(Received for publication, May 25, 1996, and in revised form, August 20, 1996)
From the Section of Endocrinology, The University of
Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 and
¶ Department of Neurological Surgery, University of
Washington, Seattle, Washington 98195
Progression of astrocytes from a benign to a
malignant phenotype is accompanied by a change in the RNA processing of
the fibroblast growth factor receptor 1 (FGFR-1) gene. The level of a
high affinity form of the FGFR-1 is dramatically elevated as a result
of -exon skipping during RNA splicing. In this paper we have been
able to duplicate this tumor-specific RNA processing pathway by
transfection of a chimeric minigene containing a 4-kilobase fragment of
the human FGFR-1 gene (including the
-exon) into a variety of cell lines. In a transfected human astrocytoma cell line,
-exon skipping was consistently observed for RNA transcripts derived from both the
chimeric minigene and endogenous gene expression. This exon skipping
phenotype was dependent on the size of the flanking intron as deletions
which reduced the introns to less than ~350 base pairs resulted in
enhanced
-exon inclusion. Increased exon inclusion was not
sequence-specific as exon skipping could be restored with insertion of
nonspecific sequence. Cell-specific exon recognition was maintained
with a 375-nucleotide sequence inclusive and flanking the
-exon,
provided that intron size was maintained. These results identify the
minimal cis-regulatory sequence requirements for exclusion
of FGFR-1
-exon in astrocytomas.
There are several mechanisms known to be involved in the malignant transformation of cells. One mechanism frequently overlooked is the disruption of pathways involving regulated RNA processing. Several different genes in multiple tumor types have demonstrated alterations in their RNA splicing pathways. Alterations in RNA processing fall into two broad categories, those involving cis effects where a specific gene is mutated and those believed to be trans-related because they lack a detectable mutation. The p53, BRCA1, hMLH1, and hMSH2 genes are examples of the former (1, 2, 3, 4, 5, 6, 7). Mutations in the splice site sequences of these genes have been associated with Li Fraumeni cancer syndrome, breast and ovarian cancer, and hereditary non-polyposis colorectal cancer (1, 2, 3, 4, 5, 6, 7). Genes without definable mutations but which have RNA processing changes associated with malignancy serve primarily as tumor markers or indicators of metastatic potential. In many cases these changes are peripheral and believed to result from the transformation process rather than be a contributing factor. For example, mutations in the RET protooncogene are the initiating event in transformation of the thyroid C-cell, but one outcome of transformation is a deregulation of calcitonin/calcitonin gene-related peptide alternative RNA processing (8, 9, 10). In other genes it is possible that alterations in RNA processing pathways may specifically contribute to the initiation or maintenance of the malignant phenotype. In breast cancer, several aberrant mRNA forms are observed for the estrogen receptor gene (11, 12, 13). Exon skipping results in receptor forms without DNA-binding or ligand-binding domains. The presence of dominant negative and positive receptor forms are believed to play a key role in estrogen-dependent cell proliferation as well as chemotherapeutic intervention with anti-estrogens (11, 12, 13). Finally, the CD44 gene product is believed to play a key role in tumor cell metastasis. Products of CD44 gene created by alternative RNA splicing are associated with increase tumor invasiveness and have been shown to enhance tumorigenicity (14, 15).
The focus of this paper involves changes in the RNA splicing patterns associated with astrocyte-derived neoplasm's of the central nervous system. It is hypothesized that the genesis of these tumors occurs as a multistep progression from benign astrocytoma to anaplastic astrocytoma, and finally, to glioblastoma multiforme. In this transformation numerous and, as of now, poorly understood cytogenetic and biochemical changes take place (16). Recent studies have been directed toward defining the genes and gene products responsible for glial tumorigenesis and progression. Among the several growth factors and growth factor receptors that are activated or overexpressed in glial tumors, the fibroblast growth factors (FGFs)1 and their cognate receptors (FGFRs) are believed to play key roles in the maintenance and possibly progression of tumorigenicity (17, 18, 19, 20).
