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of Cyclic Adenosine 3',5'-Monophosphate-Dependent Protein Kinase: Two Distinct Promoters Provide Differential Regulation of Alternately Spliced Messenger Ribonucleic Acids1
Institute of Medical Biochemistry, University of Oslo (R.S., M.S., V.N., V.H., T.J., K.T.); the Department of Anesthesiology, Ullevål Hospital (M.S.); and the Hormone Laboratory, Aker Hospital (P.A.T.), Oslo, Norway
Address all correspondence and requests for reprints to: Rigmor Solberg, Ph.D., Institute for Surgical Research, Rikshospitalet-The National Hospital, N-0027 Oslo, Norway. E-mail: rigmor.solberg{at}basalmed.uio.no
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Abstract |
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gene of cAMP-dependent protein kinase and approximately 2
kilobases (kb) of the 5'-flanking region obtained by isolation and
sequencing of several phage clones from human genomic libraries. The
RI gene is composed of nine coding exons of varying lengths,
separated by introns, giving the gene a total length of at least 21 kb.
Our recent cloning of a processed RI pseudogene with a 5'-noncoding
region different from the previously reported RI complementary DNA
indicated that the RI gene may have multiple leader exons giving
rise to alternately spliced messenger RNAs (mRNAs). Reverse
transcription of human testis RNA followed by PCR identified two
different RI mRNA species (RI1a and RI1b) containing distinct
5'-sequences due to alternately splicing the gene. The previously known
RI1b mRNA revealed low constitutive expression in a human B lymphoid
cell line (Reh) and was stimulated only 4- to 6-fold by treatment with
cAMP. In contrast, very low levels of the novel RI1a mRNA were
present in untreated Reh cells, but were stimulated 40- to 50-fold by
cAMP. The 5'-flanking sequence of the RI gene was G/C rich and did
not contain any TATA box. Several putative transcription initiation
sites were identified in front of each leader exon (exons 1a and 1b) by
the 5'-rapid amplification of complementary DNA ends technique. To
determine whether the sequences 5' of both leader exons had promoter
activities, the 5'-flanking sequences of exons 1a and 1b were inserted
in front of a chloramphenicol acetyltransferase reporter gene, and
their ability to direct transcription were examined. Transfection of
these constructs into rat GH4C1 cells
demonstrated that both constructs had promoter activities, as evidenced
by high levels of chloramphenicol acetyltransferase activity. |
Introduction |
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, RIß, RII, and RIIß) and three different C subunits
(C, Cß, and C
) have been identified in man (for references, see
Ref.3). Furthermore, splice variants of C and Cß have been
reported (14, 15).
The promoter regions of the genes for porcine RI (16), mouse RIß
(17, 18), and mouse/rat RIIß (19, 20, 21) have previously been described.
Of the C subunits, the complete exon-intron structure of C as well
as the promoter region of Cß have been reported (22). The promoters
of the PKA subunit genes are TATA-less and G/C rich, and initiation of
transcription occurs at multiple sites. However, no sequence similarity
is found in the 5'-flanking regions of these genes.
To date, splice variants of R subunits of PKA have been reported only
for the Drosophila RI subunit (23). In this case, alternate
initiation at an intron 3' to the first coding exon results in
transcripts encoding truncated proteins starting at either amino acid
54 or amino acid 80. During our cloning of the human gene encoding the
regulatory subunit RI, screening for genomic clones resulted in the
isolation of clones representing a processed, intron-less RI
pseudogene flanked by long terminal repeats (24). Processed pseudogenes
are colinear with normal cellular messenger RNAs (mRNAs), starting at
the 5'-cap site and ending at an oligo(deoxyadenosine) stretch. They
are generally thought to be incorporated in the genome after
retrovirus-dependent reverse transcription of mRNAs (25, 26). The RI
pseudogene was 89% homologous to the open reading frame of the
previously reported RI complementary DNA (cDNA) and contained
several stop codons (24). However, the 5'-region of the pseudogene was
not colinear with the 5'-nontranslated area of the known cDNA, but was
homologous to an upstream area in the porcine RI gene sequence. This
raised the possibility that the RI pseudogene originated from a
previously unknown, alternately spliced RI mRNA (24). In the present
study, experiments were designed to elucidate whether such an
alternately spliced mRNA could be detected. The results show that two
distinct, alternately spliced RI mRNAs (RI1a and RI1b) exist
due to initiation at different transcription start sites, and that
these two mRNAs (encoding the same protein) are subject to differential
regulation by cAMP. Furthermore, transfection studies demonstrated that
the 5'-flanking region of both leader exons (exons 1a and 1b) reveals
promoter activities and direct expression of a chloramphenicol
acetyltransferase (CAT) reporter gene in GH4C1
cells.
