(Received for publication, September 11, 1996)
From the Nucleotide sequence analysis of
independently isolated clones from a mouse liver cDNA library
identified two additional splice variants of folylpolyglutamate
synthetase (FPGS) mRNA with novel sequence at the 5 The enzyme folylpolyglutamate synthetase
(FPGS)1 in mammalian cells has both
physiological and pharmacological significance. FPGS mediates the
anabolism of folates and their analogs to Earlier studies in several laboratories (10-12) suggest that the
expression of FPGS activity in various tissues is under stringent regulation. The basis for this regulation and details as to its mechanism are important to our understanding of cellular folate homeostasis (1-6) and also to the metabolic disposition of folate analogs (2-8) during therapy of tumors in animal models and patients. The recent cloning by Shane and co-workers (13) of human FPGS cDNA
now makes it possible to address these issues at the level of gene
expression. Other recent studies by Moran and co-workers (14) have
provided information pertaining to the most 5 We now report other results that identify two additional splice
variants found in the form of cDNA in a murine liver cDNA library. These variants incorporate distinct alternatives to exons designated as exons 1a, 1b, and 1c of the murine FPGS gene in our
previous publication (18) and encode a distinctly different 5 Full-length
cDNA clones were selected by hybridization (19) screening of a
murine liver cDNA library in A
BALB/c mouse liver genomic library in EMBL3 SP6/T7 (Clontech; Palo
Alto, CA) was screened with an exon-specific radioactive murine FPGS
cDNA probe that was prepared by random priming with [ Double-stranded DNA was sequenced in both
directions according to the dideoxy method of Sanger et al.
(24) using Sequenase version 2.0 (U. S. Biochemical Corp.).
Oligonucleotide primers based on the mouse FPGS cDNA were used
initially. Additional oligonucleotide primers were prepared on the
basis of the sequence data generated when necessary for extending the
sequencing. Exon/intron junctions were determined by direct sequencing
across these junctions using primers based upon the mouse FPGS cDNA
sequences. Intron sizes were determined by sequencing through the
region in question.
Ten pmol of an antisense
oligonucleotide primer corresponding to nucleotide sequence in the 3 Using a standard procedure,
samples of poly(A)+ RNA were blotted (26, 27) and analyzed
by radioautography using exon-specific or nonspecific cDNA probes.
The exon A1-specific probe incorporating all of the nucleotide sequence
in exons A1a and A1b (see below) was prepared by PCR using variant IV
murine liver cDNA as template. The exon B1-specific probe
incorporating all of the nucleotide sequence in exons B1a, B1b, and B1c
and intron B1c was prepared by PCR using a mouse liver genomic clone as
template (clone 3, Ref. 18). A nonspecific probe incorporating the
complete nucleotide sequence in exon 14 was prepared by PCR using
murine liver cDNA as template (variant IV). The
poly(A)+ RNA content of each blot was normalized with a
mouse 36B4 probe (acidic ribosomal phosphoprotein PO, Ref. 28).
Labeling of each probe was by random priming (Random Primers DNA
Labeling Kit, Boehringer Mannheim) using [ The relative amount of cell FPGS mRNA
transcript representing different splice variants in L1210 cells and
murine liver was determined by reverse transcriptase PCR. Prior to
cDNA synthesis (see above), poly(A)+ RNA was digested
with RNase-free DNase I, treated with phenol chloroform and
precipitated with ethanol followed by washing two times with 70%
ethanol before dissolving in H20. All poly(A)+
RNA was treated with methyl mercuric hydroxide to reduce secondary structure. Five µl of the prepared cDNA was utilized in a PCR using four different exon 1-specific sense primers and an exon 5 antisense primer. Ten pmol of an 18-mer oligonucleotide encompassing the ATG start site were used for each PCR.