Four structurally related genes encoding high affinity receptors have been identified (21, 22, 23). Features common to members of the FGFR family include a signal peptide, two or three immunoglobulin (Ig)-like loops in the extracellular domain, a hydrophobic transmembrane domain, and a highly conserved tyrosine kinase domain split by a short kinase insert sequence. Given the structural similarities, it is not surprising that multiple FGFRs can bind multiple FGFs. However, there are several reports of cell- and tissue-specific expression and responsivity to different FGF family members (21, 22, 23, 24, 25). One mechanism responsible for generating selective responsiveness to different FGF family members is alteration of the ligand binding domain by alternative RNA splicing (21, 22, 23). It is clear that for FGFR-1 and FGFR-2 alternative processing of the RNA precursor generates multiple mRNAs which produce receptors manifesting different ligand binding specificities and affinities (21, 22, 23). The role that these receptor forms play in development and cell growth is currently an active area of investigation.
Alternative splicing of the FGFR-1 RNA has recently been associated
with astrocyte malignancy (26). For this gene, changes in the number
and not the amino acid composition of the extracellular Ig-like loops
correlates with astrocyte malignancy. An examination of graded
astrocytic tumors revealed that there was a switch from a three Ig-like
domain form (FGFR-1) to a two Ig-like domain form (FGFR-1
) during
the progression to malignancy (26). The production of the two Ig-like
domain form of FGFR-1 (FGFR-1
) is the result of exon skipping (see
Fig. 1). Normal human adult and fetal brain expressed a form containing
three Ig-like disulfide loops (FGFR-1
). While the functional
consequence of a shift in alternative RNA splicing from the
-form to
the
-form of the FGFR is not entirely understood, FGFR-1
has been
shown to exhibit a 10-fold greater affinity for acidic and basic FGF
than FGFR-1
(27, 28). If this is true, then glioma cells expressing
FGFR-1
would be more responsive to FGF than nontransformed cells or
low-grade astrocytoma cells expressing primarily FGFR-1
. These types
of structural differences may impart a growth or invasiveness advantage to glioma cells expressing FGFR-1
.
A better understanding of the mechanism(s) involved in recognition of
the -exon may lead to the identification of factor(s) directly
involved in astrocyte transformation. We believe that the change in
-exon recognition results from a change in RNA processing factors
and not gene mutation. Analysis of DNA sequence derived from the SNB 19 human astrocytoma cell line failed to find mutations within the
-exon and its flanking introns. Therefore, in an effort to begin
clarification of the mechanisms involved in FGFR-1 RNA splicing, we
have established a cell culture model system which mimics the RNA
processing decisions observed in normal and malignant brain tissue.
With this system we have determined that intron size plays a key role
in the
-exon skipping phenotype and have defined a 375-bp minimal
region that is required to maintain cell-specific RNA splicing.
The following human cell lines T98G (glioblastoma), NTERA-2 cl.D1 (NT2/D1) (Pluripotent embryonal carcinoma), PFSK-1 (Primitive neuroectodermal tumor), and JEG-3 (Choriocarcinoma) were obtained from American Type Culture Collection (Bethesda, MD). These cultures were all maintained in monolayer cultures using standard methodology and conditions recommended by the ATCC. The human astrocytoma cell line SNB 19 was maintained as described previously (26).
Gene MappingAll gene mapping studies were performed on the
human genomic P1 clone DMPC-HFF#1-4609E obtained from Genome Systems,
Inc. (St. Louis, MO). The oligonucleotide primers that were provided to
Genomic Systems map to the -specific exon for FGFR-1 gene (
F and
R). DMPC-HFF#1-4609E was one of three clones received from their
screening. This clone was subjected to BamHI or
BglII digestion, and the resultant fragments were subcloned
into pGEM 4 or pUC 19 by standard methodology. Three subclones were
obtained by colony hybridization using oligonucleotide probes (R1, P1A, and
F) derived from various positions in the 5
-most end of a cDNA encoding the secreted FGFR-1 form (GenBank Accession M34188[GenBank]) (29). From this screening we characterized three nonoverlapping clones,
pGFR-1, pGFR-2, and pGFR-3, by restriction analysis and DNA sequencing.
