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J. Biol. Chem., Vol. 275, Issue 24, 18079-18084, June 16, 2000
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From the Atherosclerosis Specialty Laboratory, Department of Pathology and Laboratory Medicine, St. Paul's Hospital and University of British Columbia, Vancouver, British Columbia, V6Z 1Y6 Canada
Received for publication, December 21, 1999, and in revised form, April 3, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have previously identified a point mutation
(intervening sequence (IVS) 4: T The removal of introns from pre-mRNA involves the recognition
of the 5'-splice site, the branchpoint sequence, and the 3'-splice site
by five small nuclear ribonucleoprotein particles
(snRNPs), i.e. U1, U2, U5, and
U4/U6, and a large number of non-snRNP splicing factors (1-4). The
functional significance of the splice sites has been well established
by the studies of a number of naturally occurring mutations and of
site-directed mutagenesis at these exon/intron junctions (5-8). It is
estimated that up to 15% of all point mutations causing human genetic
disease result from mRNA splicing defects, most of which involve a
single nucleotide substitution at the splice sites (5). To date, only a
few mutations in the branchpoint consensus sequence of introns have
been reported to cause human genetic diseases (9-13). However, the
growing evidence indicates that the branchpoint sequence can be of
essential importance for accurate and efficient splicing of human
nuclear pre-mRNA.
A series of elegant genetic experiments in both yeast and mammalian
cell lines have shown that base pairs exist between the branchpoint
sequence and a conserved region (5'-AUGAUG-3') in U2 snRNA (14-16).
The branchpoint sequence is absolutely conserved in yeast
(UACUAAC; the underlined A indicates the branchpoint nucleotide). Mutations of the yeast branchpoint adenosine significantly reduce or abolish splicing (17). By contrast, the branchpoint sequence
exhibits only a weak consensus in mammals (YNYURAY; where Y = pyrimidine, R = purine, and N = any nucleotide), and
mutations of the branchpoint adenine in mammalian introns are known to
only result in moderately reduced splicing efficiency due to the
utilization of cryptic branchpoint sequences (18-20). To compensate
for the poorly conserved branchpoint sequence, most mammalian introns contain another cis-acting element, polypyrimidine tract, between the
branchpoint sequence and the 3'-splice site. Early during the
spliceosome assembly, the specific binding of U2AF to the polypyrimidine tract has been shown to promote the base pairing of U2
snRNA with the branchpoint sequence (21). However, much less is still
known about the mechanism of the selection of the branchpoint
adenosine, which is usually located 18-40 nucleotides upstream of the
3'-splice site, in the mammalian intron with respect to the base pairs
between the U2 snRNA and the pre-mRNA.
The human lecithin:cholesterol acyltransferase (LCAT) is encoded by a
single gene that is located on chromosome 16 and composed of six exons
(22, 23). Mutations in the LCAT gene have been demonstrated to underlie
either familial LCAT deficiency or fish-eye disease (FED) (24). We have
previously identified a point mutation in intron 4 of the LCAT gene in
patients with FED (25). This point mutation resides in a 100% matched
branchpoint consensus sequence (CCCTGAC versus YNYTRAY), and
it has been shown to result in the complete intron retention. Similar
results have been observed when two other novel mutations
(i.e. T In this study, we demonstrate that the mutations of the branchpoint
adenosine residue in intron 4 of the human LCAT gene, like the natural
mutation, completely abolish the splicing in the absence of cryptic
branch sites in the intron. The point mutations introduced into other
positions of the branchpoint sequence of the intron also have
significant effects on the efficiency of splicing and thus the enzyme
activity in vivo. We therefore establish the functional
significance of the branchpoint sequence of intron 4 for the splicing
of the human LCAT mRNA precursors.
Construction of Mutant LCAT Minigenes--
The LCAT minigene
containing the full length cDNA (28) and the wild type sequence of
intron 4 (25) was first released from the pUC19-LCAT-intron 4 plasmid
by the digestion of EcoRI and BamHI. The
EcoRI-BamHI DNA fragment was then subcloned into pcDNA3.1( Transient Transfection of HEK-293 Cells--
Transient
transfection of the LCAT minigene constructs into human embryonic
kidney (HEK)293 cells was performed using the calcium phosphate
co-precipitation method (31). The mammalian expression vectors,
pcDNA3.1 and pcDNA3.1-LCAT cDNA as well as pcDNA3.1-LCAT IVS4 wild type, were used as negative and
positive controls, respectively. For transfection, 28 µg of each of
the plasmids was added to 100-mm Petri dishes containing approximately 106 cells in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum. The
medium was replaced with serum-free Opti-MEM 24 h after
transfection and incubated for another 48 h. Culture medium was
collected for the LCAT activity assays, and total cytoplasmic RNA was
prepared using the RNeasy Mini Kit (Qiagen) according to the
manufacturer's protocol.