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Materials and Methods |
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Probes
Probes used for screening were a 5' 0.8-kilobase (kb)
EcoRI fragment and a 3' 0.6-kb EcoRI fragment of
the human RI cDNA (28). Furthermore, a 325-bp fragment covering
nucleotides 178–502 of the RI cDNA was amplified using PCR (primers
RIaEx3u and RIaEx4l; for all oligonucleotide sequences, see Table 1
) (29) and used to identify clones covering this
specific region. All oligonucleotides in this study were purchased from
Genosys Biotechnologies (The Woodlands, TX).
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1a mRNA based on the genomic sequence in the 5'-region of
the RI gene and correspondence to the 5'-region of the processed
RI pseudogene (24). To obtain maximal sensitivity in the detection
of RI1a mRNA, a 112-bp fragment (nucleotides -110
+2) of the
RI1a cDNA was amplified and used as a probe (primers RIaEx1au and
RIa1aRl). The oligonucleotide RIaEx1bN located in exon 1b was used to
detect the RI1b mRNA. The probe used to identify all RI mRNA
species was a 0.8-kb EcoRI fragment of the human RI cDNA
(28) covering 0.7 kb of the common RI-coding region.
Labeling of oligonucleotides and cDNA probes
Exon-specific oligonucleotides were end labeled to a specific
activity of 1–2 x 108 cpm/µg by incubation with T4
polynucleotide kinase (Life Technologies, Gaithersburg, MD) in the
presence of [-32P]ATP (5000 Ci/mmol; Amersham,
Aylesbury, UK). The 0.8- and 0.6-kb EcoRI fragments of RI
cDNA and the RI-specific PCR products were labeled to a specific
activity of 0.8–1.2 x 109 cpm/µg using
[-32P]deoxy-CTP (3000 Ci/mmol; Amersham) and standard
nick translation or random prime kits (Amersham).
Analysis of genomic RI clones
DNA from the positive genomic clones were purified by the liquid
lysate technique (30) and characterized by restriction endonuclease
mapping and Southern blotting (see below). Fragments of interest were
identified using 32P-labeled human RI probes or
exon-specific oligonucleotides. Hybridizing fragments were subcloned
into plasmid (pUC, pBluescript) or phage (M13) vectors. Sequencing was
performed by the dideoxy chain termination method (31), using Sequenase
DNA polymerase (U.S. Biochemical Corp., Cleveland, OH) and exon- or
vector-specific oligonucleotide primers. Nucleotide analogs
(7-deaza-GTP and deoxy-ITP; Pharmacia, Uppsala, Sweden) were employed
to resolve GC compressions. The nucleotide sequence of the 5'-flanking
region was determined on both strands. Production of overlapping clones
and computer analyses were performed as previously described (27).
Southern blotting
Agarose gels were denatured in 0.5 M sodium
hydroxide-1.5 M sodium chloride and neutralized in 3
M sodium acetate (pH 5.5) before blotting onto nylon
membranes (Biotrans, ICN, Irvine, CA). The membranes were then baked at
80 C for 1 h. Hybridization was performed for 16 h in 50%
(vol/vol) formamide, 5 x SSC (standard saline citrate), 5 x
Denhardt’s solution, 50 mM sodium phosphate (pH 6.5),
0.1% SDS, and 250 µg/ml single stranded salmon sperm DNA at 42 C
when cDNA probes (1 x 106 cpm/ml) were employed. For
hybridization with the PCR or oligonucleotide probes, the same
hybridization solution was used except the concentration of formamide
was 40% (vol/vol), and yeast transfer RNA (50 µg/ml; Boehringer
Mannheim, Mannheim, Germany) was added. The filters were washed twice
in 0.1 x SSC-0.1% SDS for 15 min at 50 C. Autoradiography was
performed at -50 C using Amersham Hyperfilm MP (Amersham).
PCR cloning of exon-containing gene products
Clones 1 and 4 (Fig. 1) were analyzed for the
presence of exons 3 and 4 by the use of PCR. PCR (29) was performed
using the primers RIaEx3u and RIaEx4l, 1 ng phage DNA as template, and
30 cycles of denaturation at 94 C for 1 min, annealing at 54 C for 1
min, and synthesis at 72 C for 1 min. The PCR products were subcloned
into the vector pCRII (Invitrogen, San Diego, CA) and sequenced as
described above using vector-specific primers.
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A neoplastic B precursor cell line (Reh) was maintained as described previously (32, 33). Rat pituitary GH4C1 cells (kindly provided by Dr. David D. Moore, Harvard Medical School, Boston, MA) were grown in Nutrient Mixture Ham’s F-10 Medium supplemented with 10% FCS, 146 mg/liter L-glutamine, 105 IU/liter penicillin, 100 mg/liter streptomycin, and 0.25 mg/liter fungizone.