The distance between pGFR-1 and pGFR-2 was determined to be ~900 bp
by polymerase chain reaction (PCR) analysis of the P1 clone using
insert-specific primers FP-20 (pGFR-1) and FP-21 (pGFR-2). The distance
between pGFR-2 and pGFR-3 was determined to be ~1.3 kb by PCR using
insert-specific primers FP-1 (pGFR-3) and FP-10 (pGFR-2).
pGFR-1 contains a ~6.6-kb BamHI
fragment mapping to exon 1 inserted in pGEM 4. pGFR-2 contains an
~8-kb BglII fragment containing exon 2 inserted into pUC
19. pGFR-3 contains an ~4-kb BamHI fragment containing the
-exon inserted into pGEM 4. All the above inserts were derived from
DMPC-HFF#1-4609E. The parental minigene splicing construct pFGFR-17
was created by inserting the BamHI fragment of pGFR-3
containing the
- and
-exons into the BglII site in intron 1 of a human metallothionein 2A minigene (pRSVhMT2A) (see Fig.
2A) (30). Deletions of the 3
end of the insert were created using restriction sites within the BamHI insert. The clones
pFGFR-18 (XbaI to SmaI) and pFGFR-19
(SacI to SmaI) were deletions of pFGFR-17. The
clone pFGFR-20 is a BamHI to BglII fragment
inserted into the BglII site of pRSVhMT2A. In the
"stuffer" clone pFGFR-21, the ApaI to SmaI
region containing the
-exon has been replaced with an
ApaI to SmaI fragment from pGFR-3. The
replacement sequence is derived from intronic sequence located ~1.4
kb upstream of the
-exon. The fragment is inserted in the antisense
orientation. Deletions of the 5
end of the BamHI insert
were created using the following restriction sites: BglII
(pFGFR-22), BstXI (pFGFR-23), MscI (pFGFR-24).
The stuffer sequence in clone pFGFR-25 is a BglII to
SmaI fragment from pGFR-3. The replacement sequence is
derived from intronic sequence located ~1 kb upstream of the
-exon. The fragment is inserted in the antisense orientation. The
construct pFGFR-26 has a BglII fragment derived from pGFR-3
inserted into the BglII site of pRSVhMT2A. Constructs
pFGFR-27, pFGFR-28, pFGFR-29, and pFGFR-30 all contain the same stuffer
sequence used to create pFGFR-28. A second series of stuffer clones was
made using sequence derived from the 5
-flanking region of the FGFR-1
gene (approximately
4200 to
700). The construct pFGFR-31 was made
by insertion of an ~3500-bp BamHI to ScaI
fragment of pGFR-1 into the BglII and SmaI sites
of pRSVhMT2A, followed by deletion of a HindIII site contained within vector multilinker. The plasmids pFGFR-32 and pFGFR-33
were constructed by insertion of PCR-generated fragments containing
-exon (primers FP-2 and FP-4) and exon 4 (primers FP-11 and FP-12)
sequences, respectively, into the HindIII and SmaI sites of pFGFR-31 (see Fig. 7). In some cases multiple
cloning steps and/or partial restriction digestions were required to
obtained the desired plasmid construct. Specific details describing the construction of each plasmid are available upon request.
