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Volume 16, Number 24,
Issue of December 15, 1996
pp. 7902-7909
Copyright ©1996 Society for Neuroscience
Enhanced Cleavage of an Atypical Intron of Dopamine
D3-Receptor Pre-mRNA in Chronic Schizophrenia
Claudia Schmauss
Department of Psychiatry and Brookdale Center for Molecular
Biology, Mount Sinai School of Medicine, New York, New York 10029
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The D2-class of dopamine receptors
(D2, D3, and D4) is a target for
typical and atypical neuroleptic drugs. They have been considered,
therefore, as factors that may contribute to the pathophysiology of
psychotic disorders. Interestingly, in cortical brain tissues obtained
postmortem form patients with chronic schizophrenia D3 mRNA
was found to be significantly lower than in the corresponding anatomic
regions of controls. Because the expression of a truncated D3-like mRNA (named D3nf) appeared to be
unaffected in schizophrenic brains, these findings suggest the
possibility that the loss of D3 mRNA results from an
abnormal splicing of D3 pre-mRNA in schizophrenia that is
accompanied by an increased accumulation of the truncated D3nf mRNA. To test this, three approaches were taken. (1)
Substrate D3 pre-mRNA was spliced in vitro
in HeLa nuclear extracts. Results from these experiments show that
D3nf mRNA results from the alternative removal of a short
spliceosomal intron in D3 pre-mRNA that has a noncanonical
3
splice site. (2) Substrate D3 pre-mRNA was spliced in vivo in stably transfected rat GH3 cells. Despite the
atypical 3 cleavage that is necessary to generate D3nf
mRNA, D3 and D3nf mRNA were found to be
processed at similar amounts. (3) The relative D3/D3nf splicing efficiencies were then
determined in the anterior cingulate cortex of postmortem brains
obtained from controls and from patients with chronic schizophrenia.
Significant differences were found between the relative levels of
D3 and D3nf mRNA, suggesting that an enhanced
D3nf-specific splicing of D3 pre-mRNA in
schizophrenia leads to a decreased expression of D3
mRNA.
Key words:
D3 pre-mRNA;
in
vitro splicing;
in vivo splicing;
primer extension;
S1
nuclease protection;
postmortem brain RNA
INTRODUCTION
The cloned dopamine receptor subtypes, named
D2 (Bunzow et al., 1988
; Dal Toso et al., 1989; Grandy et
al., 1989; Giros et al., 1989; Monsma et al., 1989), D3
(Sokoloff et al., 1990), and D4 (Van Tol et al., 1991), are
targets for drugs with antipsychotic efficacy and have been repeatedly
suggested as factors in the pathophysiology of schizophrenia. Although
there is at present no evidence of linkage between the D3
gene and schizophrenia, several studies suggest that a distinct
polymorphism in the first coding exon of the D3 gene
increases the susceptibility to schizophrenia (Crocq et al., 1992; Mant
et al., 1994; Asherson et al., 1996; Shaikh et al., 1996). Although the
full significance of this finding is not yet understood (see Macciardi
et al., 1994), it might suggest a contributory etiological role of the
D3 receptor in schizophrenia. Furthermore, a postmortem
study showed that D3 mRNA was lost in certain cortical
regions of brains obtained from patients with chronic schizophrenia,
whereas a truncated D3-like mRNA (named D3nf)
could readily be detected in the same anatomic regions (Schmauss et
al., 1993).
Several different truncated dopamine D3-like mRNAs that do
not encode G-protein-coupled receptors have been identified (Giros et
al., 1991; Snyder et al., 1991; Nagai et al., 1993; Schmauss et al.,
1993). Their function is unknown. The longest of these mRNAs, named
D3nf, results from a deletion of 98 nt that constitute the
C-terminal region of the putative third cytoplasmic domain of the
D3 receptor and, therefore, encodes a D3-like
protein with a different C terminus (Schmauss et al., 1993; Liu et al.,
1994). In human brain, D3nf mRNA was shown to be abundantly
expressed and also translated into protein (Liu et al., 1994),
suggesting that D3nf mRNA does not result from an RNA
processing error. Another interesting observation is that
D3nf mRNA was expressed in certain neocortical regions of
brains obtained from patients with chronic schizophrenia, regions in
which the expression of D3 mRNA was found to be lost
(Schmauss et al., 1993) .
The human genome contains a single, intron-containing
D3-encoded gene in which the 98 nt that are deleted in
D3nf are embedded within a large continuous exon (Liu et
al., 1994). Thus, to generate D3nf mRNA via alternative
splicing, these 98 nt must be recognized by the splicing machinery as
an alternative intron. Removal of this intron, however, would require
cleavage of a rare (and nonconforming) 3 splice site sequence that
lacks the penultimate AG dinucleotides. However, these dinucleotides
are known to be the most highly conserved dinucleotides of 3 splice
sites (Reed, 1989) and have been found to be essential for both lariat
formation (Reed and Maniatis, 1985) and snRNP binding (Charbot and
Steitz, 1987).
The present study shows that D3nf mRNA is indeed
generated via atypical alternative splicing and, therefore, suggests a
mechanism by which our previous results in schizophrenic brains would
be explained: that an increased splicing of D3 pre-mRNA
leads to a decrease of D3 mRNA and an increased
accumulation of D3nf mRNA.
MATERIALS AND METHODS
Splicing of substrate D3 pre-mRNA in
vitro. The sequence schematically diagrammed in Figure 1
(top), which codes for the 3 part of the putative third
cytoplasmic domain of the D3 receptor and the 5 region of
the putative 6th transmembrane-spanning domain, was cloned into the
plasmid vector pCR II (Invitrogen, San Diego, CA). A 5-capped 2.3 kb
pre-mRNA transcript (see Fig. 1, top) of this recombinant
plasmid was synthesized in vitro using 10 U of Sp6 RNA
polymerase (Stratagene, La Jolla, CA). This D3-encoded substrate pre-mRNA (20 fmol) was added to 50 µl of HeLa nuclear extract [prepared as described by Dignam et al. (1983) and
supplemented with MgCl2, ATP, and creatine phosphate as
described by Krainer et al. (1984)]. Aliquots (10.5 µl) were
incubated at 30°C for 1-4 hr. At the end of each incubation, the
splicing reaction was treated with proteinase K, extracted with
phenol/chloroform, and precipitated. First-strand cDNA was synthesized
with 200 U of Moloney murine leukemia virus (M-MLV) reverse
transcriptase (United States Biochemical, Cleveland, OH) and the primer
sequence
5-GTATTGAGAACATGGGT-3which is complementary to the most
3 sequences of exon II (see Fig. 1). The same primer, and a 5
primer5-GAGGAGAAGACTCGGAATTC-3 (identical to the most 5 sequences
of exon I; see Fig. 1)were used for the PCR amplification to
selectively amplify the ligated exons of the in vitro
splicing reaction. PCR products were separated on a 1.5% agarose/TBE
gel, transferred to membrane, and subjected to Southern blot analysis
using a 32P-radiolabeled random-primed cDNA encoding the
human D3 receptor as a probe. D3- and
D3nf-specific PCR products were cloned into the plasmid pCR
II (Invitrogen) and subjected to nucleotide sequence analysis of both
strands.