5 All radioactive isotopes used for the above
studies were obtained from DuPont NEN. Specific activities for
[ Screening of an
L1210 cell cDNA library with a murine FPGS cDNA probe (19) and
DNA sequencing originally identified (18) two different classes of
clones exhibiting complexity with regard to exon 1 of the murine FPGS
gene. The least common cDNA clone incorporates (Fig.
1A and Ref. 18) all of the nucleotide
sequence (now designated exons B1a and B1b in variant I) homologous to human exon 1 (16) spliced to exons 2-15 and encodes both a
mitochondrial leader peptide and the cytosolic form of FPGS. The most
common variant (variant II) incorporates (Fig. 1 and Ref. 18) only a
portion (exon B1b spliced to exons 2-15) of the nucleotide sequence homologous to the human exon 1 and encodes only the cytosolic form of
FPGS. A third variant was identified (Fig. 1 and Ref. 18) initially in
a liver cDNA library and subsequently in an L1210 cell cDNA
library and incorporates an alternate to exon B1a (exon B1c) which is
spliced to exon B1b plus exons 2-15. The 5
Further screening of the mouse liver cDNA library was carried out
using the same cDNA probe. In this library, cDNA
representatives of variants I, II, and III were found to be relatively
rare (Table I) compared with their relative frequency in
the L1210 cell cDNA library. Instead, the most common variant found
(variant IV) incorporates (Fig. 1A and 2) a
novel sequence in the form of alternates (exons A1a and A1b) to exons
B1a and B1b or B1c and B1b spliced to exons 2-15. These clones were
derived from mature transcripts with an appropriate poly(A) tail and
properly positioned polyadenylation signal sequence at the 3
Relative content of splice variants found in L1210 cell and murine
liver cDNA libraries
Program of Molecular Pharmacology and
Therapeutics,
end. These
variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a
encodes most of the 5
-untranslated region. Exon A1b encodes a
downstream segment of the 5
-untranslated region, two ATG start codons,
and a unique mitochondrial leader peptide as well as 15 additional
amino acids of cytosolic FPGS not encoded by all previously identified
(Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol.
Chem., 271, 23820-23827) splice variants. It was also found by
direct sequencing of genomic fragments that although exon A1b is
spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to
exon 1 are found approximately 9.5 kilobases upstream from exons B1a,
B1b, and B1c. Exons A1a and A1b are separated from each other by a
124-nucleotide intron. Sequencing of the region 5
to exon A1a revealed
a nucleotide sequence that was promoter-like and different from the
downstream promoter region in the content of putative
cis-acting elements. Primer extension analysis identified a
number of potential transcription start sites within the more 3
end of
this region. FPGS RNA transcripts incorporating exons A1a and A1b were
detected in both normal mouse tissues, particularly, liver and kidney,
and also to a varying extent in tumors; FPGS RNA transcripts
incorporating exons B1a, B1b, and B1c were detected mainly in tumors.
Thus, transcription of the FPGS gene in this tissue-specific manner
appears to reflect the different usage of alternates to exon 1 under
the control of different promoters. An unusual splice variant
identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of
the downstream promoter region along with exons B1c and B1b and exons
2-15.
-polyglutamate peptides
(1-5). Polyglutamated forms of folate coenzymes are conserved and are
more efficient as cofactors for folate-dependent biosynthetic reactions (1-5). Polyglutamylation of folate analogs, on
the other hand, renders these agents more cytotoxic (2-8). However, a
differential in anabolism of these analogs among normal tissues and
tumors often favors (8, 9) their accumulation as polyglutamates in
tumor cells and thus contributes to their therapeutic selectivity.
