(Received for publication, February 6, 1995; and in revised form, May 19, 1995)
From the
The 9G8 factor is a 30-kDa member of the SR splicing factor
family. We report here the isolation and characterization of the human
9G8 gene. This gene spans 7745 nucleotides and consists of 8 exons and
7 introns within the coding sequence, thus contrasting with the
organization of the SC35/PR264 or RBP1 SR genes. We have located the
human 9G8 gene in the p22-21 region of chromosome 2. The 5`-flanking
region is GC-rich and contains basal promoter sequences and potential
regulatory elements. Transfection experiments show that the 400-base
pair flanking sequence has a promoter activity. Northern blot analysis
of poly(A) The splicing of nuclear pre-mRNA occurs in a multicomponent
complex containing small nuclear ribonucleoproteins (U snRNP), It
has been argued that the various SR factors are interchangeable in
constitutive splicing because each is able to complement SR-deficient
extracts (for instance a cytoplasmic S100 extract) (Fu et al.,
1992; Zahler et al., 1992). Recently, it has been shown that
SF2/ASF and SC35 are able to form commitment complexes with a pre-mRNA
substrate (Fu, 1993) and that they are required for the stable
interaction of U1 snRNP with the 5` splice site (Kohtz et al.,
1994). In agreement with these results, the region of SF2/ASF
containing the two RBDs is able to recognize a typical 5` splice site
in a short transcript (Zuo and Manley, 1994). An interesting aspect
of SR factors is that they may modulate alternative splicing in a
concentration-dependent manner when several 5` splice sites are in
competition. Increasing amounts of SF2/ASF or SC35 generally result in
the preferred selection of the more proximal 5` splice site (Krainer et al., 1990b; Ge and Manley, 1990; Fu et al., 1992).
However, a more extended comparison, including SRp40, SRp55, and SRp75,
indicates that each SR factor has a differential ability to modulate
alternative splicing in vitro (Zahler et al., 1993a).
Moreover, as these factors are differentially expressed in different
tissues, Zahler et al. (1993a) proposed that the SR factors
may be involved in tissue-specific regulation of alternative splicing in vivo. In support of this idea, overexpression of SF2/ASF by
transfection experiments led to a modulation of alternative splicing in vivo (Caceres et al., 1994). We have isolated
recently the 9G8 factor with a molecular mass (
The blot
containing the recombinant phages DNA was probed with the 38-mer QE203
oligonucleotide or the QO60/QN140 PCR product (+682/+902),
labeled with
Figure 6:
A, structure of the different deletion
mutants used in transient transfection experiments. The 9G8 promoter
from positions -414 to +26 was linked to the bacterial CAT
gene. The StuI and NotI sites are indicated. The
different deletions are shown and the positions of the deleted
nucleotides are indicated. B, transcriptional activity of the
9G8 upstream sequences. CAT activity levels with different deletion
mutants are represented. The value corresponds to the average of
independent experiments.
Figure 1:
Intron/exon organization and partial
restriction map of the human 9G8 SR factor gene. Exons are represented
as solid boxes. Exon 8 resulting of the use of distal
polyadenylation site is depicted as an expanded open box. The
partial restriction map and the overlapping human genomic clones
Figure 2:
Southern blot analysis of 9G8 gene. Human
genomic DNA (15 µg) was digested with EcoR I or SacI restriction endonucleases and after gel electrophoresis
and blotting was hybridized using cDNA 1 (positions +265 to
+595) or cDNA 2 probes (+262 to + 971). The marker size
(in kilobases) is indicated on the left.
Figure 3:
Idiogram of distribution of signals on
chromosome 2. In situ hybridization of human metaphase
chromosomal spreads were performed as described (Mattei et
al., 1993). 70.6% of grains located on chromosome 2 mapped to the
p22-21 region of short arm, with a maximum in the p21
band.