Transfections
All transfections were performed using LipofectAMINETM according to protocols suggested by the manufacturer (Life Technologies, Inc.). Briefly, cells were plated in duplicate on 100-mm dishes and allowed to grow to ~80% confluency prior to transfection. The DNA/lipofectAMINETM mixture (10 µg/40 µl) was allowed to associate at room temperature for 40 min. This mixture was then added to 4 ml of serum-free medium, which then replaced the culture medium. Transfection was allowed to proceed for 6 h prior to switching to medium containing serum. Total RNA was isolated 72 h after transfection by RNAzol B extraction as suggested by the manufacturer (TEL-TEST, Friendswood, TX). All results are representative of at least three transfections for each plasmid.
Reverse Transcription-Polymerase Chain Reactions (RT-PCR)All RT-PCR reactions were performed as described previously with minor modifications (30, 31). Both the reverse transcription and PCR steps were prepared in the same tube. Five µg of total RNA was used for each RT-PCR. The reverse transcription reaction was performed using the downstream PCR primer (Endo R, HMT3, or HMT2/3). PCR reactions were performed by diluting the RT reaction with a mixture containing 32P-end-labeled upstream primer (Endo F or DS8). Each amplification cycle consisted of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. PCR reactions were performed for 16 amplification cycles which were empirically determined to be in the linear range for the reaction primers and target sequence. PCR products were analyzed by polyacrylamide gel electrophoresis and visualized by autoradiography. The identity of all PCR products was confirmed by both restriction enzyme analysis and DNA sequencing.
PrimersF, GGAGCCCCTGTGGAAGTGGA;
R,
CTCCTCCCCTGTGATGCGGG; R1, GAACCCAAGGACTTTTCTC; P1A,
CGAGCTCACTGTGGAGTATCCATG; FP-1, CCCAAGAGAATGCAGCAAAG; FP-2,
GGGGAAGCTTGGCCAGCGTAATTCCCT; FP-4, GGGGAGTACTGGCTACCAACCTGAAACA; FP-10,
CTGGAACCTGGGGGCTGAAG; FP-11, GGGGAAGCTTGCAAGACACCTCCAGGT; FP-12,
GGGGAGTACTACACGTACCTTGTAGCC; FP-20, AACTCAGATTCTTCAGGCCT; FP-21,
TGCCCCATCCTTATATGTCC; Endo F, CCACGGCGGACTCTCCCGAG; Endo R,
TGGCAGCCGGCACTGCATGC; DS8, TTGACCATTCACCACATTGGTGTGC; HMT3, ATCTGGGAGCGGGGCTGT; HMT2/3, GCAGCAGGAGCAGCAGCTTT.
The fibroblast growth factor receptor 1 (FGFR-1) RNA precursor is
known to undergo several alternative RNA processing events affecting
both the extracellular and intracellular domains of the protein. The
production of a receptor containing three extracellular Ig-like binding
domains results from the inclusion of a single exon termed .
Deregulation of this RNA splicing has been observed in astrocyte
malignancy with a predominant
-exon skipping pathway observed (Fig.
1A) (26). Exon skipping is likely to result
from changes in trans-factor composition as analysis of
glioblastoma DNA failed to demonstrate mutations within the
-exon
and flanking intron sequence (data not shown). The goal of these
studies was to develop a model for examining the regulatory event(s)
involved in
-exon recognition. While much of the structure of the
FGFR-1 gene has been well characterized, the genomic organization of the 5
end of the human gene as well as the sequences surrounding the
-exon remain to be defined. Because the genomic structure and
intronic sequences frequently play a role in alternative RNA processing
decisions, we sought to first clarify the genomic organization in this
region.
A P1 clone was
obtained from Genome Systems, Inc., using PCR primers specifically
mapping to the -exon (
F and
R). Southern analysis of the P1
clone using a primer mapping to the 5
-most end of a cDNA (R1)
suggested the presence of the transcription start site in our clone
(31). Three nonoverlapping subclones were obtained from the P1 clone by
colony hybridization using cDNA-derived oligonucleotide probes (R1,
P1B, and
F) (Fig. 1A). Comparison of genomic sequence
with reported cDNA sequence confirmed the location of the
-exon
boundaries and, like mouse, two exons are located upstream of the
-exon (32) (Fig. 1). DNA sequence obtained from the 3
end of exon 1 allowed the determination of the exon/intron boundary. Our genomic DNA
sequence diverged from the cDNA sequence 641 bp from the predicted
transcription start site. As expected, this new intron sequence began
with a 5
splice site consensus (33). This observation would place the
translation initiation site in exon 2, as has been reported for the
mouse gene (32). The size of the introns flanking exon 2 are estimates based on Southern and PCR analysis of the P1 clone (data not
shown).