Fig. 1.
Splicing of D3 pre-mRNA in
vitro. Schematic diagram of the structure of the substrate
pre-mRNA, the products, and D3nf-specific intermediates of
in vitro splicing. The D3-encoded genomic
locus (cloned into the plasmid pCR II) contains exon sequences that code for the 3 part of the putative third cytoplasmic domain and the
5 part of the putative transmembrane spanning domain 6 of the human
D3 receptor.
[View Larger Version of this Image (14K GIF file)]
Primer extension analysis of intermediates and products of
in vitro-spliced wild-type and mutant substrate
D3 pre-mRNA. For these experiments, a 5 capped 223 nt
T7 RNA polymerase (Stratagene) transcript of the wild-type or mutant
exon I sequences (see Fig. 1) was spliced in vitro in HeLa
nuclear extracts as described above. 3 splice site mutants were
generated in vitro using the Chameleon Double-Stranded,
Site-Directed Mutagenesis kit (Stratagene). Briefly, exon I was cloned
into the plasmid pRC II, which served as the target plasmid DNA to
which two oligonucleotide primers were simultaneously annealed to one
of the strands. The selection primer was identical for all mutations.
To introduce mutations into the D3nf-specific alternative
3 splice site, the following oligonucleotide primers were used:
Wild-type sequence:
5-CCCCTGCAACCTCGGG
GTGCCACTTC- GGGAGAAG-3
Mutant 1:
5-CTGCAACCTCGGG
GTGCCACTTCGGGAG-3
Mutant 2:
5-CTGCAACCTCGGG
GTGCCACTTCGGGAGA-3
Mutant 3: 5-CCCCTGCAACCTCGGG - - - - GCCACTTCGG-
GAGAAG-3.
Extended and digested plasmid DNA was then transformed into the
repair-deficient Escherichia coli strain XLmutS,
and plasmid DNA extracted from these colonies was transformed into
Epicuran Coli XL1-Blue competent cells. The correctness of each
mutation was verified by nucleotide sequencing of the entire exon
I.
In vitro-spliced wild-type or mutant RNA was precipitated
and annealed to 5 × 105 cpm of a
32P-end-labeled oligonucleotide sequence
(5-CAAGCACAATGGCCACC-3) that is complementary to the most 3 sequence
of exons I and Ib (see Fig. 1) at 52°C for 10 min and then incubated
with 2 U/µl AMV reverse transcriptase (Boehringer Mannheim,
Indianapolis, IN) for 1 hr at 42°C. The primer extension products
were separated on a 7 M urea/6% acrylamide gel.
Splicing of substrate D3 pre-mRNA in vivo. A
D3 minigene schematically diagrammed in Figure 1
(top) was cloned into the expression vector plasmid pCEP4
(Invitrogen), which also encodes the hygromycin-resistance gene. The
recombinant plasmid (pCEP4/D3mg) was transfected into rat GH3 cells via
lipofection as described previously (Liu et al., 1994).
Hygromycin-resistant cells were isolated by ring cloning and expanded
to cell lines. The cytoplasmic abundance of spliced transcripts of the
minigene was analyzed by S1 nuclease protection analysis as described
below using 10 µg of total cytoplasmic RNA extracted from each
hygromycin-resistant clone.
RT-PCR analysis of D3 and D3nf mRNA
expression in D3 minigene-expressing GH3 cells and in human
cingulate cortex. First-strand cDNA was generated from 10 µg of
total RNA extracted from transfected GH3 cells using an
oligo-dT15 primer in conjunction with 200 U of M-MLV
reverse transcriptase. The cDNA was then amplified by PCR using a 3
primer (5-GTATTGAGAACATGGGT-3) that is complementary to the most 3
sequence of exon II (see Fig. 1) and a 5 primer (5-
GAGGAGAA-3) that is identical to the 5
sequence of the transcript derived from pCEP4/D3mg (the underlined
sequence is derived from the transcribed vector sequence immediately
upstream of the 5 sequence of exon I; see Fig. 1). Thus, this primer
pair specifically enables the amplification of cRNA that is derived from the primary transcript of the transfected plasmid pCEP4/D3mg. PCR
products were separated on 1.5% agarose/TBE gels, transferred to
membrane, and subjected to Southern blotting using a
-32P-radiolabeled oligonucleotide probe
(5-CAAGCACAATGGCCACC-3) that is complementary to the most 3 sequence
of exon I (see Fig. 1).
For amplification of D3 and D3nf mRNAs
expressed in the anterior cingulate cortex of control and schizophrenic
brains, first-strand cDNA (generated as described above) was amplified
by PCR using the primer pair D3S5: 5-TACCTGCCCTTTGGAGT-3 and
D33: 5-CTCCCTC- AGCAAGACAG-3. This pair of primers allows the
simultaneous amplification of the C-terminal halves of both
D3 and D3nf cDNA. Details of this PCR
amplification were described previously (Schmauss et al., 1993). PCR
products were separated on ethidium bromide-stained 1% agarose gels.
The relative quantities of D3- and
D3nf-specific amplification products were compared to the
quantities of ethidium bromide-stained amplification products that
resulted from parallel experiments with plasmid DNA templates
consisting of a mixture of equal amounts of the plasmids pRC/CMV/D3 and
pRC/CMV/D3nf (see Schmauss et al., 1993) ranging from 0.5 fg to 0.5 ng.