Figure 4:
Primer
extension analysis. A 5` end-labeled 38-mer oligonucleotide QM94 was
annealed to 1 µg of poly(A)
Figure 5:
Sequence of the upstream region of 9G8
transcription unit. Sequences of the upstream region of 9G8 gene from
positions -414 to +26 are represented and the transcription
start site is designated +1. The potential binding sites for
various transcription factors are indicated on the corresponding
sequences by arrows, which give also the orientation of these
elements (Locker and Buzard, 1990; Faisst and Meyer, 1992). The natural StuI and NotI sites are indicated with open boxes and the artificial HindIII and BamHI sites (see
``Materials and Methods'') with solid
boxes.
Figure 7:
Northern blot analysis of the 9G8 mRNA
species in human fetal tissues. On the upper panel, analysis
performed with human multiple fetal tissue Northern blot (Clontech) is
represented. The tissues are indicated above each lane and on the top of each blot, we have indicated the probe used (see
``Materials and Methods'' for the description of the probes).
The molecular weight markers are indicated on the right, and
the sizes of the different 9G8 mRNA species are inscribed on the left of each blot. The scheme of each mRNA species is given in
the lower panel. Exons are represented by open boxes,
and coding regions are indicated by black boxes. The intron 3
is delineated by a hatched box.
To
further analyze the different isoforms, the blot was hybridized with
DNA probes specific for (i) the 3`-noncoding region between the two
potential poly(A) signals (3` UTR; Fig. 7, lanes
5-8). We have used this probe because the distance observed
between the two potential polyadenylation signals ( The 9G8 gene is divided into eight exons and seven introns,
and the coding sequence is highly divided, since it starts in exon 1
and stops in exon 8. Thus, its exon/intron organization is very
different from those of the two other SR factors SC35/PR264 (Sureau and
Perbal, 1994) or RBP1 (Kim et al., 1992), since in these
genes, the coding sequence is contained only in two exons. Previous
comparison of amino acid sequences of RBD has shown that 9G8 presents a
good homology with SRp20 and RBP1 (Cavaloc et al., 1994),
suggesting that several SR factors may originate from a common
ancestral gene, as already proposed (Birney et al., 1993).
However, the very different organization of 9G8 and RBP1 genes
indicates that profound modifications have occurred after ancient gene
duplication. It has been proposed that the intron sequences frequently
demarcate important functional or structural domains of proteins. We
observe indeed that the RNA binding domain of the 9G8 factor covers
precisely exon 2 sequences, whereas the middle region containing the
specific CCHC zinc knuckle is located in the third exon. Finally, the
SR domain of 9G8 is distributed in the exons 4-8, but a specific
region of this domain, not found in the other SR factors, is encoded
precisely in exon 5. This region contains four conserved repetitions of
the consensus RRSRSXSX (Cavaloc et al.,
1994) and might originate from intragenic recombination occurred during
evolution. We have shown that the 9G8 gene is located in human on the
chromosome 2p22-21. This region is the site of several known genes,
including T cell leukemia virus enhancer factor (HTLF) (Li et
al., 1992), and translocations of chromosome 2p22-16 with
chromosome 11p23 have been reported in human leukemia (Bloomfied and de
la Chappelle, 1987). Examination of the 400-bp region upstream of
the 9G8 gene shows several interesting features. The G + C content
of 57% and the CpG content of 7% are indicators that the promoter
region of the 9G8 gene is in a CpG island (Larsen et al.,
1992). However, the presence of a TATA box and many potential
regulatory elements does not allow us to classify this gene as a
typical housekeeping gene (Locker and Buzard, 1990; Faisst and Meyer,
1992). The promoter of the 9G8 gene has been shown to be functional in
JEG-3 cells and to respond to several trans-acting factors. In this
respect, the 9G8 promoter resembles the SC35/PR264 gene which contains
several Myb-responsive elements (Sureau et al., 1992). In
fact, 9G8 expression, similar to that of the other SR factors, is
likely ubiquitous, but it has been shown that the SR factors recognized
by mAb104 (SRp20, SRp30, SRp40, SRp55, and SRp75) are expressed at
different levels in various calf tissues (Zahler et al.,