sequence of the human
FPGS gene which included the sequence of a putative promoter-like
region. Preliminary functional studies reported recently (15) provided
evidence of promoter activity for this region. The earlier (14) studies
also suggested alternate transcription start sites as a basis for the
regulation of synthesis of both mitochondrial and cytosolic forms of
human FPGS with the former requiring the inclusion of a
NH2-terminal leader peptide encoded by nucleotide sequence
at the 5
end of exon 1. This same group (16) and Shane and co-workers
(17) provided detailed information on the structural organization of
the human FPGS gene in the form of 15 exons spanning approximately 11 kb of DNA sequence. Shane and co-workers (17) also provided evidence
for the existence of alternate splice variants of exon 1. Recent
studies of our own (18) characterized the murine FPGS gene, a
promoter-like region with somewhat different characteristics than the
human promoter and revealed an interesting complexity with regard to exon 1. This was expressed in the form of three splice variants that
differed in their content of sequence homologous to human exon 1. As
only some variants incorporated a nucleotide sequence encoding both a
mitochondrial leader peptide as well as cytosolic FPGS, the results of
these studies raised the possibility that the regulation of synthesis
of mitochondrial and cytosolic forms of FPGS could occur as a result of
alternate splicing.
end and
proximal open reading frame within the mRNA transcript. Hybridization screening of DNA restriction fragments showed that these
new alternatives to exon 1 exist in the genome >10 kb upstream from
the previously designated exons 1a, 1b, and 1c, and their transcription
along with exons 2-15 also incorporated in these variants, appears to
be under the control of a different promoter. We have putatively
identified such a promoter in the sequence immediately 5
of these new
alternates to exon 1. We also report on data that show that
transcription of these various splice variants that incorporate
alternatives to exon 1 occurs in a tissue-specific manner reflecting
the alternate usage of different promoters.
Selection of Murine FPGS cDNA Variants
gt11 using an L1210 cell FPGS
cDNA (19) as a probe. The hybridization screening of the cDNA
library was carried out under conditions described earlier (20). The
radioactive FPGS cDNA probe was prepared by random priming
(Boehringer Mannheim) with [
-32P]dCTP and the L1210
cell FPGS cDNA as a template. Isolation of poly(A)+ RNA
was prepared as discussed earlier (21) and utilized in the synthesis of
cDNA by a standard procedure (22).
-32P]dCTP (Boehringer Mannheim). The nucleotide
sequence of this FPGS cDNA was submitted to GenBank (GenBank/EMBL
Accession U59517[GenBank]). After screening of a large number of plaques and
purification several positive clones were obtained. The DNA insert from
these clones was purified and characterized by restriction mapping and Southern hybridization (23). One of these nonidentical clones designated clone 10 was selected by the above procedure for further analysis because of the large size of its insert (~18 kb).
Restriction fragments of these clones generated with SacI or
EcoRI were selected for further study on the basis of
hybridization with the region-specific probes. The fragments of
interest after extraction were subcloned into Bluescript
SK+ (Stratagene) for sequencing.
end of exon A1b (5
-GGAGGCAGTCTTAGCTTCGTAAG-3
) of the murine FPGS gene
was end labeled (Promega, Madison, WI) at the 5
terminus using T4
polynucleotide kinase and [
-32P]ATP. The labeled
primer was hybridized to 5 µg of poly(A)+ RNA isolated
from mouse liver cells which was first treated with methyl mercuric
hydroxide and the primer extension reaction carried out as well with a
standard kit as specified (Promega). An mRNA preparation derived
from a 1.2-kb kanamycin-resistant plasmid cDNA with an antisense
primer was used as a control. Electrophoresis on an 8% polyacrylamide
gel was carried out by a standard protocol (25).
-32P]dCTP
and a 10-20-ng insert.