Based on our genomic clones and previously published
data (34), we estimate the FGFR-1 gene to contain 20 exons that map to
a region in excess of 30 kb. Therefore, it is impractical to perform
cis element mapping in the intact gene. To overcome this problem we employed a strategy similar to that used to map the cis-regulatory regions of the human calcitonin gene (30,
31). The ~4-kb insert of pGFR-3 was inserted into intron 1 of the
human metallothionein 2A (MT 2A) splicing reporter gene construct to create pFGFR-17 (Fig. 2A). Transcription of
this minigene is driven by the Rous sarcoma virus LTR
enhancer/promoter. This provides both a high level of expression and a
means of distinguishing transcripts derived from the minigene from
endogenous MT 2A expression. The construct maintains the alternatively
spliced downstream -exon but replaces the upstream exon 2. pFGFR-17
was transiently transfected into several cell lines (primarily of
central nervous system origin) in order to determine whether RNA
processing of the minigene transcripts mirrored the processing observed
for endogenous FGFR-1 precursor RNA. Splicing pattern was determined by
quantitative RT-PCR analysis for both endogenous FGFR-1 and pFGFR-17
RNA transcripts. As seen in Fig. 2 the pattern of
-exon inclusion
for transcripts derived from the minigene was similar to those observed
for endogenous RNA precursor splicing. The glioblastoma-derived cell
lines, T98G and SNB 19, showed almost exclusive
-exon skipping. The
neuroectodermal-derived cells PFSK-1 also exhibited a predominant
-exon skipping pattern for both the endogenous and transfected
FGFR-1 gene transcripts. In contrast, the embryonal carcinoma cell line
(NTERA-2 cl.D1) and choriocarcinoma cell line (JEG-3) both displayed
predominant
-exon inclusion pathways. The level of endogenous gene
expression did not differ between the cell lines, except for the JEG-3
cells which had significantly lower levels. Differences between the level of transcript derived from the minigene likely result from differences in transfection efficiency.
The observed RNA processing pattern
for the pFGFR-17 minigene in several cell lines confirmed that minigene
transcripts are capable of mimicking the RNA processing pathway
observed for the endogenous gene. However, the FGFR-1 gene insert size
of this construct exceeds 4 kb. A series of 3 end insert deletions
were introduced into the pFGFR-17 construct to determine the role of downstream intron and
-exon sequence on
-exon skipping (Fig. 3). Constructs pFGFR-17, pFGFR-18, pFGFR-19, and
pFGFR-20 were transiently transfected into the SNB 19 glioblastoma cell
line. RNA splicing pathways were determined by 16 cycle RT-PCR (see "Experimental Procedures"). As previously observed pFGFR-17-derived transcripts predominantly excluded the
-exon (Fig. 3, lane
1). The stepwise removal of downstream sequence resulted in a
gradual increase in the level of
-exon inclusion, with the highest
level observed for pFGFR-20. This effect appeared to be nonspecific with no significant increase coinciding with the removal of the
-exon (Fig. 3, lane 3 versus lane 4). To confirm the
nonspecific effect, the downstream intron size was expanded with
antisense sequence derived from intron 2 (pFGFR-21). Transfection of
pFGFR-21 into SNB 19 cells demonstrated that the intron expansion was
capable of restoring the RNA splicing phenotype (compare lanes
1 and 5). These results suggest that downstream intron
size and not the presence of specific sequence or the
-exon plays a
role in
-exon selection. The results obtained above from the 3
end
deletion clones suggest that while the ~950 bp of deleted sequence is
necessary for appropriate
-exon skipping, the specific sequence is
not required as it can be substituted.