1993a). We show in this paper that the expression of the 9G8 mRNA is
the target of different regulations such as alternative splicing and
alternative polyadenylation, leading to five well detectable species
from 1.3 to 3.8 kb. One interesting feature is the retention of the
entire or a part of the intron 3, because it leads to the translation
of a truncated form of the 9G8 protein devoid of the SR domain by
introducing a stop codon downstream of the exon 3/intron 3 junction (Fig. 8). In fact, although there are no important variations in
the total amounts of 9G8 transcripts within the tested fetal tissues (Fig. 7), some important changes within the distribution of each
species occur. The existence of the alternative splicing of intron 3,
most likely due to the existence of a suboptimal 3` splice site at the
end of this intron (see the sequence in Fig. 8) seems puzzling
if it is thought that maximal levels of SR factors are required for an
efficient splicing machinery. However, we have to take into account
that levels of SR factors are variable (Zahler et al., 1993a)
and may participate to the modulation of alternative splicing, as
proposed by these authors. Moreover, our data raise the interesting
possibility that the splicing of intron 3 might be submitted to
regulation, because its 3` splice site is similar to other weak 3`
splice sites, such as the female-specific 3` splice site of double-sex
pre-mRNA (Ryner and Baker, 1991; Hedley and Maniatis, 1991) or the
fibronectin ED1 exon (Caputi et al., 1994). In this respect,
purine-rich motifs of the form CAGGAGGAA, CAGCAGGAG, and CAGGGACGAAG,
located downstream within the exon 4 of this gene, resemble cis-acting
motifs found in the M2 exon of IgM gene (Tanaka et al., 1994),
troponin (Xu et al., 1993), fibronectin (Lavigueur et
al., 1993; Caputi et al., 1994), or bovine growth hormon
gene (Dirksen et al., 1994). They may be important for the
excision of the whole intron 3. In fact, a mRNA variant containing a
retained intron had also been previously described for the ASF/SF2
protein (Ge et al., 1991), but the amount of this species was
small (<5%). Interestingly, the
Figure 8:
Representation of the splicing events
occurring on intron 3 and incidence on the primary sequence of the 9G8
protein. A, the intron 3 and the flanking exons are
represented. Exonic sequences are boxed, and the different
splice sites are indicated by an arrow. Under the diagram the
amino acid sequence of each splice variant is indicated (stop codons
are indicated by an asterisk). B, the sequence of the
two alternatively used 3` splice sites are designed. The exonic
sequences are written in uppercase letters and intronic
sequences in lowercases letters.
We
also show that the 9G8 pre-mRNA is alternatively processed in its
3`-untranslated region. Looking at polyadenylation signals within the
genomic primary sequence, we find one ATTAAA motif at position 1433 and
one AATAAA motif at position 2334 that are used in vivo. It
had been observed that SC35/PR264 pre-mRNA was submitted to an
alternative polyadenylation coupled to alternative splicing within its
3`-untranslated region (Sureau and Perbal, 1994). Differences in the
stability of the SC35/PR264 mRNA species have been demonstrated in
agreement with studies showing that sequences contained in the
3`-untranslated region are involved in the stability of the mRNA or in
the control of translation (Sachs, 1993). In conclusion, we have
identified splice variants of the 9G8 transcript that may allow the
synthesis of significant and variable amounts of 9G8 SR factor deleted
of the SR domain. It will be interesting to determine whether the
differential expression of the 9G8 factor may be involved in the
modulation of alternative splicing in vivo.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
RNA isolated from human fetal tissues has
allowed us to identify five different species, generated by alternative
splicing of intron 3, which may be retained or excised as a shorter
version, as well as the use of two polyadenylation sites. We also show
that the different isoforms are differentially expressed in the fetal
tissues. The persistence of sequences between exon 3 and 4 results in
the synthesis of a 9G8 protein lacking the SR domain which is expected
to be inactive in constitutive splicing. Thus, our results raise the
possibility that alternative splicing of intron 3 provides a mechanism
for modulation of the 9G8 function.