Preparation of RNA and Northern analyses
Extraction of total RNA from cells and tissues and Northern
analyses were performed as described previously (32), except that
hybridization with oligonucleotides was performed in a solution
containing 40% formamide and with addition of yeast transfer RNA (50
µg/ml; Boehringer Mannheim). Densitometric analyses were performed on
suitably exposed autoradiograms using an Omnimedia 6cx/s scanner (X-Ray
Scanner Corp., Torrance, CA) and a Visage 4.60 software analysis
package (Bio Image Products, Millipore Corp., Ann Arbor, MI). Controls
were assigned the relative value of 1, and the data are presented
relative to control levels.
PCR cloning of RI1a and RI1b cDNA
First strand cDNA from oligo(deoxythymidine)
[oligo(dT)]-primed human testis total RNA (see above) was synthesized
employing a reverse transcriptase kit (Superscript preamplification
system, Life Technologies) according to the instructions of the
manufacturer. PCR of RI1a and RI1b cDNA was performed employing
exon-specific upper primers (RIaEx1au and RIaEx1bu) designed from
sequence information in the 5'-area of the RI gene corresponding to
the RI pseudogene 5'-region (exon 1a) and the human RI cDNA
5'-region (exon 1b) (28), respectively. A lower primer located in the
common coding region (RIacoml) was used in both PCR reactions. The PCR
mixtures were initially denatured at 94 C for 5 min before the addition
of Taq polymerase (Perkin-Elmer/Cetus Corp., Norwalk, CT)
and PCR proceeded by 30 cycles of denaturation at 94 C for 1 min,
annealing at 58 C for 1 min, and synthesis at 72 C for 1 min, followed
by 10-min elongation at 72 C after the last cycle.
PCR products were identified by gel electrophoresis in 10% polyacrylamide gels followed by ethidium bromide staining. Amplified DNA fragments were subcloned in the vector pCRII (Invitrogen) and sequenced (see above).
5'-Rapid amplification of cDNA ends (5'-RACE)
5'-RACE was performed using a 5'-AmpliFINDER RACE Kit (Clontech)
and 5'-RACE-Ready cDNA from human lung (Clontech). The AmpliFINDER
anchor primer was used in combination with two nested RI-specific
primers (RIacoml and RIa1aRl or RIa1bRl) to amplify either the RI1a
or the RI1b cDNA 5'-ends. The primary reaction was initially
denatured at 95 C for 5 min before adding the primers, followed by 30
cycles of denaturation at 94 C for 1 min, annealing at 58 C for 1 min,
and synthesis at 72 C for 1 min. Secondary PCR was performed using
either RIaEx1al or RIaEx1bl and 2 µl of a 1:10 dilution of the
primary PCR reaction mixture. Before adding Taq polymerase,
the reaction mixture was heated to 95 C for 4 min and continued as
described above using 55 C as the annealing temperature. The PCR
products were analyzed by Southern blotting (see above), using either
RIaEx1aN or RIaEx1bN as probes, before they were subcloned to pCRII
(Invitrogen) and sequenced.
CAT reporter constructs
A 572-bp DNA fragment corresponding to nucleotides 1509–2080 of
the human RI 5'-flanking sequence covering the putative promoter
upstream of exon 1a was PCR amplified [30 cycles: 1 min at 94 C, 1 min
at 48 C, 1 min at 72 C in the presence of 10% dimethylsulfoxide
(DMSO)] using primer RIaEx1aN and an outside primer (M13 forward
primer). Similarly, a 486-bp DNA fragment corresponding to nucleotides
1985–2470 of the 5'-flanking sequence of the RI gene covering the
putative promoter upstream of exon 1b was amplified (30 cycles: 1 min
at 94 C, 1 min at 48 C, 1 min at 72 C) using primers RIaEx1au and
RIaEx1bN. PCR products were subcloned in both orientations in front of
a CAT reporter gene in the vector pCATbasic (Promega Corp., Madison,
WI), purified by two cesium chloride gradients, and sequenced to verify
correct orientations.
Transfections and CAT assays
Transient transfections were carried out employing the calcium
phosphate precipitation method as described by Guérin et
al. (34). GH4C1 cells were plated in 60-mm
culture dishes and grown in 5 ml transfection medium (1 x DMEM,
0.1% NaHCO3, 2 mM L-glutamine,
10% FCS, and antibiotics as described above). Fifteen micrograms of
RI-CAT DNA and 5 µg control plasmid DNA (pXGH5) directing the
expression of human GH (hGH) (35) were added to the cell cultures.
After transfection for 18 h, cells were treated for 2 min with
10% DMSO in PBS, and cultures were subsequently continued in
transfection medium in the absence or presence of 100 µM
8-(4-chlorophenylthio)cAMP (8-CPTcAMP). After 48 h of incubation,
cells were harvested in lysis buffer [15 mM Tris-HCl (pH
8.0), 60 mM KCl, 15 mM NaCl, 2 mM
EDTA (pH 8.0), 0.15 mM spermine, 1 mM
dithiothreitol, and 0.4 mM DMSO) (36), and CAT activities
were measured according to the organic phase extraction method (37)
with some modifications (36). Results were normalized against the
levels of hGH directed from the cotransfected plasmid pXGH5. Triplicate
determinations were performed in each experiment, and the data
presented represent the mean of five to eight separate experiments.