RNA extraction and S1 nuclease protection assays. RNA was
extracted from stably transfected GH3 cells (107 cells per
clone) or from 0.5 gm of each postmortem tissue using the guanidine
isothiocyanate/CsCl ultracentrifugation methods as described previously
(Schmauss et al., 1993). To protect D3- and
D3nf-specific mRNAs from ribonuclease digestion, 10 µg
(cell lines) or 20 µg (brain tissues) of total cytoplasmic RNA was
hybridized to 5 × 105 cpm of
32P-end-labeled 39-mer antisense oligonucleotide at 50°C
overnight. The nucleotide sequences of these oligonucleotides are
either complementary to sequences found in both D3 and
D3nf mRNAs (D3ALL: 5-TCCACCCAAGGCAGTGTCCTGGCAGAT- GCTGTAGTAACG-3) or they are
complementary to sequences found only in D3 mRNA (D3-AS1:
5-GAGCTTAGGCGCTATGGTGGGAC- TCAGGGAATTCCGAGT-3) or in
D3nf mRNA (D3nf-AS1: 5-GCCTTC-
TTCTCCCGAAGTGGCACTCAGGGAATTCCGAGT-3). In parallel, sense
oligonucleotides were incubated with RNA to control for the specificity
of the RNA protection from ribonuclease digestion. Furthermore, 10 µg
of total cytoplasmic RNA extracted from each brain tissue was also
hybridized to a 215-nt-long [32P]UTP-labeled antisense
RNA encoding the 5-untranslated sequence of the human snRNP-associated
protein N (Schmauss et al., 1989) before digestion with S1 nuclease.
The N-encoded antisense riboprobe is an Sp6 RNA-polymerase transcript
of the EcoRI-linearized plasmid pCRII-N. Sense-N-encoded
mRNA (a T7 RNA-polymerase transcript of the same plasmid
linearized with HindIII) was used in parallel experiments to
control for the specificity of the RNase protection experiments.
Hybridized RNA was digested with 500 U of S1 nuclease (Life
Technologies, Gaithersburg, MD) at 37°C for 30 min. Protected fragments were precipitated and separated on 7 M urea/6%
acrylamide gels. Triplicate experiments were performed for each
sample.
Tissues sources. Normal control tissues [n = 8; age (mean ± SD) 60 ± 11.4 years; postmortem interval
[PMI (mean ± SD) 11.7 ± 3.6 hr] were obtained from the
National Neurological Research Specimen Bank (VAMC, Los Angeles, CA;
sponsored by NINDS/National Institutes of Health, National Multiple
Sclerosis Society, Hereditary Disease Foundation, Comprehensive
Epilepsy Program, Tourette Syndrome Association, Dystonia Medical
Research Foundation, and Veterans Health Services and Research
Administration, Department of Veterans Affairs). Age-matched tissues
from long-term hospitalized patients with chronic schizophrenia were
obtained from the Schizophrenia Brain Bank of the Department of
Psychiatry at Mount Sinai School of Medicine. Details of the
recruitment and assessment of these patients and their brains were
reported previously (Schmauss et al., 1993).
RESULTS
To clarify whether D3nf mRNA is indeed generated via
atypical splicing of D3 pre-mRNA, in vitro
splicing experiments were done. A 2.3 kb in
vitro-synthesized RNA transcript of the D3-encoded genomic locus (shown in Fig. 1) was incubated with HeLa nuclear extracts, and the products and intermediates of this substrate pre-mRNA-spliced in vitro were analyzed.
In the first set of experiments, the spliced RNA was precipitated and
used as a template for first-strand cDNA synthesis that was primed with
a 17-mer oligonucleotide sequence complementary to the most 3 sequence
of the distal exon of the substrate pre-mRNA (exon II; see Fig. 1). The
cDNA was then amplified by PCR using the same 3 primer in conjunction
with a 5 primer whose sequence is identical to the most 5 sequence of
the proximal exon of the pre-mRNA (exon I; Fig. 1). A Southern blot of
the PCR products is shown in Figure
2A. The lengths of the three
hybridizing products correspond to the lengths of the unreacted
pre-mRNA (2.3 kb), the D3-specific ligated exons (226 nt),
and the D3nf-specific ligated exons (128 nt).
The nucleotide sequences of the latter two products were found to be
identical to D3- and D3nf-specific sequences expressed in vivo (10) (Fig.
2B). Thus, in vitro splicing of the
substrate D3 pre-mRNA resulted in the removal of the 2.1 kb
intron to allow religation of the proximal exon (I) and distal exon
(II) to generate D3 mRNA (constitutive splicing; see Figs. 1, 2). In addition, the proximal exon (exon I) of some of the pre-mRNA
is further cleaved to remove the 98-nt-long spliceosomal intron
[resulting in the consecutive religation of the two short proximal
exons (exons Ia and Ib) and the distal exon II] to yield D3nf mRNA (alternative splicing; see Figs. 1, 2). These
results suggest that the sequence UGA:
in exon I is
recognized as a (typical) 5 splice site, whereas the sequence
G:GU serves as an AG-independent 3 splice site. Because
such a 3 splice site sequence is indeed atypical, it should be
stressed that the possibility of a sequencing compression at the
boldfaced G of the 3 splice site (GA:GT), which would make
canonical splicing quite feasible (:GT), has been
excluded by analyzing multiple sequencing reactions of both strands
using 7-deazaGTP and dITP.
Fig. 2.
Analysis of the products and intermediates of
in vitro-spliced D3 pre-mRNA.
A, Southern blot of PCR-amplified products of the
in vitro-spliced substrate pre-mRNA. The time points
shown on top of each lane indicate the
length of the incubation of the substrate pre-mRNA with HeLa nuclear
extracts at 30°C. Time point 0 corresponds to a 30 min incubation at
4°C. The blot was exposed to film for 10 min. The PCR amplification
allowed the detection of ligated D3- and
D3nf-specific exons after a 1 hr incubation of the
pre-mRNA with nuclear extracts. B, Nucleotide sequence of ligated D3- and D3nf-specific exons.
The sequences of D3- and D3nf-specific
PCR amplification products shown in A precisely match
the sequence of D3 and D3nf mRNA expressed in
human brain (see Schmauss et al., 1993). The splice-junctional
sequences resulting from both D3nf-specific splicing
events are indicated by arrowheads. The lower
arrowhead indicates the position of the splice junction resulting from cleavage of an atypical 3 splice site (*) in the alternative intron.