Measurement of LCAT Activity--
LCAT activity in the collected
media was determined using proteoliposome substrate as described
previously (26). The substrate contained apoA-I,
[3H]cholesterol, and phosphatidylcholine at a molar ratio
of 0.8:12.5:250. The reaction mixture containing 100 µl of HEK-293
transfection medium was incubated at 37 °C for 2 h, and the
reaction was terminated by adding 1 ml of absolute ethanol.
Unesterified cholesterol and cholesteryl esters were separated using
thin-layer chromatography in petroleum ether/diethyl ether/acetic acid
(70:12:1, v/v), and the radioactivity was determined by liquid
scintillation counting.
Reverse Transcription-PCR Amplification--
First-strand
cDNA was synthesized from 1.0 µg of total cytoplasmic RNA in a
20-µl reverse transcription mixture containing 50 mM
Tris-HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl2, 1 mM dNTPs, 200 ng of
oligo(dT)12-18, and 200 units of Moloney murine leukemia virus reverse transcriptase (RT; Life Technologies, Inc.) at 42 °C
for 1 h. After reverse transcription, 10% of the reaction product was used for PCR amplification in a 50-µl final reaction volume (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.4 µM of primers that
were derived from sequences of exon 2 (5'-GCT ACC GCA AGA CAG AGG
AC-3') and exon 5 (5'-GGC CAA TGA GGA AGA CAG GC-3') of the LCAT gene,
respectively, and 2 units of Taq DNA polymerase). To
determine the efficiency of transient transfection, another primer pair
complementary to the sequences (sense primer, 5'-GGG GTT CGA AAT GAC
CGA CC-3'; antisense primer, 5'-CAG CTG GCA CGA CAG GTT TC-3') within
the neomycin resistance gene of the pcDNA3.1 vector were utilized
to serve as an internal control. Thirty-five cycles of amplification
were performed using a Perkin-Elmer DNA thermal cycler (System 2400).
Each cycle consisted of a 30-s denaturation at 95 °C, a 30-s
annealing at 62 °C, and a 30-s extension at 72 °C. The PCR
products were separated on a 3% agarose gel, and the bands were
visualized by ethidium bromide staining.
Generation of Mutations in the Branchpoint Sequence of Intron 4 of
the LCAT Gene--
To study the effects of various mutations in the
branchpoint consensus sequence of intron 4 on the efficiency of LCAT
pre-mRNA splicing in vivo, we first cloned the human
LCAT minigene into the mammalian expression vector, pcDNA3.1 (Fig.
1A). Intron 4 of the human
LCAT gene is small and consists of only 83 base pairs (Fig.
1B). The putative branchpoint sequence (CCCTGAC)
is located
Most of the site-directed mutagenesis involved the nucleotide
transversion according to the consensus sequence YNYTRAY. We also made
one plasmid construct in which the wild type branchpoint sequence
CCCTGAC was replaced by the absolutely conserved yeast branch site
sequence TACTAAC to test whether the invariant branchpoint sequence is
the most preferred sequence for mammalian pre-mRNA splicing
(32).
Mutations of the Branch Site Adenine Completely Abolish Splicing of
Intron 4 of the LCAT Gene in Vivo--
The retention of intron 4 of
human LCAT gene caused by the substitution of the thymine to other
nucleotides in the putative branchpoint sequence (25, 26) suggested
that the mutations of the branchpoint adenosine residue also result in
the defective splicing for this particular intron. As shown in Table
II, the LCAT cDNA exhibited the
highest LCAT activity. When the wild type intron 4 was inserted into
cDNA, the enzymatic activity decreased to approximately 20-25% of
the original, which was most likely due to the influence of the
splicing process on the expression of the gene. However, like the
natural mutation, IVS4-MUT, and its two related mutants, IVS4-MUT-1 and
-MUT-2, substitution of the branchpoint adenine with a cytosine
resulted in the absence of LCAT activity as compared with control
transfections with the empty pcDNA3.1 vector. A similar result was
also obtained when the branchpoint adenine was changed to guanosine or
thymine. To determine further the effect of the branchpoint mutations
on the LCAT pre-mRNA splicing in vivo, RT-PCR was
performed. The wild type intron was efficiently spliced out of the
pre-mRNA (Fig. 2, lane 3)
giving a spliced RNA band corresponding to the LCAT cDNA
(Fig. 2, lane 2), whereas no spliced RNAs could be detected for either the IVS4-MUT, -MUT-1, and -MUT-2 (Fig. 2, lanes
4-6) or the branchpoint mutants, IVS-20c, -20g, and -20t (Fig. 2,
lanes 7-9). These observations are consistent with previous
reports (32, 33) that mutations of the highly conserved thymine at the
fourth position of the branchpoint consensus sequence have a similar
effect on splicing as do mutations at the branch site adenosine.