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Results |
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gene cDNA or PCR probes resulted in several positive clones. Five
independent clones (clones 1, 4, 5, 8, and 29) were selected for
further analyses. Each clone was plaque purified, and the inserts were
characterized by restriction mapping, Southern blotting, and DNA
sequencing (Fig. 1
).
The isolated clones show that the RI gene comprises at least 21 kb
of human genomic DNA. In addition, the clones contain approximately 8
kb of 5'- and 7 kb of 3'-flanking sequences. All exons were sequenced,
whereas the intron lengths were determined by either sequencing, PCR
(Table 1), or restriction mapping. As shown in Fig. 1, the RI gene
contains 11 exons. The exact number of base pairs in each exon and
their locations relative to the RI cDNA are summarized in Table 2. The introns vary in size from a few hundred to
several thousand base pairs. The sizes of the different introns as well
as the exon-intron border sequences are shown in Table 3. With two exceptions, the exon-intron splice junctions
follow the consensus rule for such sequences, where the 5'-intron
border starts with GT, and the 3'-intron border terminates with AG
(38).
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cDNA gene indicated that an
alternately spliced mRNA, from which the RI pseudogene (24) had been
derived, existed in human testis. Furthermore, another oligonucleotide
(RIaEx1bN) specific to the previously known RI 5'-cDNA sequence also
identified a mRNA in testis (data not shown). To determine whether the
former signal represented an alternately spliced functional mRNA, a
strategy was designed to clone the putative alternately spliced
transcript from human testis (Fig. 2A). This strategy
involved first strand cDNA synthesis using reverse transcriptase and
oligo(dT)-primed human testis total RNA as a template. PCR
amplification of this cDNA using an upper primer (RIaEx1au) specific to
the 5'-nontranslated region of the putative alternately spliced RI
(1a) mRNA and a lower primer (RIacoml) in the coding region of the
RI cDNA would yield a PCR product with an expected size of 373 bp
based upon the sequence of the pseudogene. A control reaction using an
upper primer (RIaEx1bu) specific to the 5'-region of the previously
reported RI (1b) mRNA (28) and the same lower primer should yield a
351-bp fragment after PCR.
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shows the experimental results of this strategy. PCR products
of the expected sizes were detected using the indicated RI1a- or
RI1b-specific primers. When reverse transcriptase was omitted from
the reaction mixtures during cDNA synthesis, no DNA fragments could be
detected after PCR, excluding the possibility that the amplification
products originated from genomic DNA (i.e. from the RI
pseudogene) contaminating the RNA preparation. Southern blot analysis
of DNA from the same reaction mixtures as those shown in Fig. 2B
revealed that only the longer PCR product amplified by the exon
1a-specific upper primer hybridized to an exon 1a-specific
oligonucleotide (RIaEx1aN), and vice versa, only the shorter
PCR product amplified using the exon 1b-specific upper primer
hybridized to an exon 1b-specific oligonucleotide (RIaEx1bN; data not
shown).
DNA fragments from the PCR mixtures were cloned into a cloning vector,
and two individual clones from each amplification were sequenced. The
amplified cDNAs were identical in the nucleotide sequences downstream
of the splice junction of exon 1/exon 2 (Fig. 3).
However, 5' of this splice junction the two cDNAs differed. The cDNA
denoted RI1b (containing exon 1b) was identical to the previously
reported RI cDNA (28). In contrast, the cDNA amplified by the primer
RIaEx1au had a 5'-nontranslated region originating further upstream in
the 5'-flanking region of the RI gene. The diverging parts of the
distinct RI1a and RI1b cDNAs are 5' to the exon 1/exon 2 splice
junction, and thus, the RI1a and RI1b mRNAs originate from
alternate splicing of two different leader exons of the RI gene
(exons 1a and 1b, respectively).
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1a and RI1b
cDNAs, RACE was conducted on first strand cDNA from human lung. Several
clones of varying lengths were subcloned, sequenced, and shown to
extend 30–50 nucleotides 5' of the reverse transcription-PCR products.
Composite 5'-cDNA sequences based on reverse transcription-PCR and
5'-RACE clones are presented in Fig. 3. In addition, Fig. 3 shows where
the exon/intron splice sites (numbered I1a/b to I9) are located in the
cDNA sequence, including the nucleotides at the borders of each
intron.