(
)splicing factors and hnRNP proteins (for reviews,
see Green(1991) and Moore et al. (1993)). Numerous splicing
factors have been characterized in both lower (mainly the PRP factors)
and higher eukaryotes. In higher eukaryotes, a unique set of factors,
which belong to the family of the splicing factors (called the SR
factors) rich in serine and arginine residues, is involved in the first
steps of splice sites recognition. At present, seven SR factors have
been identified and characterized: SF2/ASF (Krainer et al.,
1990a, 1990b; Ge and Manley, 1990), SC35/PR264 (Fu and Maniatis, 1990,
1992; Vellard et al., 1992), SRp20 (also named X16) (Zahler et al., 1992; Ayane et al., 1991), SRp55 (Roth et
al., 1991; Mayeda et al., 1992), SRp75 (Zahler et
al., 1993b), RBP1 (Kim et al., 1992), and the 9G8 factor
(Cavaloc et al., 1994). All these factors share two common
characteristics: one or two RNA binding domains (RBD) near the amino
terminus and a domain rich in serine/arginine (the SR domain) at the
carboxyl terminus. The SR factors range in mass from 20 to 75 kDa and
the best characterized are the 30-kDa SF2/ASF and SC35 (Krainer et
al., 1990a, 1990b, 1991; Ge and Manley, 1990; Ge et al.,
1991; Fu and Maniatis, 1990, 1992; Zahler et al., 1992).
30 kDa) similar to
those of SF2/ASF and SC35 (Cavaloc et al., 1994). However, its
primary sequence in the RBD is only
40% conserved relative to
SF2/ASF and SC35. In addition, 9G8 presents some specific features
since it contains an RRSRSXSX consensus sequence
repeated six times in the SR domain and a CCHC zinc knuckle motif in
its median region (Cavaloc et al., 1994). The occurrence of a
large family of SR splicing factors which are differentially expressed
in organisms raises questions related to the structure of their genes,
the existence of a common ancestral gene, and the molecular basis of
their modulated expression. In contrast with the abundant data on the
SC35/PR264 gene (Sureau et al., 1992; Sureau and Perbal,
1994), very little is known about the genomic structure and the
expression of these factors. We report here the isolation and
characterization of the 9G8 gene, the determination of the exon/intron
organization, a succint analysis of the promoter, and the determination
of the structure of the mRNA isoforms produced by alternative splicing
and polyadenylation.
Isolation of Human 9G8 Gene
A 5` P-labeled 38-nt probe spanning positions
+153/+190 of the cDNA (QE203) or a
P-labeled
random priming cDNA PCR product using the QO60 and QN140
oligonucleotides (+682/+902) have been used to screen a human
placental genomic library in
GEM 12. Duplicate plaque lifts were
prepared and probed as described (Sambrook et al., 1989).
Three positive genomic clones containing inserts of 17 (
9G8-I), 15
(
9G8-II), and 17 kb (
9G8-III) were isolated from 8.10
recombinant phages.
Design of Probes
cDNA 1 probe, a 330-bp BstBI-BglII fragment (265/595) and cDNA 2 probe, a
709-bp EcoRI-EcoRI fragment (262/971) were obtained
from the 9G8 cDNA clone 3 (Cavaloc et al., 1994);
3`-untranslated region, a 383-bp NdeI-AvaII fragment
(1003/1386), was obtained from the 3.2-kb SacI subclone of
9G8-III. The probes used for the recognition of intron 3 were
obtained by an amplification by PCR of a fragment of 1064 bp containing
the total intron 3 and short sequences of the surrounding exons using
the primers QM95 (5`-TTTGATAGACCACCTGCC-3`) and QP101
(5`-TTCGTCCCCTGCTCCTGCTGC-3`). The resulting fragment was then cleaved
with RsaI and XbaI, and two DNA bands of 450 bp (IVS
3 up) and 327 bp (IVS 3 down) were gel-purified. Each probe was labeled
with [
P]dCTP by random priming.