-CCAAGTGATGATGAAAGC-3
was used as the A1b-specific sense
primer, and 5
-TAAGACTATGTCGCTGGGC-3
was used as the
B1a-specific sense primer. 5
-GAGCCGGGCATGGAGTAT-3
was used
as the B1b-specific sense primer, and
5
-ACAAAGATGGCCGTGATA-3
was used as the B1c-specific sense
primer. 5
-TTCAGGCCGTAATTCCGC-3
was used as the exon 5-specific
antisense primer. Initially, reactions were run with different amounts
of cDNA and different PCR cycles to obtain conditions that ensured
that amplification was in the linear range at the time the amount of
product of each primer-specific reaction was compared. As a control,
the cDNA reaction mixture without reverse transcriptase was used to
initiate a PCR to make sure that there is no genomic contamination
(data not shown). As an internal standard, sense
(5
-CCTGGTGTCCCTGCCTTC-3
and antisense (5
-TCCTATGGATGAAACCTC-3
primers of FPGS representing exons 9 and 14, respectively, were used in
a parallel reaction. The experimental reaction was run for 25 cycles
using a standard procedure (28), and the relative amount of product
generated with each set of primers was determined by 1.5% agarose gel
electrophoresis and staining with ethidium bromide (29).
-32P]dCTP, [
-35S]dATP, and
[
-32P]ATP were 3,000, 1,000, and 3,000 Ci/mmol,
respectively. DNA restriction enzymes were purchased from Boehringer
Mannheim. Amplitag DNA polymerase was obtained from Perkin-Elmer.
Nitrocellulose was purchased from Schleicher & Schuell, and
oligonucleotide primers were synthesized by Genelink. Solutions for
polyacrylamide sequencing gels were obtained from National Diagnostics.
All other materials were reagent grade.
Novel Splice Variants of the Murine FPGS Gene
nucleotide sequences of
these variants aligned with respect to the sequences in exon 2 are
given in Fig. 2 where it can be seen that variants I and
III incorporate different 5
ends and a different nucleotide sequence
within the proximal segment of the open reading frame.
Fig. 1.
Organization of the murine FPGS gene showing
a restriction map, exon/intron junctions, and different spice variant
formation. The composition of the different splice variants is
shown to scale in panel A. The three genomic clones shown in
panel B had inserts of 17 (clone 3), 12 (clone 5), and 18 kb
(clone 10), respectively. The lengths of the exons and introns are
shown to scale. The details of the methodology employed are provided
under "Experimental Procedures."
[View Larger Version of this Image (32K GIF file)]
Fig. 2.
Nucleotide sequence in the 5 region of
various murine FPGS cDNAs. The nucleotide sequences of the 5
region of variants I, II, III, and IV are aligned with respect to exon
2. The details of the methodology employed are provided under
"Experimental Procedures."
[View Larger Version of this Image (48K GIF file)]
end. The
length and sequence of the 5
-untranslated region of variant IV, the
position of the two ATG start codons, and the encoded mitochondrial
leader peptide (MMKSTRSLPMSWPVREKFW) were distinctly different from
those encoded by either exons B1a and B1b or exons B1c and B1b. The
nucleotide sequence and the encoded amino acid sequence of all of these
splice variants are compared in Fig. 2. These data show that the exons included in these splice variants differ considerably in their composition. Variants I, III, and IV all have in-frame upstream and
downstream ATG start codons with properly positioned Kozak (29)
consensus sequences. Variant II incorporates only the downstream ATG.
In addition to the untranslated region incorporated in exon B1a, exons
B1a and B1b in variants I encode different segments of a mitochondrial
leader peptide. Along with a shorter untranslated region incorporated
in exon B1c, exons B1c and B1b in variant III incorporate different
segments of another mitochondrial leader peptide larger in size than
that encoded by variant I. By contrast, exon A1a in variant IV only
incorporates a untranslated region much longer than that found in
variants I and III, whereas exon A1b encodes a different mitochondrial
leader peptide in its entirety as well as 15 additional amino acids of
cytosolic protein not encoded in variant I, II, or III. The results of
quantitative reverse transcriptase PCR obtained earlier (18) and in the
current studies (data not shown) using poly(A)+ RNA from
L1210 cells and mouse liver were consistent with the relative frequency
of these variants observed in the L1210 cell and mouse liver cDNA
libraries summarized in Table I.