Several individual clones from each PCR reaction were sequenced. The
noncoding region of the RI1a cDNA product was identical to a region
in the 5'-flanking sequence of the RI gene. Four clones diverged
from the previously reported cDNA sequence at nucleotide 87 (G instead
of A; Fig. 3) (28, 39). Sequencing of the corresponding region in RI
genomic clones as well as resequencing of the previously reported RI
cDNA (28, 39) revealed that this sequence has to be corrected. This
correction (third position of a codon triplet) has no implications for
the translated peptide sequence.
Exon-intron splice junctions compared to protein domain
structure
In Fig. 4, the predicted protein domain structure
of RI is compared to the positions of the exon-intron splice
junctions of the RI gene. As shown in the figure, there is no direct
relationship between the protein domain organization of RI and the
positions of the exon-intron junctions. However, the dimerization
domain is encoded within exon 2, whereas the hinge region is encoded by
exon 3. Furthermore, cAMP-binding domain A is encoded by four exons
(exons 4, 5, 6, and 7), and cAMP-binding domain B is encoded by three
exons (exons 8, 9, and 10). Thus, the A and B domains involved in cAMP
binding are joined together at the junction between exons 7 and 8.
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1a and RI1b mRNAs by cAMP mRNA by cAMP
in the human B lymphoid cell line Reh (33). Figure 5
shows time-dependent changes in the levels of RI mRNAs after
incubation of Reh cells for various time periods (0–48 h) with 100
µM 8-CPTcAMP. Both the RI1a mRNA (Fig. 5A) and the
RI1b mRNA (Fig. 5B) were induced by 8-CPTcAMP with similar kinetics
(maximal stimulation at 4–8 h). However, the magnitude of the
stimulation differed greatly. The RI1b mRNA was expressed at low
levels in the untreated cells and increased 5- to 6-fold by cAMP. In
contrast, the RI1a mRNA was expressed at very low levels in
untreated cells, but was strongly induced (40- to 50-fold) by cAMP.
Hybridization with an RI cDNA probe common to both mRNAs (Fig. 5C),
revealed a cAMP-mediated stimulation (5- to 6-fold) of the total RI
mRNA level similar to that of the RI1b mRNA.
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mRNA levels were
observed (data not shown). Furthermore, inhibition of protein synthesis
by cycloheximide (5 µg/ml) differentially influenced the levels of
expression of RI1a and RI1b mRNAs. Combined treatment with
cycloheximide and forskolin resulted in a superinduction of RI1a
mRNA, whereas cycloheximide by itself had no effect. No synergism by
cycloheximide in combination with cAMP was observed for the RI1b
message (data not shown).
5'-Upstream region of the RI gene
Figure 6A shows a 2600-bp sequence of the 5'-end of
the human RI gene. A schematic representation of the same gene
region is presented below (Fig. 6B). Two leader exons (exons 1a and 1b)
were identified in the 5'-flanking region, based on computer analysis
of the 5'-gene sequence vs. the RI cDNA (28), the RI
pseudogene (24), and the alternately spliced RI1a cDNA (Fig. 3).
Nucleotides -108 to -7 of the previously known RI1b cDNA (termed
exon 1b) correspond to nucleotides 2392–2493, whereas nucleotides
-144 to -7 of the novel RI1a cDNA (termed exon 1a) span
nucleotides 1945–2087 in the gene sequence (Fig. 6A). A consensus
5'-intron border (GT) was located 3' of both exons 1a and 1b (Fig. 6A).
This shows the presence of two leader exons in the human gene that give
rise to alternately spliced mature RI mRNAs.
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gene did not contain any classical
TATA boxes. Two inverted CAAT boxes were identified in front of exon 1b
(Fig. 6A). There was 72% G/C in the area preceding exon 1a (termed
promoter 1a) and 61% G/C in the area preceding exon 1b (termed
promoter 1b). The locations of exon 1a, exon 1b, and the various DNA
elements in their respective promoter regions are indicated in Fig. 6B.
Computer analysis (40) of the 5'-gene region identified several GC
boxes (potential Sp1 binding sites) (41) located upstream of both
leader exons (Fig. 6). Eight recognition sites for the activator
protein-2 (AP-2) (42) were clustered in front of exon 1a, and one AP-2
site was located in front of exon 1b. Furthermore, two potential AP-1
sites and two potential nuclear factor-1 (NF-1) sites were located in
the 5'-flanking region. In addition, a cAMP response element sequence
(CRE) (43) was located 5' of exon 1b (nucleotides 2326–2333), whereas
a CRE half-site was identified inside exon 1a (nucleotides 1950–1954;
Fig. 6A).
The 5'-sequence of the human RI gene was compared with the
corresponding region of the porcine RI gene (16). The similarity
starts at nucleotide 1776 in the human 5'-gene sequence (Fig. 6A),
which aligns to nucleotide 2 in the porcine 5'-gene sequence. The
overall nucleotide identity between the two genes was 80% in the
5'-flanking region, when gaps were introduced to allow maximal
alignment. The novel leader exon 1a of the human gene contained 80%
A/G and aligned to an A/G-rich sequence reported in the porcine gene
(16). Furthermore, the exon 1a 3'-splice donor site was conserved
between the human and the porcine gene, indicating that the porcine
RI gene may also have two leader exons. The region corresponding to
exon 1b of the human gene was similar to exon 1 in the porcine gene.