Southern Blot Analysis and Subcloning of DNA
Fragments
For the Southern blot analysis of the recombinant
phages, 2 µg of each DNA were digested with SacI or EcoR I restriction enzymes. One µg was run on 5%
polyacrylamide gel and then transferred to a nylon membrane filter
Hybond N+ with 0.4 M NaOH as described by the supplier.
The phage DNA digested by SacI was used to subclone, by
shotgun technique, the different fragments produced. For the genomic
Southern analysis, 15 µg of human placental DNA digested by EcoRI and SacI were electrophoresed on 0.8% agarose
gel and transferred to a nylon membrane (Hybond N+).P. The blot containing human placental DNA
was probed with the
P-labeled random priming cDNA 1 and
cDNA 2 probes. The hybridization was performed at 42 °C for 16 h in
the hybridization solution (2
SSC, 0.1% SDS, 1
Denhardt's solution, 30% formamide, and 10 µg/ml salmon sperm
DNA), except for the QE203 probe where formamide was omitted. The
filters were then washed with 0.2
SSC and 0.1% SDS at 45 °C
(QE203 probe) or at 60 °C (other probes) and subjected to an
overnight autoradiography.
Primer Extension Analysis
The 5` P
labeled QM94 (5`-GCAGCGCCCAGGGCTCGAGTGAC-3`) or QO14
(5`-GTAACGCGACATGATGACAGACC-3`) were annealed overnight with aliquots
of 1 µg of 293 cells poly(A)
RNA in a solution 1
NPES (250 mM NaCl, 40 mM Pipes, pH 6.4, 5
mM EDTA, 0.2% SDS). After precipitation and washing, the
extension reaction was performed with 10 units of avian myeloblastosis
virus reverse transcriptase for 30 min at 42 °C. The RNA was then
degraded by 0.2 M NaOH at 42 °C and the extension DNA
product was electrophoresed on a 6% denaturing polyacrylamide gel
containing 8 M urea.
Construction of Reporter and Expression
Plasmids
Artificial restriction sites HindIII and BamHI were inserted by PCR technique at positions -420
and +25, respectively. After endonuclease digestion, the PCR
product was inserted in the corresponding sites of the pBLCAT3 (Luckow
and Schtz, 1987), giving the 9G8
``full-length'' construct (9G8FL) (Fig. 6B).
This region contains one StuI site at position -205 and
a NotI site at position -38. Deletions mutants
9G8H/S and 9G8
S/N were obtained by releasing respectively HindIII-StuI and StuI-NotI
fragments from the 9G8FL vector, blunting, and religating the vector
backbone. Deletion mutant 9G8
S/-72 was obtained by releasing
the StuI-NotI fragment from the 9G8FL vector and
inserting the phosphorylated double-stranded 36-mer oligonucleotide
corresponding to the +72/NotI fragment of the wild type
promoter. The 9G8
-88/-34 mutant was generated by
cutting the 9G8FL construct with SphI and digesting with
Bal-31 for various times. DNA was then blunted with the Klenow fragment
of DNA polymerase and religated. All constructs were confirmed by
restriction analysis and sequencing. The pE1A, pSVCREM
, and
pSVBmyb clones that express the 293-amino acid protein of adenoviral
E1A unit, CREM
, and c-Myb, respectively, were described
previously (Leff et al., 1984; Foulkes et al., 1992;
Sureau et al., 1992).
Transfections and CAT Assays
JEG-3 human
choriocarcinoma cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and were
transfected by calcium phosphate coprecipitation technique. Cells were
plated at a density of 10 cells/10-cm plate and transfected
with 10 µg of total plasmid DNA. 3 µg of reporter plasmid was
included in each transfection sample together with 1 µg of
pSVCREM
, pE1A, or pSVBmyb expression plasmids. CAT activity was
assayed as described previously (Sassone-Corsi et al., 1988)
and was quantified by PhosphorImager counting. The activity values
correspond to the percentage of chloramphenicol modified by the
chloramphenicol acetyltransferase.