Intron 4 of the LCAT Gene Lacks Cryptic Branchpoint
Sequence--
In our study, we have not observed any alternative
splicing of LCAT pre-mRNA containing the natural and the
branchpoint mutations. Examination of the intron sequence upstream from
the 3'-splice site (Fig. 1B) does not reveal any potential
branch site that matches the mammalian YURAY consensus sequence, and
this may be the reason why we observe a complete abolishment of
splicing. To test this hypothesis, we inserted a normal branchpoint
sequence CCCTGAC into the intronic sequence just six bases upstream of the mutated branch sites (Fig. 3). The
results of the insertion of the branchpoint sequence are shown in Fig.
4. The introduction of a normal branch
site totally restored the enzyme activities from the natural and the
branchpoint mutants, respectively. Interestingly, the insertion of the
branch site into the branchpoint mutant IVS4-20t resulted in even
higher LCAT activity than did the wild type intron. However, the
insertion had no obvious effect on the intron 4 wild type (Fig.
4A). The efficiently spliced RNAs from the natural and the
branchpoint mutants were revealed by RT-PCR (Fig. 4B, lanes 6 and 8, respectively), which further
confirmed the effect of the insertion of a normal branch site into the
intron sequences on the splicing of these mutated LCAT mRNA
precursors. These results suggested that the inserted branch site was
utilized when the normal branchpoint sequence contained a mutation.
The Natural Mutation of Intron 4 Can Be Partially Suppressed by
Changing G Single Base Changes in the Branchpoint Sequence of Intron 4 Affect
Splicing of LCAT pre-mRNA in Vivo--
To demonstrate further the
functional role of the branchpoint sequence, we identified the effects
of mutations of other nucleotides in the branchpoint consensus sequence
on the efficiency of LCAT pre-mRNA splicing by LCAT activity
assay and RT-PCR (Fig. 6). The mutants
IVS4-19g and IVS4-21c (Fig. 6A, bars 4 and
5) were associated with the lowest LCAT activities followed
by IVS4-19a (Fig. 6A, bar 3). A dramatic
reduction in LCAT enzyme activity was also observed with IVS4-23g (Fig.
6A, bar 8). The mutants IVS4-21t and IVS4-23a
(Fig. 6A, bars 6 and 7) also had
markedly decreased LCAT activities compared with that of the wild type. However, the C
The effects of these intron mutations on the efficiency of RNA splicing
were further determined by RT-PCR. As expected, the normally spliced
RNA could be detected in all of these mutants, but the efficiency of
the splicing was completely different from one another. The mutant
IVS4-19g and IVS4-21c had the least spliced RNAs (Fig. 6B,
lanes 6 and 7) followed by IVS4-19a and IVS4-21t (Fig. 6B, lanes 5 and 8). The
densities of the spliced RNAs for the mutants IVS-23a and 23g were less
than that of wild type (Fig. 5B, lanes 9 and
10), and the levels of spliced RNA in IVS4-24a and -24t were
similar to those of wild type control (Fig. 5B, lanes
11 and 12). It was interesting to note that the RNA
splicing of mutants IVS-25a and -25g was much more efficient than that of the wild type (Fig. 5B, lanes 13 and
14), approximately 50% of the transcripts were spliced. All
of these observations are consistent with the corresponding LCAT
activities for these mutants. Therefore, it appears that the efficiency
of splicing is well correlated with the activity of LCAT secreted from
the transfected HEK-293 cells into the medium. Another surprising
observation in this study was that the exchange of the wild type
branchpoint sequence of intron 4 with the invariant TACTAAC box of
yeast did not increase the efficiency of splicing but had a mildly
decreased spliced RNA (Fig. 6B, lane 4) and LCAT
activity (Fig. 6A, bar 2).
We have previously shown that a point mutation in a lariat
branchpoint sequence of intron 4 of the LCAT gene (IVS4:T-22C) resulted
in the intron retention causing a human inherited disorder, fish-eye disease (25). We have also demonstrated that the thymine residue at the fourth position of the branchpoint sequence in which the
natural mutation occurs is crucial for the pre-mRNA splicing (26).