The 3'-splice donor site of exon 1b/exon 1 (human/porcine) was also
conserved.
Sequencing of several RACE clones demonstrated some variability in the
5'-extension of the different clones. The 5'-end of individual RACE
clones indicated the approximate transcription initiation sites in the
RI gene and show that both leader exons have several putative
initiation sites (Fig. 6A). Several transcription initiation sites have
also been found in the porcine gene (16) (Fig. 6A).
Two inverted CAAT boxes were found in the human RI gene (Fig. 6A).
One CCAAT sequence was also present in the porcine gene and had exactly
the same spacing to exon 1b as that in the human gene. The other CAAT
box was also conserved and overlapped the CRE sequence found in both
genes. There was a GA repetitive sequence located at nucleotide 90–145
in the human gene. This sequence was difficult to resolve due to
nucleotide compressions; however, we were able to identify 28 GA
repetitions in a row.
Comparison of the mouse RIß promoter region (17, 18) with the
5'-flanking region of the human RI gene revealed a 71% similarity
over 153 bp in the region upstream of exon 1a of the human gene (data
not shown). Although the sequences in the 5'-region of these two genes
diverge, all exon/intron splice sites of the entire RI and RIß
genes are conserved, except for the exon 1/exon 2 splice site.
The two alternately spliced RI mRNA species (RI1a and
RI1b) originate from alternate initiation at two distinct
promoters
The two RI mRNAs may originate from one long primary transcript
initiated upstream of both leader exons in the RI gene.
Alternatively, the RI1a and RI1b mRNAs may result from alternate
initiation at the respective leader exons. To determine which of the
two mechanisms involved in the alternate splicing of the RI
transcripts, genomic DNA fragments (572 and 486 bp, respectively)
flanking both leader exons of the RI gene were inserted in both
orientations in front of a CAT reporter gene in the vector pCATbasic.
This vector has no endogenous promoter activity. The resulting
constructs were transfected into rat pituitary
GH4C1 cells (Fig. 7). The
5'-flanking region upstream of exon 1a directed high levels of CAT
activity. When this fragment was inserted in the opposite orientation,
an approximately 50% reduction in activity was observed. The region
flanking exon 1b directed a 2-fold higher basal CAT activity than the
exon 1a promoter. In this case, insertion of the promoter in the
reverse orientation completely abolished the activity. Treatment of the
transfected cell line with 100 µM 8-CPTcAMP for 48 h
had no effect on the CAT activity directed from either construct (data
not shown). The lack of responsiveness of RI-CAT constructs to
8-CPTcAMP in GH4C1 cells was in line with
results observed on RI mRNA expression in these cells by Northern
analyses (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene. We also show that the
RI gene contains two noncoding leader exon (exons 1a and 1b), giving
rise to two alternately spliced mRNAs (RI1a and RI1b), and that
these two exons are directed by distinct promoters. Examinations of the
previously characterized 5'-promoter regions of R and C subunit genes
from vertebrates, including porcine RI (16), mouse RIß (17, 18),
human RII (44), and mouse/rat RIIß (19, 20, 21), as well as the C
and Cß subunit genes (22), reveal that they all lack the TATA
sequences typical for many promoters of eukaryotic genes. Instead, they
contain G/C-rich sequences and several copies of the hexanucleotide
GGGCGG (GC box) binding the mammalian transcription factor Sp1 (41).
G/C-rich and TATA-less promoters are found in many mammalian genes and
are typical features of constitutively expressed genes (45), although
it is now established that transcription of such promoters can be
regulated in a tissue-specific manner (46, 47). Indeed, using probes
specific to exons 1a and 1b, respectively, both probes revealed low
level expression in all human tissues tested, but with higher levels in
endocrine tissues (adrenal, thyroid, testis, and ovary; data not
shown). In lower eukaryotes, RI-like genes have been described in Dictyostelium discoideum (48), Caenorhabditis elegans (49), and Drosophila melanogaster (23). Furthermore, an RII-like gene is present in Saccharomyces cerevisiae (50) and Blastocladiella emersonii (51). The promoters of all of these genes are G/C-rich and TATA-less, like the promoters of the higher eukaryotic R subunit genes.
In this study, we have examined the presence of an additional exon 1
(1a) in the RI gene giving rise to an alternately spliced mRNA.