[View Larger Version of this Image (42K GIF file)]
For mammalian introns, AG-independent 3 splice sites are extremely
rare. Thus, to verify further the cleavage of the noncanonical 3
splice site GA:GU, primer extension experiments were performed with
wild-type substrate pre-mRNA corresponding to exon I (see Fig. 1) and
various mutants thereof with altered or lacking 3 splice site
sequences. These substrate pre-mRNAs were spliced in vitro
in HeLa nuclear extracts at 30°C for 2 hr, precipitated, and
subjected to primer extension analysis using a primer sequence that is
complementary to the most 3 17 nt of exon I (see Fig. 1). Primer
extension products were expected to correspond in size to the unspliced
substrate (D3) RNA (238 nt; the 5 ends of each extension
product are determined by the transcription start site of T7 RNA
polymerase in pCR II/exon I, which is located 66 nt upstream of the
first nucleotide in exon I), the religated exons Ia and I b (140 nt),
and a splicing intermediate resulting from cleavage of the 5 splice
site and release of the 3 exon attached to the branched 98-nt-long
intron. As shown in Figure 3, when wild-type exon I is
spliced in vitro, prominent extension products of 238 nt
(corresponding in size to unspliced D3 RNA) and of 140 nt
(corresponding in size to the religated exons Ia and Ib after splicing
of the alternative 98 nt intron) are indeed obtained. This result is is
consistent with the results shown in Figure 2.
Fig. 3.
Determination of the length of the splicing
intermediates and products of in vitro-spliced wild-type
and mutant substrate pre-mRNAs by primer extension. A T7 RNA polymerase
transcript of exon I (see Fig. 1) was spliced in vitro
in HeLa nuclear extracts for 2 hr at 30°C. The 238 nt extension
product corresponds in size to the unspliced D3 mRNA, and
the 140 nt extension product corresponds to the spliced and religated
D3nf-specific exons Ia and Ib (see Fig. 1). In
contrast to wild-type pre-mRNA (lane 2), the
D3nf-specific splicing of mutant 1 (:GU) substrate pre-mRNA appears to be less efficient
(lane 1). D3nf-specific alternative splicing of mutant 2 (
:GU) pre-mRNA appears to be
indistinguishable from that of wild-type pre-mRNA (lane
3). In mutant 3, the 3 splice site sequence GA:GU is deleted,
and no D3nf-specific splicing is observed
(lane 4). Although the precise nature of the 131 and 81 nt extension products is presently unknown, it is likely that the 98 nt extension product resulted from the extension of the splicing
intermediate exon Ib/branched intron (see Results). Primer-extension products were separated on 7 M urea/6% polyacrylamide
gels. Gels were exposed to film for 15 hr.
[View Larger Version of this Image (56K GIF file)]
In addition, three mutant substrate pre-mRNAs were spliced in
vitro and subsequently analyzed by primer extension. Mutant 1 contains the consensus AG (:GU) dinucleotides, rather
than the wild-type GA (:GU) dinucleotides, penultimate to
the 3 cleavage site. Interestingly, although alternative splicing of the 98 nt intron is still obtained, the efficiency of this alternative splicing appears to be reduced (rather than enhanced) as indicated by
the decreased signal of the primer extension product that corresponds in length to the D3nf-specific religated exons Ia
and Ib (Fig. 3, lanes 1, 2). Furthermore,
when mutant 2, which carries a single nucleotide change from
A:GU (wild-type) to 
A:GU, is spliced in
vitro, the D3nf-specific alternative splicing
appears to be unaffected, i.e., the signal intensity of the extension
product that corresponds in length to the
D3nf-specific religated exons Ia and Ib is similar
to those obtained for the spliced wild-type RNA (Fig. 3, lanes
2, 3). In another mutant substrate RNA (mutant 3), all of the four nucleotides (GA:GU) that constitute the 3 splice
site are deleted. As expected, no D3nf-specific
splicing could be detected (Fig. 3, lane 4).
Compared to experiments with wild-type and mutant 1 or 2 substrate RNA,
less RNA was recovered after splicing of mutant 3 in vitro.
However, it is apparent that the smaller amount of the mutant 3 RNA
cannot account for the lack of visible D3nf-specific
primer extension products, because other extension products are visible
that do not correspond in size to any of the specific
extension products seen in lanes 1-3 of Figure 3.
Additional extension products of 81, 98, and 131 nt were also detected
after splicing of wild-type and mutant 1 or 2 substrate RNAs. The 131 nt extension product could perhaps have resulted from a strong
impediment for reverse transcriptase that is provided by the 5
sequence (derived from the plasmid pRC II) of the extended RNA
template. Alternatively, T7 RNA polymerase may have used two different
transcription start sites, one located 9 nt downstream of the other. If
the latter scenario applies, however, one has to assume that the
splicing of mutant 1 is as efficient as the splicing of the wild-type
RNA sequence (see Fig. 3, lanes 1, 2).
Because the branchpoint of an intron is known to be an impediment for
reverse transcriptase, either the 98 or the 81 nt extension products
could have resulted from the extension of exon Ib/branched intron that
results after cleavage of the 5 splice site. Because the 81 nt
extension product terminates at a cytosine nucleotide (which would be
atypical for a branchpoint), it is more likely that the 98 nt extension
product indicates the position of the branchpoint. This would be an
adenosine located 49 nt upstream of the 3 cleavage site. This putative
branchpoint adenosine is preceded by a short pyrimidine-rich
sequence.
In conclusion, the analysis of the intermediates and products of
D3 pre-mRNA spliced in vitro revealed clearly
that D3nf mRNA is derived from D3 pre-mRNA via
removal of a short alternative spliceosomal intron that has a
noncanonical 3 splice site. However, these experiments did not
directly address the relative D3/D3nf splicing efficiencies. Therefore, additional in vivo
splicing experiments were performed with transfected rat GH3 cells that stably express the D3 minigene that is schematically
diagrammed in Figure 1 (top). To test whether transcripts of
the transfected D3 minigene are processed in GH3 cells to
yield cytoplasmic D3- and D3nf-specific
mRNA sequences, RNA was extracted from stable transfectants and
analyzed by RT-PCR analysis. PCR primers were used that specifically
enable the amplification of the expression vector pCEP4/D3mg-derived
transcripts (see Materials and Methods). These amplification products
are expected to be 235 nt (D3 mRNA) and 137 nt
(D3nf mRNA) in length. Figure
4A shows a Southern blot of the
PCR-amplified products. In 2 of 4 hygromycin-resistant GH3 cell clones,
D3- and D3nf-specific amplification
products were obtained; the other two clones did not express the
minigene. Nucleotide sequence analysis (data not shown) revealed that
the sequences of these two PCR products are identical to those shown in
Figure 2B. Thus, as in HeLa nuclear extracts, in the
neuroendocrine cell-derived GH3 cells D3 pre-mRNA is also
processed constitutively and alternatively to yield D3 and
D3nf mRNA.