In this report, we extend our studies into the whole region of the
branchpoint sequence and further demonstrate that the branchpoint
sequence of intron 4, especially the branchpoint adenosine residue and
the nucleotides around it, is essential for the efficient splicing of
LCAT pre-mRNA.
Previous studies have shown that the branchpoint sequence is not
essential for splicing of mammalian introns, because deletion or
mutation of the branchpoint sequence does not abolish splicing both
in vitro and in vivo (18-20). This paradox has
been explained by the utilization of a relatively inefficient nearby
cryptic branchpoint sequence (20). However, our results reported here clearly demonstrate that this is not always the case. The single base
substitutions of the adenosine reside in the branchpoint region of
intron 4 of LCAT pre-mRNA completely abolish splicing as assayed by
the enzyme activity and RT-PCR. Newman et al. (34) has
previously shown that mutation of the adenosine residue in the yeast
branchpoint sequence abolishes the RNA splicing by preventing the
cleavage at the 5'-splice site. In this respect, the mutations of the
branchpoint A as well as the natural mutation in intron 4 of the LCAT
gene might have the same effect on the cleavage at the 5'-splice site.
Because the intronic sequence contains a stop codon signal, the failure
to detect any LCAT activity and the truncated protein (data not shown)
in the culture medium indicates that the translation products are
likely rapidly degraded within the cells (26). Recently, another
example of the branchpoint mutation that disrupts mRNA splicing in
intron 9 of the low density lipoprotein receptor gene has been
described in a patient with familial hypercholesterolemia. This
mutation disrupts the branchpoint consensus sequence and also causes
intron retention (10). These observations, therefore, suggest that the
branchpoint sequence plays an important role in the selection of the
branchpoint nucleotide, although it is poorly conserved in mammals.
To investigate the possible mechanisms for the defective splicing, we
first inserted a normal branchpoint sequence CCCTGAC into the natural
and the branchpoint mutants (IVS4-22c and IVS4-20t, respectively) six
nucleotides upstream of the mutated branch sites (Fig. 3). The purpose
of the insertion is to see whether this sequence could rescue the
natural or the branchpoint mutation. As expected, we observed not only
the complete restoration of the LCAT activities of both the mutants but
also an increase in splicing efficiency of the branchpoint mutant
compared with the intron 4 wild type construct. These results suggest
that the sequence of intron 4 itself does not contain any potential
cryptic branch sites. Once the branchpoint sequence had been mutated,
there was no alternative to back up the splicing machinery so that the
consequence of the natural and the branchpoint mutations was retention
of the intron. At the moment, we do not know the reasons why the introduction of the normal branch site into the intronic sequence of
the branchpoint mutant increased the efficiency of LCAT pre-mRNA splicing. It is possible that two potential branch sites within a
single intron might compete with each other (33). For the intron wild
type and the natural mutant, either of the two potential branchpoint
sequences could be screened as the branch site signal, but the
one that mostly matches the consensus sequence would be preferentially
utilized. In the case of the branchpoint mutant, the inserted branch
site becomes the only branchpoint sequence to be used in the
pre-mRNA splicing, which may be one of the possible explanations
for the increased efficiency of pre-mRNA splicing.
The base pairing interaction between the branchpoint sequence and the
sequence AUGAUG in U2 snRNA is one of the early essential events in
pre-mRNA splicing (14-16). In an attempt to test the hypothesis
that the natural mutation causes intron retention as the result of
interference with the binding of U2 snRNA to the branchpoint sequence
(Fig. 5A), we changed the naturally mutated branchpoint
sequence from CCCCGAC to
CCCCAAC (the underlined C is the natural
mutation, and the bold letters represent the mutation we made, G This conclusion was further supported in the mutational analysis of the
functional significance of other specific nucleotides in the
branchpoint consensus sequence. In addition to the mutations at the
fourth position of the branchpoint sequence, the alterations of the
nucleotides, especially around the branchpoint A, i.e. IVS4-19a, -19g, and -21c have a dramatic effect on the splicing of the
intron, probably because these mutations greatly reduce the ability of
potential base pairing with the U2 snRNA. And if the branchpoint
adenosine residue was not flanked by a base-paired residue on either
its 3'- or 5'-site, such as in the case of IVS4-19a, -19g, and -21c,
the branch nucleotide would not be properly bulged and, therefore,
would affect the splicing. Recently, Pascolo et al. (35)
demonstrated that U2 snRNA does indeed base pair, with the nucleotides
preceding and following the branchpoint adenosine residue that is
constrained into a bulged conformation. The mutants IVS4-23g, -21t, and
-23a also have significant effects on the splicing efficiency, probably
through a similar mechanism. Interestingly, in contrast to these
downstream mutations, the substitution of the last nucleotide from C
Another unexpected observation made in this study is that the
substitution of the normal branchpoint sequence CCCUGAC with the yeast
branch site UACUAAC sequence did not increase the efficiency of LCAT
pre-mRNA splicing but was modestly decreased. This result is
surprising, because it is in conflict with a previous study (33) in
which the data showed that the optimal branchpoint sequence is the
yeast UACUAAC sequence for mammalian introns. Although the UACUAAC has
perfect complementarity with the sequence AUGAUG in U2 snRNA, our
results from the transfection experiments do not seem to support the
concept that the perfect match improves the efficiency of pre-mRNA
splicing in vivo. However, the observation that the yeast
UACUAAC box did not increase the efficiency of LCAT pre-mRNA splicing
is consistent with the results that the mutations of the last position
of the branchpoint sequence markedly increase the splicing efficiency
even though the changes reduce the match within the consensus
sequence. This would, at least in part, explain why splicing is less
dependent on base pairing of the pre-mRNA with U2 snRNA in mammals.