Significant amounts of RI1a mRNA (3.0 kb, but not 1.5 kb) and
RI1b mRNA (3.0 and 1.5 kb) are present in human testis (data not
shown). For this reason, human testis RNA was used as a template for
PCR-directed cloning of two distinct RI cDNAs. Sequencing revealed
that these two cDNAs are different upstream of the splice junction of
exon1/exon 2, but are identical downstream of this splice site. We
demonstrate that two different RI mRNAs (RI1a and RI1b) are
formed as a result of alternate splicing of primary transcripts from
two distinct untranslated leader exons. Transcription of the leader
exons of the RI gene (exons 1a and 1b) is directed by two different
promoters (1a and 1b promoters, respectively) that both display
activity in a CAT reporter assay. The alternate splicing of two
different leader exons in the RI mRNA does not affect the amino acid
sequence of RI, as the translation initiation codon (ATG) is located
in exon 2. The RI1a cDNA has no in-frame, upstream initiation codons
in the 5'-nontranslated region that can lead to modification of the
peptide sequence translated from the RI1a mRNA. Alternate
transcriptional initiation sites and differential splicing have also
been shown for the RI gene in Drosophila (23). However, in
this case the initiation occurs in an intron 3' to the first coding
exon, and alternate splicing results in transcripts encoding truncated
proteins starting at amino acid 54 or 80.
Numerous mammalian genes are regulated by cAMP (11, 52). Such genes
frequently contain CRE, which bind members of the CRE-binding
protein/CRE modulator/activating transcription factor family (52, 53).
Furthermore, AP-2 can mediate transcriptional activation in response to
protein kinase C- and PKA-dependent phosphorylation (54). The RI
5'-upstream region contains a consensus CRE, a CRE half-site, as well
as several AP-2 sites, which may all be involved in the cAMP-mediated
increase in the levels of RI mRNAs.
In several cell types, the RI mRNA is stimulated by cAMP (36, 55).
In a human B lymphoid cell line (Reh), we previously demonstrated a
4-fold stimulation of RI mRNA that was associated with a similar
increase in transcriptional activity in nuclear run-on analyses (33).
When the regulation of RI mRNAs in this cell line was reexamined
with probes specific to either the RI1a or the RI1b mRNA, it was
evident that the two alternately spliced mRNAs are both subject to
regulation by cAMP with similar kinetics, but with different
magnitudes. One of these mRNAs (RI1b) is constitutively expressed at
low levels and is only moderately stimulated by cAMP (5- to 6-fold). In
contrast, the levels of the other splice variant (RI1a) are very low
in untreated cells, but increase 40- to 50-fold during cAMP treatment.
We have also seen that forskolin has similar effects as cAMP on the
levels of RI1a and RI1b mRNAs (data not shown). By comparing the
relative levels of RI1a and RI1b mRNAs to those detected with an
RI probe common to both transcripts, it is evident that although
untreated cells contain primarily RI1b mRNA, cAMP-treated cells also
contain significant amounts of RI1a mRNA. Furthermore, differential
effect by inhibition of protein synthesis on the cAMP-mediated
regulation of the RI1a and RI1b mRNAs (data not shown) indicate
that distinct mechanisms are involved in the regulation of these two
mRNAs.
Sequencing of the 5'-flanking region of both exons 1a and 1b and
mapping of transcription start sites indicated that the RI gene may
have two distinct promoters. Transfection studies in
GH4C1 cells demonstrated that the RI1a
5'-flanking region had promoter activity directing relatively high
levels of a downstream CAT reporter. The RI1b 5'-flanking region
directed an even 2-fold higher activity. Both promoters were direction
dependent, but whereas the RI1b promoter lost all activity in the
reverse orientation, the RI1a promoter retained about 50% of its
activity. The observed basal activities of RI1a and RI1b are in
agreement with basal levels of mRNAs on the Northern blots presented.
In contrast to this, we were not able to reproduce the cAMP
responsiveness observed on Northern blots using relatively short
5'-flanking sequences by transfection in GH4C1
cells (data not shown), even though the 1a construct contained multiple
AP-2 sites and a CRE half-site, and the 1b construct contained an AP-2
and a consensus CRE site. In agreement with this, no cAMP regulation of
RI mRNA was observed in Northern analyses of
GH4C1 cells. Attempts to transiently transfect
the highly responsive Reh cells with CAT constructs were unsuccessful.
However, the molecular mechanisms involved in conferring cAMP
responsiveness to the RI1a and RI1b promoters are beyond the
scope of this study and will have to await a more detailed
characterization of upstream elements in the RI 5'-flanking region.
The discrepancy between the cAMP responsiveness of RI in Reh cells
and that in GH4C1 cells may also be due to the
inherited difference in the two cell lines.