Fig. 4.
In vivo splicing of D3
pre-mRNA transcripts derived from a D3 minigene stably
inserted into the genome of GH3 cells. A, Southern blot
of RT-PCR-amplified cytoplasmic D3- and
D3nf-specific mRNA sequences. PCR products were
separated on 1.5% agarose/TBE gels. The Southern blot was probed with
a -32P-radiolabeled oligonucleotide probe (5-CAAGCA-
CAATGGCCACC-3) that is complementary to the most 3 sequence of
exon I (see Fig. 1). Two of four randomly selected stable GH3
transfectants express both D3 and D3nf mRNA
sequences that are derived from the transfected plasmid pCEP4/D3 mg
(lanes 1, 3). B,
Determination of the relative abundance of cytoplasmic D3
and D3nf mRNA sequences in stable GH3 transfectants by S1
nuclease protection assays. Ten micrograms of total cytoplasmic RNA
extracted from GH3 clones 1 and 3 were hybridized to either
D3 (a)- or D3nf
(b)-specific antisense oligonucleotides (see Materials
and Methods) and subsequently digested with S1 nuclease. Protected
fragments were separated on 7 M urea/6%
polyacrylamide gels. The gel was exposed to film for 1 hr.
[View Larger Version of this Image (25K GIF file)]
To analyze further the relative
D3/D3nf splicing efficiencies, S1
nuclease protection assays were performed with RNAs extracted from the
two D3 minigene-expressing cell clones (see Fig.
4A). For these experiments, antisense
oligonucleotides that are either complementary to a region of the
sequence of D3 mRNA (D3-AS1) that is missing in
D3nf or complementary to the splice-junctional sequence of
D3nf mRNA (D3nf-AS1), which is not present in
D3, were used (see Materials and Methods). Thus,
D3 and D3nf mRNAs are separately protected from
S1 nuclease protection, allowing their relative abundance to be
compared. As shown in Figure 4B, in both clones that
were shown to express transcripts of the D3 minigene (see
Fig. 4A) the relative abundance of cytoplasmic
D3/D3nf mRNA sequences is approximately
equal. The sense sequence of both oligonucleotides did not specifically
protect the respective RNAs from ribonuclease digestion (data not
shown).
As shown previously, D3nf mRNA is abundantly
expressed in human brain (Liu et al., 1994), indicating that in
vivo the atypical alternative intron described here is spliced
efficiently. Interestingly, whereas both D3 and
D3nf mRNAs are expressed in normal neocortical tissues,
only D3nf mRNA was detected by RT-PCR in motor and parietal cortices of chronic schizophrenics (Schmauss et al., 1993). One possible explanation for these results is that D3 pre-mRNA
is generally decreased in chronic schizophrenics. Another possibility, especially given the above findings, is that the loss of D3
mRNA in chronic schizophrenia results from enhanced
D3nf-specific splicing of the D3-encoded
primary transcript. The first possibility would predict a decreased
abundance (relative to controls) of both D3 and
D3nf mRNAs, whereas the abundance of D3nf mRNA
should be unaltered or increased if the second possibility is obtained.
To test these two possibilities, S1 nuclease protection assays were
performed on total cytoplasmic RNA extracted from the anterior
cingulate cortex of postmortem brains derived from the same population
of patients that were examined previously (Schmauss et al., 1993), as
well as from matched controls (see Materials and Methods). This
particular anatomic region was chosen because a nonquantitative RT-PCR
analysis demonstrated coexpression of D3 and
D3nf in both diagnostic groups (see below).
In a first series of experiments, RNA extracted from 8 control and 8 schizophrenic tissues was subjected to S1 nuclease protection assays
using a 215-nt-long antisense riboprobe that codes for the
5-untranslated region of mRNA encoding the snRNP-associated protein N
(Schmauss et al., 1989). This initial control experiment was performed
to test for the integrity of each RNA preparation. N-encoded mRNA was
chosen for this control experiment because it is exclusively expressed
in neurons, and it is found in all major neuronal
populations throughout the brain (Schmauss et al., 1992). As shown in
Figure 5A, the expression of the abundant
N-encoded mRNA (protected from S1 nuclease digestion by an Sp6 RNA
polymerase transcript of the EcoRI-linearized plasmid
pCRII-N; see Materials and Methods) could readily be detected in all
schizophrenic and control brains. Although the levels of N mRNA vary
between samples, no significant differences were found in the range of
N mRNA levels between both diagnostic groups. A sense N-encoded
riboprobe (a T7 RNA polymerase transcript of the
HindIII-linearized plasmid pCRII-N) did not protect RNA from
S1 nuclease digestion.
Fig. 5.
Expression of D3 and D3nf
mRNAs in the cingulate cortex (Brodmann areas 24/25) of control brains
and brains from patients with chronic schizophrenia. A,
Determination of the expression of N-encoded mRNA (left)
and of the sum of D3 and D3nf mRNA
(right). Ten micrograms of total cytoplasmic RNA were
hybridized to a 215-nt-long antisense riboprobe encoding the human
snRNA N mRNA, which is known to be expressed exclusively and at high
levels in all major neuronal populations of the brain. The gel was
exposed to film for 5 hr. Twenty micrograms of the same RNA samples
were hybridized to an antisense oligonucleotide (D3ALL)
that is complementary to a sequence common to both D3 and
D3nf mRNA. The gel was exposed to film for 12 hr. Sense
N-encoded riboprobe and the sense sequence of the oligonucleotide
D3ALL did not specifically protect the respective mRNAs
from ribonuclease digestion (lanes marked asterisks). B, Separate detection of cytoplasmic D3 and
D3nf mRNA sequences. S1 nuclease protection analysis of 20 µg of the same total cytoplasmic RNA samples hybridized to either a
D3-specific (a) or a
D3nf-specific (b) 39-mer antisense
oligonucleotide (see Materials and Methods). The gels were exposed to
film for 24 hr and developed simultaneously. The individual samples
were loaded in the same order on all gels. C,
Amplification of D3 and D3nf cDNAs by PCR.