Variant
L1210 cells
Murine liver
%
%
I
5
<5
II
50
<5
III
20
<5
IV
20
80
V
<5
5
During further screening, another splice variant (variant V) was eventually identified (Fig. 1) in the liver cDNA library which was also rare and extremely unusual. This cDNA was larger (2.67 kb in length) than the other variant cDNAs and incorporated not only exons A1a and A1b but also a portion (identified as exon B1d) of the putative promoter region described previously (18) along with exon B1c and B1b and exons 2-15.
Genomic Location of Exons A1a and A1bUsing a cDNA probe
derived from the novel sequence in exons A1a and A1b, screening of
genomic clones (clones 3 and 5) incorporating the nucleotide sequence
in the previously published (18) murine FPGS gene showed that these new
alternatives to exon 1 were not located within or near the confines of
that region of the gene (18). However, the nucleotide sequence was
subsequently found by hybridization within another clone (Fig.
1B) bearing an 18-kb insert (clone 10) which was identical
to the novel sequence in the 5 end of variant IV. The 3
end of this
clone overlaps with the 5
end of clone 3. By restriction mapping and
sequencing, the location of these new exons was found (Fig.
1B) to be approximately 9.5 kb upstream from exon B1a. Also,
these two exons are separated by an intron 124 nucleotides in length.
The exon compositions of all of the splice variants found so far in
this mouse liver cDNA library are compared in Fig. 2. The
nucleotide sequence at the intron/exon junctions pertaining to all of
the alternatives of exon 1 are given in Table II. These
junctions match those for published (25) consensus splice
junctions.
|
Sequencing of DNA 5 of exon A1a identified a 2-kb region
with stretches of the sequence that included a number of putative binding sites for various cis-acting factors known to affect
transcription. The characteristics of this region (Fig.
3) are quite different from that of the promoter-like
region 5
of exon B1a (18). In contrast to this downstream region,
there are no putative SP-1 binding sites. However, in addition to a
GATA/TATA box, there are putative sites for 15-20 different
transcription factors not found in the downstream promoter-like region,
notably MyoD, Myc, Myb, and p53.
Enzymatic primer extension analysis was carried out using a primer
matching the 3 end of exon A1b and methyl mercuric hydroxide-treated poly(A)+ RNA from murine liver. The data suggest (Fig.
4) multiple transcription start sites distributed within
this region spanning approximately 200 nucleotides (Fig. 3) upstream
from the location of the primer. Start sites were found at +52, +55,
+62, and +64 nucleotides. Major start sites were found at +53 and
especially at
184. The usage of only the latter would result in the
formation of a transcript encoding the putative mitochondrial leader
peptide. The results of primer extension analysis pertaining to the
downstream promoter-like region using exons B1b- and B1c-specific
primers have already been reported (18).
Tissue-specific Expression of FPGS in the Form of Alternate RNA Transcripts
Using antisense probes specific for the A1 and B1
alternates of exon 1, a series of Northern blots was performed with
poly(A)+ RNA from a variety of normal and neoplastic murine
tissues. Poly(A)+ RNA from these same tissues was also
blotted with a nonspecific probe incorporating the nucleotide sequence
from a downstream exon. All of these blots were normalized as well by
blotting with a 36B4 cDNA probe. The data in Fig. 5
show that FPGS mRNA transcripts in the range of 2.3 kb could be
detected in all of the tissues examined using the exon nonspecific
probe. In contrast, the detection of FPGS mRNA transcripts with the
A1- and B1-specific probes was highly tissue-specific. With the
nonspecific probe there was considerable variability in the relative
amount of 2.3-kb FPGS mRNA detected depending upon the tissue.