Multiple initiation of untranslated leader exons giving rise to
alternately spliced mRNAs have been demonstrated to be involved in
cell- and development-specific expression of several genes. This is the
case for the tyrosine kinase p56lck (56, 57),
the aldolase A gene (58), the liver/bone/kidney alkaline phosphatase
gene (59, 60), and the insulin-like growth factor I and II genes (61, 62). Furthermore, alternate initiation at multiple leader exons is
shown to represent a mean of differential regulation of gene expression
by hormones and second messengers. cAMP-mediated regulation of alkaline
phosphatase in fibroblasts is observed with one exon (1B) and not the
other (1A) (59). Similar exon-specific regulation has been reported for
protein kinase C-mediated regulation of p56lck
(56) and in GH-regulated expression of insulin-like growth factor I
(61). Different, alternately spliced, 5'-nontranslated regions of an
mRNA may also contribute to a differential regulation of mRNA stability
(63, 64, 65). Furthermore, the 5'-nontranslated region of a mRNA may have
consequences for mRNA translation due to formation of stem-loop
structures that inhibit translational initiation. Such mechanisms are
operative during translation of the ornithine decarboxylase mRNA (66),
and there is evidence that specific, insulin-regulated initiation
factors may be involved in regulation of translation of this mRNA by
melting the stem-loop structure (67). Potential stem-loops structures
can be predicted in the 5'-nontranslated regions of both the RI1a
and RI1b mRNAs (data not shown). Whether this is important for the
translation of the RI mRNAs remains to be shown.
A repetitive (GA)28 sequence was identified in
the RI 5'-flanking region. A similar homopurine/homopyrimidine
repeat sequence, (CTT)33, has been reported for the mouse
RIß 5'-flanking region (18). Such sequences are thought to generate a
triple helix conformation that promotes DNA-protein interactions and
possibly influences gene expression (68).
With two exceptions, donor and acceptor splice sites for exons in the
RI gene follow the consensus sequences for such splice sites (38).
Several of the vertebrate R subunit genes, for which the 5'-flanking
sequence and the first exons have been reported, reveal an organization
that involves an untranslated leader exon. This is the case for the
porcine RI gene (16), the mouse RIß gene (17), and the B.
emersonii RII gene (51). Whether any of these genes reveals a
mechanism of alternate initiation and splicing remains to be shown.
However, the position of exon 1a in the human RI gene sequence
coincides exactly with a known G/A-rich sequence in the 5'-flanking
sequence of the porcine RI gene (16). It is quite possible that this
region also represents an alternate leader exon in the porcine
gene.
All exon/intron junctions, except those between exons 1 and 2, are
conserved between the human RI and the mouse RIß (18) genes.
Furthermore, the exon 2/exon 3 splice junction is conserved between the
human and porcine RI genes and the B. emersonii RII gene.
This intron splits the sequence encoding the dimerization domain and
the hinge region into separate exons. A similar exonic contribution to
these two domains is found in the C. elegans and
Drosophila R genes (23, 49), although the location of the
splice junction is not exactly conserved. This is not surprising
because these domains are the most variable regions in R subunits. The
two cAMP-binding sites are highly conserved between species. In this
region, conservation of exon-intron splice sites is found. The splice
site between exons 7 and 8, which divides the two cAMP-binding domains,
is similarly located in human RI, mouse RIß, human RIIß (Øyen,
O., and T. Jahnsen, unpublished data), and C. elegans RI,
but not in the RI gene from Drosophila. Furthermore, the
splice junction between exons 5 and 6 is conserved among all four
species. The human and C. elegans RI genes also have
similarly positioned, although not exactly conserved, junctions between
exons 6 and 7, and exons 9 and 10. Although some splice junctions are
similarly positioned in relation to protein domains, the C.
elegans gene is quite different from the human RI gene. It
comprises 8 kb of genomic DNA and is composed of 8 exons, whereas the
human RI gene presented here is composed of 11 exons and constitutes
at least 21 kb of genomic DNA. However, the mouse RIß gene has 11
exons and spans more than 75 kb (18).
In summary, the present report reveals the complete structure of the
human RI subunit gene. We also provide evidence that two different,
alternately spliced RI mRNAs (RI1a and RI1b) exist due to
alternate initiation from two distinct promoters in the RI gene. The
RI1b mRNA is expressed at relatively low constitutive levels,
whereas the expression of the RI1a mRNA is very low, but strongly
stimulated by cAMP. This is the first demonstration of alternate
splicing as a mechanism of conferring differential regulation of R
subunit mRNAs of PKA. The results presented add to the complexity of
regulation of RI, which probably involves a wide array of regulatory
mechanisms at the level of transcription as well as mRNA and protein
stability.
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Acknowledgments |
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Footnotes |
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Received June 10, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Cß
subunits of mouse cAMP-dependent protein kinase. J Biol Chem 263:5739–5744 of cAMP-dependent protein kinases and its sublocalization
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Chem 268:23483–23489 regulatory subunit of
cAMP-dependent protein kinase from human testis are due to the use of
different polyadenylation site signals. Biochem Biophys Res Commun 167:323–330[CrossRef][Medline]
of cAMP-dependent protein kinase. Biochim Biophys Acta, in
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