Upstream Intron Size Also Plays a Key Role in
The intronic sequence upstream of the -exon in the
pFGFR-17 clone accounts for the majority of the FGFR-1 insert. In order to define further the sequence requirements for regulated
-exon recognition, additional deletions were made in this upstream intron sequence. As above, series of 5
stepwise deletions of ~1500 bp (pFGFR-22), ~2250 bp (pFGFR-23), and ~2300 bp (pFGFR-24) were introduced into pFGFR-17. The constructs were then transfected into SNB
19 cells and total RNA analyzed by RT-PCR for splicing products (see
"Experimental Procedures"). For these constructs, removal of
intronic sequence resulted in a dramatic increase in
-exon inclusion
(Fig. 4, lanes 2-4). The average ratio of
-exon inclusion/exclusion was 5.5 for pFGFR-23 and 8.1 for pFGFR-24 compared with 0.2 observed for pFGFR-17 (as determined by measurement using a PhosphorImager, Molecular Dynamics). This increase in
-exon
inclusion suggested that an inhibitory element preventing exon
recognition may have been deleted in these clones. To test this
possibility the deleted sequence was replaced with a nonspecific stuffer to create pFGFR-25. Analysis of splicing from products derived
from this clone showed
-exon skipping was restored to the wild-type
level (compare lanes 1 and 5). Therefore, as
above, these results again suggest a role for intron size and not
specific sequence, with a dramatic increase in
-exon recognition
occurring when the intron was reduced below ~330 bp.
Defining the Intronic Sequence Requirements for
Deletion of intronic sequence flanking the -exon
suggested that individually neither sequence was specifically required
for exon inclusion and that only intron size was important. In order to
define further the sequence requirements and test for redundancy of
elements, additional constructs were created. The construct pFGFR-26
(Fig. 5) combines the 5
and 3
deletions of pFGFR-20 and pFGFR-22. As observed for the individual deletions, there was an
enhancement of
-exon inclusion during RNA processing relative to
pFGFR-17 (compare lanes 1 and 2, Fig. 5).
However, the level of
-exon inclusion was not significantly greater
than that observed for the pFGFR-22 deletion construct. Therefore, any
redundancy or synergism involving downstream elements would involve
sequences 5
of the pFGFR-22 deletion point. To test this possibility,
the same 5
deletions introduced into pFGFR-17 were created in pFGFR-21 (constructs pFGFR-27, pFGFR-28, and pFGFR-29). As previously observed, there was a dramatic increase in the level of
-exon inclusion with
the deletions leaving 151 (pFGFR-28) and 95 (pFGFR-29) nucleotide of
intron sequence preceding the
-exon, even when stuffer sequence was
included downstream (compare lanes 5 and 6 with
lane 4, Fig. 5). Finally, a construct containing replacement
sequence both 5
and 3
of the
-exon (pFGFR-30, Fig. 5) was made to
confirm the role of intron size and rule out the possibility of
redundant elements flanking the
-exon. Like pFGFR-21 (lane
3, Fig. 5) and pFGFR-25 (Fig. 4), exclusion of the
-exon was
maintained in the absence of specific flanking sequence. This would
suggest that the
-exon with 95 nucleotides of 5
-flanking intron and
191 nucleotides of downstream intron provides sufficient sequence
information for regulated exclusion when provided with large flanking
introns.
The splicing pathway observed for pFGFR-30 transcripts in transfected
SNB 19 cells could result either from regulated skipping of -exon or
deletion of constitutive processing signals. To determine if the
pFGFR-30 construct maintained sequences required for cell-specific
-exon inclusion, this construct was transfected into the five cell
lines described in Fig. 2. Total RNA was isolated 48 h
post-transfection, and the processing pathways were determined by
RT-PCR analysis (see "Experimental Procedures") (Fig.