D3 and D3nf cDNAs, derived either from a
mixture of equal amounts of D3- and
D3nf-encoded plasmid DNA templates at concentrations
ranging from 0.5 fg to 0.5 ng or from RNA extracted from the anterior
cingulate cortex of brains from five controls and five schizophrenics,
were simultaneously amplified by PCR. A single pair of PCR primers (see
Materials and Methods) results in the coamplification of the C-terminal halves of D3 (619 nt) and D3nf (521 nt) cDNAs.
PCR products were separated on ethidium bromide-stained agarose gels.
To be able to visualize the small amount of D3-specific
amplification products of the schizophrenic samples on such gels,
products of a 35 cycle PCR amplification are shown here.
[View Larger Version of this Image (54K GIF file)]
A second series of experiments, performed on the same RNAs, involved
the simultaneous protection of D3 and D3nf mRNA
via hybridization of a 39-mer antisense oligonucleotide
(D3ALL; see Materials and Methods) that is complementary to
a sequence that codes for the N terminus of the putative third
cytoplasmic domain of D3. This probe, therefore, protects
both D3 and D3nf mRNAs from S1 nuclease digestion. As shown in Figure 5A, the signals obtained for
the sum of D3 and D3nf mRNAs were also found to
be similar in the control and schizophrenic tissues. The sense sequence
of the oligo-D3ALL did not protect these mRNAs from S1
nuclease digestion.
It is noted that the variability of the sum of D3 mRNA
expression between samples is substantially less than that of N-encoded mRNA. This is perhaps because of differences in the turnover rates (stabilities) between both mRNAs. The abundance of the 1.6 kb N-encoded
mRNA has been shown to be similar to that of the very abundant
actin-encoded mRNA (Schmauss and Lerner, 1990). In contrast, D3 mRNA (8.3 kb) is known to be expressed at very low
levels (Sokoloff et al., 1990). It is possible that an mRNA with a high
transcription rate also has a relatively short turnover rate and that
differences in the postmortem intervals (PMIs) between samples (the SDs
of the PMIs of the samples analyzed here are ~3 hr), therefore,
result in the detection of different levels of N mRNA. This is
supported by the observation that higher levels of N-encoded mRNA were
detected in samples with shorter PMIs. Thus, if D3 mRNA
transcripts are substantially more stable than N mRNA, the magnitude of
the differences between the individual PMIs of the samples analyzed
here may not be large enough to result in the detection of similarly
different levels of D3 mRNA.
A third series of experiments targeted D3 and
D3nf sequences separately, using D3- and
D3nf-specific antisense 39-mer oligonucleotides. As
shown in Figure 5B, in 5 of 8 cingulate cortices obtained
postmortem from normal controls D3nf mRNA was more abundant
than D3 mRNA, although D3 mRNA was clearly
detected in all 8 individuals. In contrast, no D3 mRNA
could be detected in cingulate cortical tissues obtained from 8 long-term hospitalized patients with chronic schizophrenia, whereas in
all 8 tissues D3nf mRNA was found to be expressed at levels
that, in most cases, are higher than the corresponding levels found in
control tissues.
Differences in the expression of D3 mRNA in control and
schizophrenic brains are also apparent in RT-PCR experiments in which D3 and D3nf cDNAs are simultaneously amplified.
This is demonstrated in Figure 5C. PCR experiments on the
five control samples that have a higher ratio of
D3nf/D3 mRNA (see Fig. 5B)
also indicate that the amplification of D3 cDNA is 5- to
10-fold less than that of D3nf if one compares ethidium
bromide-stained signals of these PCR products to the corresponding ones
obtained from PCR amplifications, performed in parallel, of a mixture
of equal amounts of D3- and D3nf-encoded
plasmid DNA templates that range from 0.5 fg to 0.5 ng (Fig.
5C; see Materials and Methods). Both D3- and
D3nf-specific amplifications are also obtained from
five randomly selected schizophrenic samples, indicating that not all
of the D3 pre-mRNA is spliced in a
D3nf-specific manner. However, for the schizophrenic
samples the difference in the amounts of D3nf and
D3 amplification products is at least 100-fold (Fig.
5C).
In summary, the analysis of the relative abundance of
D3 and D3nf mRNA by RNase protection as well as
by RT-PCR revealed a significant decrease of D3 mRNA in the
cingulate cortex of brains of patients with chronic psychosis. In the
same anatomical region, however, D3nf mRNA is abundantly
expressed and this can explain why the sum of D3 and
D3nf mRNAs (see Fig. 3A) is similar between control and schizophrenic brains. Altogether, these data support the
suggestion that the loss of D3 mRNA in some neocortical
regions obtained postmortem from patients with chronic schizophrenia
(Schmauss et al., 1993), and the decrease of D3 mRNA in the
cingulate cortex found in this study, results from an enhanced
D3nf-specific splicing of the D3-encoded
primary transcript.
DISCUSSION
The results of in vitro and in vivo splicing
experiments shown here demonstrate that the truncated
D3-like mRNA (D3nf) results from D3
pre-mRNA via removal of a short alternative spliceosomal intron that is
retained in D3 mRNA. This intron is flanked by a
noncanonical 3 splice site sequence (:GU).
Although in general one would predict that the in vivo
splicing efficiency of such a rare and nonconforming alternative 3 splice site is significantly lower than the cleavage of constitutive and/or canonical 3 splice sites, results from in vivo
splicing experiments with GH3 cells that stably express RNA transcripts of a transfected D3 minigene revealed that both
D3 (constitutive)- and D3nf
(alternative)-specific splicing events occur with similar frequency.
Furthermore, in human brain the expression levels of D3nf
mRNA are either equal to or higher than the corresponding levels of the
constitutively spliced D3 mRNA (Liu et al., 1994) (this
study). Interestingly, substitution of the penultimate GA (:GU) dinucleotides of the
D3nf-specific 3 splice site of substrate D3 pre-mRNA with the consensus AG (:GU)
dinucleotides does not increase the D3nf-specific
alternative splicing efficiency. Somewhat unexpectedly, substitution of
the boldfaced G (GA:GU) of the wild-type 3 splice site with
a uridine (UA:GU) also does not affect the 3 cleavage (see Fig. 3).
Thus, the recognition signals for cleavage of the noncanonical 3
splice site of the D3nf-specific intron are
obviously not determined by the sequence of the 3 splice site
(:GU) alone.