Among normal tissues examined, FPGS mRNA content was highest in
kidney and liver and lowest in spleen, lung, and small intestine. Using
the same probe, FPGS mRNA was readily detectable in all of the
tumors except the hepatoma. This also included the Ehrlich tumor, which
had less mRNA in the sample employed than was assumed (see also the
blot with the 36B4 probe). With the exon A1-specific probe, relative
levels of FPGS mRNA detected among these different tissues were
similar to that obtained with the nonspecific probe. However, with the
B1-specific probe, the level of FPGS mRNA detected was highest in
the tumors with the exception of the mouse hepatoma. The level of
mRNA detected by this probe in this tumor was relatively low by
comparison and extremely low in kidney and small intestine and
virtually undetected in spleen, lung, and liver. The low intensity
overall of the blot obtained with this probe most likely reflects the
relatively high GC content of this probe compared with the other probes
used in this study. In addition to the tumors identified in Fig. 5,
poly(A)+ RNA from several other murine tumors was also
probed with these two cDNA probes with similar results (data not
given). These included B16 melanoma, T241 fibrosarcoma, P388 lymphoma,
EO771 mammary carcinoma, and taper liver tumor.
These studies provide further evidence for substantial heterogeneity at
the 5 end among murine FPGS RNA transcripts. Our earlier (18) studies
documented 5
end heterogeneity in the form of a splice variant
(variants I and III) incorporating alternatives of exon B1 (exons B1a
and B1c). The current studies extend these findings and document
additional heterogeneity in the form of a transcript (variant IV) which
incorporates two previously undescribed exons (A1a and A1b) which are
located approximately 9.5 kb upstream of the exon B1 alternates. Like
variants I and III, variant IV encodes a different mitochondrial leader
peptide and unique amino acid sequence at the NH2-terminal
end of cytosolic FPGS.
Our results also appear to show that the generation of 5 end
heterogeneity during transcription of the murine FPGS gene in part
reflects the activity of different promoters regulating transcription of this gene in the mouse. These promoters differ in their nucleotide sequence, the number and putative identity of various
cis-acting elements, and the pattern of potential
transcription start sites contained therein. Additional studies at a
functional level will be necessary to characterize these promoters with
regard to the extent that cis-elements, and
trans-acting factors may initiate, enhance, or suppress
transcription in either case. Of relevance to this question were other
findings from Northern blotting using exon A1- and B1-specific probes,
which strongly suggest that the activation of these promoters occurs in
a tissue-specific manner. Activation of promoter A appears to occur in
both normal mouse tissues and tumors and results mainly in the
formation of transcripts incorporating exon A1a plus A1b spliced to
exons 2-15. In contrast, activation of promoter B appears to occur
primarily in tumor tissue resulting mainly in the formation of
transcripts incorporating exons B1a plus B1b, exons B1c plus B1b, or
only exon B1b spliced to exons 2-15. The relative transcriptional
activity of promoter A appears to vary considerably among the various
normal tissues examined with activity apparently highest in liver and
kidney. In contrast, the relative transcriptional activity associated with promoter B appeared to be less variable among the different tumors
examined.
5 end heterogeneity associated with differential promoter usage has
been documented or suggested to occur in the case of a variety of
mammalian genes. These include rodent genes for
-glutamyl transferase (30), acyl-CoA synthetase (31), and human genes for
aminopeptidase N (32), carbonic anhydrase I (33), and phosphofructokinase (34). Despite this precedence, it is not clear why
multiple promoters for the murine FPGS gene have evolved. However, in
view of the widespread distribution of FPGS in different tissues (4)
and evidence for the regulation of FPGS during development (19-12) the
regulation of transcription of this gene may necessitate multifactorial
control.
The functional significance, if any, of our finding pertaining to variant V, which incorporates a large portion (exon 1d) of promoter B, is not evident and may, in fact, reflect a splicing anomaly. However, the incorporation of a downstream promoter region within a mature RNA transcript while highly unusual is not without precedence. A similar finding has been reported in the case of a multidrug resistance gene (35).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U33557[GenBank], U54793[GenBank], and U59517[GenBank].