Northern Blot Analysis
The human fetal multiple
tissue Northern blot, containing 2 µg of poly(A) RNA from each tissue was obtained from Clontech (catalog number
7761-1). The membrane was prehybridized and hybridized in
pre/hybridization solution (5
SSC, 10
Denhardt's
solution, 45% formamide, 1.5% SDS, and 100 µg/ml salmon sperm DNA)
at 42 °C. After a 20-h hybridization with radiolabeled probe (2
10
cpm/ml), the blot was washed 30 min at 55 °C
in 0.1
SSC and 0.1% SDS and exposed 16 h with Kodak X-Omat film
or was quantified by PhosphorImager counting.
Isolation and Structural Organization of the Human 9G8
SR Splicing Factor Gene
We previously cloned a cDNA encoding
the 9G8 SR factor (Cavaloc et al., 1994). To isolate the
corresponding genomic clone, a 38-nt oligonucleotide probe encompassing
an amino acid sequence of the RBD not present in the other SR factors
was used to screen a placental human genomic library in GEM12. We
obtained two genomic clones
9G8-I and -II containing inserts of 17
and 15 kb, respectively (see also Cavaloc et al., 1994).
However, preliminary analysis indicated that they do not cover the
entire open reading frame of the 9G8 mRNA. Therefore, a 211-bp PCR
product, from positions 692 to 902 of the 9G8 mRNA, covering the
C-terminal region of the 9G8 factor, allowed the isolation of another
17-kb clone (
9G8-III). Analysis of the three clones by restriction
endonuclease mapping and Southern blotting using the specific probes
mentioned above revealed that the inserts overlap and together cover
the entire open reading frame of the 9G8 gene (Fig. 1). The
5.5-kb SacI fragment at the 3` terminus of the clone I insert,
as well as the 2.6-kb internal fragment and 3.2-kb fragment at the 3`
terminus of the clone III insert were subcloned into pBluescript
SK+ and used for further characterization and sequencing analysis.
9G8-I, -II, and -III are shown below the schematic representation
of the 9G8 gene. The different restriction enzymes sites are
represented as follows: H, HindIII; S, SacI; X, XhoI; and E, EcoR
I. The complete genomic sequence is available in GenBank®
(accession number L41887).
Sequencing of the Human 9G8 Gene and Its Genomic
Organization
Complete exon-intron organization of the 9G8 gene
was determined by sequencing the totality of the gene. Using the
previously cloned 9G8 cDNA sequence as the reference mRNA sequence, we
have determined that the 9G8 gene is 7745 nucleotides long and contains
8 exons and 7 introns (Table 1). The exons range from 36 (exon 7)
to 1572 bp (last exon), and the intron sizes vary from 308 to 1298 bp (Table 1). From the sizes of exons and introns as well as the
splice site sequences which fulfill the GT-AG rule (Breathnach and
Chambon, 1981), we deduced that the 9G8 gene exhibits features typical
of many eukaryotic genes. The translated sequence of 9G8 is highly cut
up as the open reading frame is distributed over the 8 exons.
Interestingly, the RBD is contained in exon 2, and the exon 3 encodes
for the zinc knuckle motif (Cavaloc et al., 1994). In
contrast, the SR domain which is 110 amino acids in size covers exons
4-8. A study of the DNA sequence downstream the stop codon
reveals two polyadenylation signals at 610 and 1511 bp downstream of
the stop codon.
Southern Analysis and Chromosomal
Localization
Southern blot analysis of human genomic DNA
digested by EcoR I or SacI was performed using the
two cDNA probes cDNA 1 (positions 265/595) covering exons 2 and 3 and
cDNA 2 (positions 262/971) covering exon 2 to exon 8 (Fig. 2).
The short cDNA probe revealed a single band after digestion with either EcoRI (2.7 kb) or SacI (7.5 kb), in agreement with
what was expected from structure of genomic clones (Fig. 1). In
contrast, the extended probe (cDNA 2) detected an additional fragment
in the EcoRI (8 kb) and SacI (2.5 kb)
restriction digests (Fig. 2), consistent with the structure of
the
9G8-III clone shown in Fig. 1. Thus, these results
confirm the structural organization of the 9G8 gene. They indicate also
that 9G8 is encoded by a single copy gene and that no pseudogenes are
present in the genome. The chromosomal location of the 9G8 gene was
determined by in situ hybridization using the cDNA 2 probe.