6). The level of
-exon inclusion in each cell line
was similar to that observed for pFGFR-17 (compare with Fig.
2B). The
-exon was predominantly excluded in T98G and SNB
19 glioblastoma cell lines, while predominantly included in NT-2 and
JEG-3 cell lines. This observation strongly suggests that the key
elements required for cell-specific
-exon recognition are contained
within the 553-nucleotide sequence inclusive and flanking the
-exon.
However, it does not rule out the possibility that the stuffer sequence
may contain a cell-specific regulatory element. This might result
fortuitously or because the stuffer sequence used to create the
pFGFR-30 construct is derived from intron upstream of the
-exon. A
palindromic element might still function in the antisense orientation.
To address these concerns and narrow the regulatory region, additional
constructs were created. The construct pFGFR-32 contains a 375-bp
insert, deleting an additional 180 bp of the downstream intron while
leaving the 5
splice site intact. The new stuffer sequences are
derived from the 5
-flanking region of the FGFR-1 gene (Fig.
7A). To control for the possibility of an
unforeseen regulatory element contained within this stuffer sequence,
the
-exon and its flanking sequence in pFGFR-32 was replaced with
analogous FGFR-1 exon 4 sequence to create pFGFR-33 (Fig.
7A). FGFR-1 exon 4 was chosen because like the
-exon it encodes Ig loop sequence but, unlike the
-exon, is not involved in
cell-specific splicing (21-23, and data not shown).
The constructs pFGFR-17, pFGFR-32, and pFGFR-33 were transfected into
SNB 19 and JEG-3 cells to compare the RNA processing of transcripts
derived from these constructs. As was previously observed, transcripts
derived from constructs pFGFR-17 showed -exon exclusion in SNB 19 cells (~80%) and inclusion in JEG-3 cells (~70%) (Fig.
7B). The RNA processing of transcripts derived pFGFR-32
construct displayed a similar cell-specific splicing pathway (~70%
exclusion in SNB 19 cells and ~70% inclusion in JEG-3 cells, as
determined by measurement using a PhosphorImager, Molecular Dynamics).
Therefore, cell-specific
-exon exclusion and inclusion was both
maintained with the smaller 375-bp fragment and occurred independently
of the stuffer sequence used. Finally, when
-exon was replaced with
exon 4, transcripts derived from this construct (pFGFR-33) no longer
displayed cell-specific exon recognition (Fig. 7B). While
exon 4 was not efficiently recognized in the two cell types, it was
included at a similar ratio (~60% by as determined by measurement
using a PhosphorImager, Molecular Dynamics). These results clearly
indicate that the sequence contained within and flanking the
-exon
is sufficient to mediate cell-specific recognition of this exon.
However, they cannot rule out the possibility that additional elements
located outside the defined region might play some role in modulating
this event.
The alternative recognition of exons during RNA processing not only allows creation of genetic diversity but regulated processing decisions provide key switches to several developmental and tissue-specific processes. Changes in cell-specific exon recognition have been correlated to numerous cellular events including cell differentiation and the cellular transformation associated with tumorigenesis (10, 11, 12, 13, 14, 35, 36, 37, 38). Correlations involving a change in RNA processing are most often recognized by examination of a single gene. However, it is easy to imagine that the identification of a single aberrant RNA processing event provides a mechanism for monitoring a change which would have a global effect and possibly lead to cellular transformation.
In this report we have focused on alternative RNA processing of FGFR-1
transcripts. Processing of this gene's RNA precursor is quite complex
and known to alternatively recognize at least six different exons
(21, 22, 23, 29, 39, 40). While the expression of the FGFR-1 gene is
widespread, occurring in almost all cell types, little is known about
the distribution of the alternative RNA forms. It is important to note
that these RNA processing changes greatly impact upon the functionality
of the protein, altering ligand affinity and specificity, subcellular localization, and tyrosine kinase activity. For the -exon, inclusion in the final transcript encodes for the production of a receptor with
three Ig-like domains in the extracellular domain, while exclusion
encodes a receptor with only two domains. This change has no effect on
ligand specificity but has been demonstrated to reduce FGF-1 binding
affinity (27, 28). The impact of the change in this splicing decision
on glial cell growth is unclear and currently under investigation.