Because the frequency of D3nf-specific
splicing of D3 pre-mRNA ultimately determines the level of
D3 mRNA, this alternative splicing could function as an
important regulator of D3-receptor expression. It is of
interest, therefore, to note that different relative ratios of
D3/D3nf mRNA were observed in the
anterior cingulate cortex of controls and chronic schizophrenics.
Whereas D3 mRNA levels are lower in schizophrenics compared
to controls, D3nf mRNA levels are increased. This suggests
that the D3nf-specific splicing activity is higher
in brains of the chronic schizophrenic population studied here (and in
Schmauss et al., 1993).
It is unlikely that the results of the S1 nuclease protection assays
and RT-PCR experiments shown here reflect different stabilities of
D3 and D3nf mRNA in schizophrenic and normal
brains (which could obscure estimates of the relative
D3/D3nf-specific splicing activities). First, in schizophrenia, D3 and
D3nf mRNAs change in opposite directions and the level of
the sum of both mRNAs is not different from the corresponding level
found in control brains (see Fig. 5). Second, in stably and
tetracycline-regulated D3- and
D3nf-expressing GH3 cells (see Howe et al., 1995)
the stabilities of both cDNA-derived RNAs (determined with
transcription inhibition experiments) were identical
(t1/2 = 80 min; C. Schmauss, unpublished
observation). Although this result does not exclude the possibility
that other destabilizing or stabilizing factors operate on full-length
D3 and D3nf mRNAs, it clearly shows that the
absence or presence of the alternative 98 nt intron has no significant
effect on the stability of either RNA.
In summary, the results reported here demonstrate that it is possible
to reconstitute both in vitro and in vivo an
alternative splicing pathway of D3 pre-mRNA that involves
cleavage of a noncanonical 3 splice site to generate the truncated
D3nf mRNA. A comparison between the relative levels of the
products of this splicing (D3 and D3nf mRNA) in
the anterior cingulate cortex of schizophrenics and controls suggests
an abnormal post-transcriptional processing of D3 pre-mRNA
in schizophrenia. This result may implicate altered activities of
post-transcriptional regulators of gene expression in this disease.
Clearly, it will now be of interest to search for other introns with a
3 splice site sequence specificity similar to the one described here
and to analyze the corresponding splicing pattern in schizophrenics and
controls. It is possible that the D3nf-specific
alternative intron is a member of a novel class of minor introns. In
this respect, it is of interest to note that recent studies on another
novel group of minor introns, called the AU-AC introns (which have
atypical, but evolutionarily highly conserved, 5 and 3 splice sites),
have shown that a deviation of consensus-sequence splice sites of
major-class introns is accompanied by the assembly of different snRNPs
(Hall and Padgett, 1996; Tarn and Steitz, 1996). Four pre-mRNAs are
presently known that have such AU-AC introns. Importantly, these
introns are spliced by a U11/U12 and U5 snRNP-containing spliceosome
that is completely different from the U1, U2, and U6 snRNP-containing
spliceosome known to be essential for cleavage of all major-class
introns (GU-AG introns). It is possible, therefore, that the cleavage of the alternative D3nf-specific intron requires the
presence of unique splicing factors and that an altered expression of
such splicing factors may underlie the observed abnormality in the D3 pre-mRNA processing. If unique splicing factors indeed
mediate the cleavage of atypical introns like the one described here, they are likely to be found in the common nuclear repertoire of snRNPs.
This assumption is supported by the observation that, in addition to
neuronal cells, D3 pre-mRNA is also spliced both constitutively and alternatively in two completely different cell types, human HeLa cells and rat GH3 cells.
It is also important to note that the patient population studied here
and previously (Schmauss et al., 1993) represents a rather unique group
of schizophrenic patients that were chronically and severely ill,
therapy-resistant, and hospitalized long-term. Future studies will show
whether a similar alteration of D3 and D3nf mRNA expression can be detected in brains of
schizophrenic patients with a less severe course of the disease.
Another unresolved issue is whether the altered splicing of
D3 pre-mRNA is an outcome of neuroleptic treatment, which
is the most common therapeutic strategy for schizophrenia. It is
impossible, at present, to address this issue with studies on tissues
obtained from drug-naive schizophrenic patients because of an extremely
limited availability of such brains. However, the reconstitution of
D3 pre-mRNA splicing in vivo in transfected
cells that express D2 and/or D2-like receptors (D3, D4) will now allow us to test directly
whether different pharmacological manipulations of these receptors have
effects on the splicing of D3 pre-mRNA. If neuroleptic
drugs can alter the splicing pattern of D3 pre-mRNA, this
would not necessarily imply that another side effect of such drugs has
been discovered. It could, in fact, be a mechanism by which
neuroleptics mediate their antipsychotic effect.
FOOTNOTES
Received Aug. 12, 1996; revised Sept. 20, 1996; accepted Oct. 1, 1996.
This work was supported by a National Alliance for Research on
Schizophrenia and Depression Young Investigator Award. I thank the
staff members of the National Neurological Research Specimen Bank
(Veterans' Administration Medical Center, Los Angeles, CA) and of the
Schizophrenia Brain Bank of the Department of Psychiatry at Mount Sinai
School of Medicine (New York, NY) for providing the brain tissues and
the corresponding diagnostic and neuropathological evaluations for this
study. Dr. Boris Skryabin and Julie Yoon assisted in the generation of
mutant plasmids and the preparation of nuclear extracts,
respectively.
Correspondence should be addressed to Dr. Claudia Schmauss, Mount Sinai
School of Medicine, Box 1229, One Gustave L. Levy Place, New York, NY
10029.
REFERENCES
-
Asherson P,
Mant R,
Holmans P,
Williams J,
Cardno A,
Murphy K,
Jones L,
Collier D,
McGuffin P,
Owen MJ
(1996)
Linkage, association and mutational analysis of the dopamine D3 receptor gene in schizophrenia.
Mol Psychiatry
1:125-132.[ISI][Medline]
-
Bunzow JR,
Van Tool HM,
Grandy DK,
Albert P,
Salon J,
Christie M,
Machida CA,
Neve KA,
Civelli O
(1988)
Cloning and expression of a rat D2 dopamine receptor cDNA.
Nature
336:783-787 .
[Medline]
-
Charbot B,
Steitz JA
(1987)
Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.
Mol Cell Biol
7:281-293.