Analysis of 100 metaphase cells revealed a total of 237 silver grains
on chromosomes, and 51 of these (21.5%) were located on chromosome 2.
Analysis of the grain distribution indicated that 36 out of 51 (70.6%)
of these mapped to the p22-21 region on the short arm of chromosome 2,
with an intense localization in the p21 band (Fig. 3). This
result allows to unequivocally assign the 9G8 gene to the p22-21 region
of human chromosome 2.
Identification of the Transcription Initiation
Site
To determine the transcription initiation site, we
performed a primer extension analysis (Fig. 4). Primer extension
on poly(A) RNA isolated from 293 cells, using two
23-mer oligonucleotides QM94 (upstream the AUG codon) and QO14
(encompassing the AUG codon) resulted in the synthesis of cDNAs of
72-70 residues (Fig. 4) or 117-115 residues (not
shown), respectively. Comparing the extension products with a sequence
ladder generated by extending the same primers from a plasmid
containing incomplete 9G8 cDNA localized the initiation site at a G
residue, downstream from the CT-rich sequence CTCTTCCTC/G + 1. It
is not known if the cDNA beginning 2 residues downstream of the longer
cDNA (Fig. 4) corresponds to a true initiation site at an A
residue or to a premature stop of the primer extension.
or poly(A)
RNA of 293 cells or 2.5 µg of tRNA of Escherichia
coli. Extension with reverse transcriptase was as described under
``Materials and Methods.'' The same primer was employed for
dideoxy sequencing (lanes, A, C, G, and T) in which
the template was an incomplete cDNA clone. The primer extended and the
sequence products were electrophoresed on a 6% polyacrylamide gel. The
DNA band representing the major transcription start site, 72 nt
upstream the 5` end of the QM94 primer, is denoted by an arrow.
Structure of the 9G8 Upstream Region
Analysis of
the sequence of the 5`-flanking region of the 9G8 gene contained in the
5.5-kb SacI subfragment reveals a high GC content (57%) and
several promoter elements (Fig. 5). A TATA motif (TATATAA) is
present at position -29, and three potential SP1 binding sites
(GGCGGG) are found at positions -87, -148, and -224.
Computer search reveals also putative regulatory elements (Locker and
Buzard, 1990; Faisst and Meyer, 1992). Two sequence motifs for
liver-specific factors A1 (LFA1), TGAACC and TGACCC, are present at
positions -149 and -345, and one possible AP-2 motif
(GCCTGGg), which deverges from the AP-2 consensus by one nucleotide, is
located at position -298. In addition, an ATGACGcA sequence,
which exhibits a good match with the consensus ATF site is present at
position -59 and overlaps a TGACGcat sequence, with a significant
homology to the CRE motif. Sequences identical to the core consensus
for Ets (GGAAPu) are also present at positions -266, -261,
-208, and -115. Finally, minimal consensus sites for Myb
(AACNG) are located at positions -96, -121, -239, and
-375.
