 3'-end of the constant
C2 sequence and were included in the densitometric quantitation.
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Fig. 6.
RT-PCR analysis of FGFR2 minigene expression
by transfection into premalignant prostate epithelial cells (DTE).
DTE cells were transiently transfected with the same minigenes and by
the same methods described in Fig. 4A. Cloning and sequence
analysis confirmed the identity of all bands except for the weak band
appearing at 400 bp following EcoRV digestion in the
MIIIb-stop lane.
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Fig. 7.
RT-PCR analysis of FGFR2 minigene expression
in DTE cells after stable transfection. Stable cell lines were
selected after permanent transfection of DTE cells as described under
"Experimental Procedures." Total RNA was isolated and analyzed as
described in Fig. 4. Cloning and sequence analysis confirmed the
identity of major bands.
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Fig. 8.
Ribonuclease protection analysis with the
IIIc probe of FGFR2 minigene expression in transiently transfected DTE
cells. The band at 185 nt from the CIIIc-stop has not been
identified, was variable, and is thought to be partially degraded IIIc
probe. The illustration presented is one of three experiments for all,
except the MIIIc-resc, which is representative of two independent
experiments. The quantitative data are the mean of all
experiments.
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Fig. 9.
Ribonuclease protection analysis with the
IIIb probe of FGFR2 minigene expression in transiently transfected DTE
cells. The illustration presented is representative of three
experiments, except MIIIb-resc, which is representative of two
independent experiments. The quantitative data are the mean of all
experiments.
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Fig. 10.
Ribonuclease protection analysis with the
IIIc probe of FGFR2 minigene expression in stably transfected DTE
cells. The band at 170 nt was residual -actin probe and was
excluded from the quantitative densitometric analysis. The illustration
presented is representative of three experiments, except MIIIb-resc,
which is representative of two independent experiments, and the
quantitation data presented are the mean of those experiments.
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Fig. 11.
Ribonuclease protection analysis with
the IIIb probe of FGFR2 minigene expression in stably transfected
DTE cells. The residual -actin probe at 170 nt was excluded
from the densitometric analysis. The illustration presented is from one
of three experiments, except MIIIb-resc, which is representative of two
independent experiments. The %IIIb-C2 for the displayed lane for
MIIIc-stop was 55%. The quantitative data are the mean of all
experiments.
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Fig. 12.
RT-PCR analysis of cells expressing
minigenes with restored translational reading frames.
A, DTE cells were transfected with minigene MIIIb-resc in
which the reading frame was restored by a substitutional mutation into
the coding sequence for exon IIIb within the MIIIb-stop, and products
were analyzed as described in Fig. 4. B, AT3 cells were
transfected with minigene MIIIc-resc in which the reading frame was
restored by a substitutional point mutation into the coding sequence
for exon IIIc within MIIIc-stop and products analyzed as described in
Fig. 4.
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Fig. 13.
Ribonuclease protection analysis with the
IIIc probe of expression of FGFR2 minigenes containing PTCs in
transiently transfected DTE cells. The control MWt contained
native exons IIIb and IIIc; MIIIc-stop contained a 4-bp mutation
causing a PTC in exon IIIc; MIIIc1bp-stop contained a 1-bp mutation in
exon IIIc causing a PTC, and MIIIc-1bprev contained a 1-bp
"revertant" mutation, which restored the reading frame in the
MIIIc1bp-stop. The data presented are one of two independent
experiments, and the quantitative data were the mean.
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Fig. 14.
Detection of the C1-IIIb-IIIc-C2 product
with paired primers specific for exon IIIb and IIIc. RT-PCR
analysis of mRNA from either nontransfected (NT) or
following transfection of MWt into DTE and AT3 cells using 5'-primer
(brescup1) (aaagaattcagcactcggggataaatagctcca) and 3' primer
(cconslo) (gagggatccgatatcccgatagaattacccgc). Products were then run on
2% agarose gel without digestion (N) or following digestion
with HpaI (H). The band at 281 bp correlates with
the predicted size of the C1-IIIb-IIIc-C2 product, whereas the bands at
169 and 112 bp are the predicted sizes of products resulting from
treatment with HpaI.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Mariano Garcia-Blanco and Russ Carstens for critical review of the manuscript, Khalid Mohamedali for helpful comments, and Lynn Maquat for advice and discussion during the course of this work.
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FOOTNOTES |
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* This work was supported by United States Public Health Services Grants DK40739 and DK35310 from the NIDDK, National Institutes of Health (NIH), and CA59971 from the NCI, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF147757.
¶ Current address: Dept. of Applied Science for Biological Resources, School of Agriculture, Gifu University, 1-1 Yanagido, Gifu City 501-1193, Japan.
Current address: Central Research Laboratories, Zeria
Pharmaceutical Co., Ltd., 2512-1 Oshikiri, Kohnan-machi, Ohsato-Gun, Saitama 360-0111, Japan.
** To whom correspondence should be addressed: Institute of Biosciences and Technology, 2121 W. Holcombe Blvd., Houston, TX 77030-3303; Tel.: 713-677-7522; Fax: 713-677-7512; E-mail: wmckeeha@ibt.tamu.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M006151200
2 R. B. Jones, F. Wang, Y. Luo, C. Yu, C. Jin, T. Suzuki, M. Kan, and W. L. McKeehan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; RT-PCR, reverse transcriptase-polymerase chain reaction; RPA, ribonuclease protection assay; nt(s), nucleotide(s); bp, base pair(s); NMD, nonsense-mediated decay; PTC, premature termination codon.
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