[Abstract/Free Full Text]
-
Crocq MA,
Mant R,
Asherson P,
Williams J,
Hode Y,
Mayerova A,
Collier D,
Lannfelt L,
Sokoloff P,
Schwartz JC,
Gill M,
Macher JP,
McGuffin P,
Owen MJ
(1992)
Association between schizophrenia and homozygosity at the dopamine D3 receptor gene.
J Med Genet
29:858-860 .
[Abstract]
-
Dal Toso R,
Sommer B,
Ewert M,
Herb A,
Pritchett DB,
Bach A,
Shivers B,
Seeburg PH
(1989)
The dopamine D2 receptor: two molecular forms generated by alternative splicing.
EMBO J
8:4025-4034 .
[ISI][Medline]
-
Dignam JD,
Lebovitz RM,
Roeder RG
(1983)
Acurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res
11:1475-1489 .
[Abstract/Free Full Text]
-
Giros B,
Sokoloff P,
Martres MP,
Riou JF,
Emorine LJ,
Schwartz JC
(1989)
Alternative splicing directs the expression of two D2 dopamine receptor isoforms.
Nature
342:923-926 .
[Medline]
-
Giros B,
Martres MP,
Pilon C,
Sokoloff P,
Schwartz JC
(1991)
Shorter variants of the D3 dopamine receptor produced through various patterns of alternative splicing.
Biochem Biophys Res Commun
176:1584-1592 .
[ISI][Medline]
-
Grandy DK,
Marchionni MA,
Makam H,
Stofko RE,
Alfano M,
Frothingham L,
Fischer JB,
Burke-Howie KJ,
Bunzow JR,
Server A,
Civelli O
(1989)
Cloning of the cDNA and gene for a human D2 dopamine receptor.
Proc Natl Acad Sci USA
86:9762-9766 .
[Abstract/Free Full Text]
-
Hall SL,
Padgett RA
(1996)
Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns.
Science
271:1716-1718 .
[Abstract]
-
Howe JR,
Skryabin BV,
Belcher SM,
Zerillo CA,
Schmauss C
(1995)
The responsiveness of tetracycline-sensitive expression system differs in different cell lines.
J Biol Chem
270:14168-14174 .
[Abstract/Free Full Text]
-
Krainer AR,
Maniatis T,
Ruskin B,
Green M
(1984)
Normal and mutant human
-globin pre-mRNAs are faithfully and efficiently spliced in vitro.
Cell
36:993-1005 .
[ISI][Medline]
-
Liu K,
Bergson C,
Levenson R,
Schmauss C
(1994)
On the origin of mRNA encoding the truncated dopamine D3-type receptor D3nf and detection of D3nf-like immunoreactivity in human brain.
J Biol Chem
269:29220-29226 .
[Abstract/Free Full Text]
-
Macciardi F,
Verga M,
Kennedy JL,
Petronis A,
Bersani G,
Pancheri P,
Smeraldi E
(1994)
An association study between schizophrenia and the dopamine receptor genes DRD3 and DRD4 using haplotype relative risk.
Hum Hered
44:328-336 .
[ISI][Medline]
-
Mant R,
Williams J,
Asherson P,
Parfitt E,
McGuffin P,
Owen MJ
(1994)
Relationship between homozygosity at the dopamine D3 receptor gene and schizophrenia.
Am J Med Genet
54:21-26 .
[ISI][Medline]
-
Monsma F,
McVittie LD,
Gerfen CR,
Mahan LC,
Sibley DR
(1989)
Multiple D2 dopamine receptors produced by alternative RNA splicing.
Nature
342:926-929 .
[Medline]
-
Nagai Y,
Ueno S,
Saeki Y,
Soga F,
Yanagihara T
(1993)
Expression of the D3 dopamine receptor gene and a novel variant transcript generated by alternative splicing in human peripheral blood lymphocytes.
Biochem Biophys Res Commun
194:368-374 .
[ISI][Medline]
-
Reed R
(1989)
The organization of 3 splice-site sequences in mammalian introns.
Genes Dev
3:2113-2123 .
[Abstract/Free Full Text]
-
Reed R,
Maniatis T
(1985)
Intron sequences involved in lariat formation during pre-mRNA splicing.
Cell
41:95-105 .
[ISI][Medline]
-
Schmauss C,
Lerner MR
(1990)
The closely related snRNP polypeptides N and B/B are distinguishable by antibodies as well as by differences in their mRNA and gene structures.
J Biol Chem
265:10733-10739 .
[Abstract/Free Full Text]
-
Schmauss C,
McAllister G,
Ohosone Y,
Hardin JA,
Lerner M,
R
(1989)
A comparison of the snRNP-associated Sm-autoantigens human N, rat N and human B/B.
Nucleic Acids Res
17:1733-1744 .
[Abstract/Free Full Text]
-
Schmauss C,
Brines ML,
Lerner MR
(1992)
The gene that encodes the snRNP-associated protein N is expressed at high levels in neurons.
J Biol Chem
267:8521-8529 .
[Abstract/Free Full Text]
-
Schmauss C,
Haroutunian V,
Davis KL,
Davidson M
(1993)
Selective loss of dopamine D3 receptor mRNA expression in the parietal and motor cortex in patients with chronic schizophrenia.
Proc Natl Acad Sci USA
90:8942-8946 .
[Abstract/Free Full Text]
-
Shaikh S,
Collier DA,
Sham PC,
Ball D,
Aitchison K,
Vallada H,
Smith I,
Gill M,
Kerwin RW
(1996)
Allelic association between a Ser-9-Gly polymorphism in the dopamine D3 receptor gene and schizophrenia.
Hum Genet
97:714-719 .
[ISI][Medline]
-
Snyder LA,
Roberts JL,
Sealfon SC
(1991)
Alternative transcripts of the rat and human dopamine D3 receptor.
Biochem Biophys Res Commun
180:1031-1035 .
[ISI][Medline]
-
Sokoloff P,
Giros B,
Martres MP,
Bouthenet ML,
Schwartz JC
(1990)
Molecular cloning and characterization of a novel dopamine receptor (D3) as a targets for neuroleptics.
Nature
347:146-151 .
[Medline]
-
Tarn WY,
Steitz JA
(1996)
A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro.
Cell
84:801-811 .
[ISI][Medline]
-
Van Tol HHM,
Bunzow JR,
Guan HC,
Sunahara RK,
Seeman P,
Niznik HB,
Civelli O
(1991)
Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine.
Nature
350:610-614.
[Medline]
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