The goal of this study was to develop a model system with which the
mechanisms involved in alternative -exon recognition might be
elucidated. Similar model systems have been developed to examine RNA
processing for several alternatively regulated genes, including FGFR-2
(41, 42). In the process of defining the human FGFR-1 gene structure,
we found that like the mouse gene the first two introns are
disproportionately large relative to other introns in the gene (the
next largest, intron 10, is ~2100 bp) (Fig. 1) (34). Similar sized
introns are observed for other receptor genes in this family. For
example, the platelet-derived growth factor receptor has been found to
have a 23-kb first intron (43). Therefore, it is possible that the size
of these introns play a role in regulation of
-exon recognition.
This concept is supported by our observation that a reduction in the
size of the intron preceding the
-exon in the chimeric minigene had
such a dramatic effect on RNA splicing (Fig. 4). However, cell-specific splicing can be restored by insertion of nonspecific sequence. Therefore, this regulatory mechanism is likely to be nonspecific.
Cell-specific RNA splicing of the -exon was maintained with only 375 bp of FGFR-1 gene sequence. The sequence is comprised of the
-exon
with 95 nucleotides of upstream and 11 nucleotides of downstream
flanking sequence. This places the specific regulatory sequences either
within or immediately adjacent to the
-exon. This finding is not
unexpected. For several alternatively recognized exons where regulatory
elements have been identified, these sequences are often found near the
splice site regions. Splicing of the FGFR-2 gene RNA transcript
provides a specific example. Mutually exclusive splicing of two exons
encoding part of the third immunoglobulin-like loop determines ligand
specificity. Inclusion of a K-SAM encoding exon in
epithelial cells produces a receptor with high affinity for
keratinocyte growth factor. Regulated splicing of this exon has been
shown to require three different elements all located within or
flanking the exon (41, 42). The three elements are suboptimal splice
sites, a splicing enhancer (recognized in epithelial cells), and an
inhibitory sequence (recognized in other cell types). For this RNA a
purine-rich exon element functions to inhibit splicing of the
K-SAM exon, whereas a pyrimidine-rich intronic element stimulates exon inclusion. Similar purine-rich sequences are present in
the 375-bp FGFR-1 fragment on the pFGFR-32 minigene construct. Whether
these sequences function in an analogous fashion remains to be
addressed.
The observation that cell-specific RNA splicing of the FGFR-1 -exon
can be maintained in a chimeric minigene provides a useful tool for the
further study of this splicing event. While a relatively small region
(375 bp) is required to maintain regulated splicing of FGFR-1
-exon,
the mechanism(s) of exon recognition may be quite complex, involving
several elements and possibly sequences outside the defined region
which might modulate the response. The failure to include the
-exon
as a result of astrocyte transformation would suggest some alteration
of factors that function through splicing enhancers. This could involve
a pyrimidine-rich element, such as that used for FGFR-2
K-SAM or some other regulatory sequence. For example, the
purine-rich exonic splicing enhancers have been identified in several
exons and regulate the splicing of several transcripts (44). Sequences
resembling the exonic splicing enhancer consensus can also be found in
the
-exon. These elements function through interactions with a class
of RNA binding proteins (SR proteins) to regulate exon inclusion (45).
It is not know if neoplastic transformation is associated with a change
in the expression of individual SR proteins, but they do show
tissue-specific distribution. These proteins, therefore, become an
attractive candidate for regulators of this splicing event. Additional
experimentation to first define the specific cis-regulatory
sequences, however, is required before a role for these proteins in
-exon splicing can be determined.