Functional Analysis of the 5`-Flanking Region
To
characterize the upstream region of the 9G8 gene, a DNA fragment
spanning from -414 to +26 (clone 9G8FL), as well as
fragments containing various deletions, were fused to the bacterial CAT
reporter gene in the pBLCAT3 plasmid (Fig. 6A). These
constructs were transiently expressed into JEG-3 cells, which express
9G8 mRNA at levels similar to those of HeLa cells and cellular extracts
were assayed for CAT activity 36 h after transfection. In Fig. 6B, we show that the upstream region was able to
induce significant CAT activity. However, a deletion of the -202
to -36 region (clone 9G8S/N), which leaves the TATA motif
intact, results in an 8-fold reduction of the CAT activity (compare the
first and the fifth bars). A similar reduction was observed with the
almost complete deletion of the 9G8 upstream region from -414 to
-36 (not shown). Moreover, two smaller deletions
-202/-72 (clone 9G8
S/-72) and
-88/-34 (clone 9G8
-88/-34), which cover
the -202 to -36 deletion (Fig. 6A),
resulted in both cases in a
2.5-fold reduction compared with the
wild type construct. In contrast, deletion of the region -414 to
-203 (clone 9G8
H/S) induced a 2-fold stimulation of the
promoter activity (compare the first and the second bars), indicating
that the most upstream sequences do not contain strong positive acting
elements and that some negative elements may be present. Thus, this
deletional analysis shows that the upstream region of the 9G8 gene is
effective in activating a CAT reporter gene and that the positive
acting elements are mainly concentrated all along the -202 to
-34 region, immediately upstream of the TATA box. The presence of
many putative regulatory elements served as a guide for testing, in a
preliminary manner, several trans-acting factors. The effect of
CREM
+PKA, Myb, and E1A factors, which are known to activate
many cellular genes, were analyzed by cotransfection experiments. We
found that each factor stimulates the transcription around three fold
(not shown), suggesting that the 9G8 promoter is able to respond to
different trans-acting factors.
The 9G8 Gene Is Alternatively Spliced
Northern
blot analysis of human fetal poly(A) RNA isolated from
the brain, liver, kidney, and lung (Clontech) was carried out using a
330-nt cDNA probe, covering exons 2 and 3 of the 9G8 RNA (cDNA 1). We
detect five mRNAs of approximately 1.3, 2.0, 2.4, 2.6, and 3.8 kb in
size, respectively, the 2.4- and 2.6-kb being very close (Fig. 7, lanes 1-4). We observed that the same
five transcripts are present in adult poly(A)
RNAs
(Multiple Tissue Northern blot 1, Clontech), suggesting that none of
the isoforms is specific for a developmental stage (not shown).
1 kb) could
account for different sizes of mRNA observed. (ii) The intron 3, since
further characterization of the cDNA clones for 9G8 isolated previously
(Cavaloc et al., 1994) indicated that two out of nine cDNA
clones contained this intron (not shown). We observed that the 3.8- and
2.6-kb mRNA species use the distal poly(A) signal (Fig. 7,
second panel), and that only the 3.8-, 2.4-, and 2.0-kb species contain
intron 3 sequences (not shown). Nevertheless, the 2.0-kb species is too
short to contain the entire intron 3. Looking for consensus signals
within this intron, we have found one potential 3` splice site in its
middle. By using DNA probes specific for the sequences downstream (Fig. 7, lanes 9-12) and upstream (Fig. 7, lanes 13-16) of this splice site, we show that the
2.0-kb mRNA is generated by the use of this alternative 3` splice site
and that the 3.8- and 2.4-kb mRNA contain the totality of intron 3
sequences. The putative structure of all the mRNA isoforms is given in Fig. 7. A quantitative analysis of the relative abundance of the
different species in various fetal tissues indicates that the 1.3-kb
species, which encodes the whole 9G8 factor, is highly predominant in
the liver, but it appears as a minor isoform in the kidney. In
contrast, the 2.4-kb isoform, which contains the entire intron 3, is
the predominant species in kidney. A quantitative estimation of the 9G8
mRNA isoforms indicates that the relative ratio of the intron 3 minus
transcripts (1.3- and 2.6-kb species) to the intron 3 plus transcripts
(2.0-, 2.4-, and 3.8-kb species) varies from about 1 to 5 between the
kidney and the liver, respectively.
SR splice variant of ASF/SF2
conserves all its ability to modulate the alternative splicing, but
loses its characteristics of constitutive splicing factor (Zuo and
Manley, 1993; Caceres and Krainer, 1993). Nevertheless, it is still
unknown whether this type of truncated SR factors is involved in the
regulation of alternative splicing pathways in vivo.
We are grateful to Dr. I. Davidson for critical
reading of the manuscript. We thank G. Hildwein for excellent technical
assistance, the cell culture group for growing cells, the photographic
staff for preparation of the manuscript, B. Chatton, N. Foulkes, and J.
Soret for the gift of clones.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.