For the mammalian introns, it is likely that other protein-RNA and
RNA-RNA interactions are required to maintain the pre-mRNA and the
U2 snRNA in the correct conformation in the absence of one or two of
these base pairs.
In conclusion, by taking advantage of our in vivo assay for
measuring the splicing efficiency, we are able to demonstrate that the
branchpoint consensus sequence of intron 4 is essential for the
splicing of LCAT pre-mRNA. With the increasing intron sequence data
available, more intron mutations far from the splice sites will be
found to be the underlying causes for the human genetic disorders.
C) in the branchpoint consensus
sequence of intron 4 of the lecithin:cholesterol acyltransferase (LCAT) gene in patients with fish-eye disease. To investigate the possible mechanisms responsible for the defective splicing, we made a series of
mutations in the branchpoint sequence and expressed these mutants in
HEK-293 cells followed by the analysis of pre-mRNA splicing using
reverse transcriptase-polymerase chain reaction as well as LCAT
activity assay. The results reveal that 1) the mutation of the
branchpoint adenosine to any other nucleotide completely abolishes
splicing; 2) the insertion of a normal branch site into the intronic
sequence of the natural (IVS4-22c) or the branchpoint (IVS4-20t) mutant
completely restores splicing; 3) the natural mutation can be
partially rescued by making a single nucleotide change (G A) within
the branchpoint consensus sequence; and 4) other single base changes,
particularly around the branchpoint adenosine residue, significantly
decrease the efficiency of splicing and thus enzyme activity.
Surprisingly, the nucleotide transversion at the last position of the
branchpoint sequence (i.e. IVS4-25a or -25g) results in a
2.7-fold increase in splicing efficiency. Therefore, these observations
clearly establish the functional significance of the branchpoint
sequence of intron 4 for the splicing of the human LCAT mRNA precursors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G or T A) were introduced into the same site
of the natural mutation (intervening sequence (IVS) 4: T C),
suggesting that the highly conserved thymidine residue in the
branchpoint sequence is essential for the LCAT pre-mRNA splicing
(26). LCAT gene has an unusually high conserved sequence (27). The
intron 4 of LCAT gene contains only 83 base pairs, and the possible
absence of cryptic branchpoint sequences in this short intron may
render it highly sensitive to any alteration within the branchpoint
region. Therefore, the branchpoint sequence in intron 4 of the LCAT
gene may act as a model to identify the functional significance of
conserved nucleotides contained within the branchpoint sequence of the
human genes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), a mammalian expression vector (Invitrogen, San Diego, CA) using standard procedures (29). The LCAT intron 4 mutants were
constructed by the overlap-polymerase chain reaction (PCR) technique
(30), and the pcDNA3.1-LCAT intron 4 wild type plasmid was used as
the template. The upstream (5'-GGG AGA CCC AAG CTG GCT A-3') and
downstream (5'-CGT CGA GGC TGA TCA GCG G-3') primers used in the
mutagenesis are complementary to sequences that flank the 5'- and
3'-sites of the polylinker of the pcDNA3.1 vector, respectively.
The sequences of site-directed oligonucleotide primers used to create
the desired intron mutations are listed in Table I. In detail, the first PCR was performed
with 100 ng of template DNA, 200 µM of each dNTP, 0.4 µM of each upstream primer and reverse mutagenic
oligonucleotide, or 0.4 µM of each downstream primer and
forward mutagenic oligonucleotide. The PCR was performed as follows: 30 cycles each of 30 s at 95 °C, 30 s at 56 °C, and
60 s at 72 °C with 2.5 units of pfu DNA polymerase (Stratagene,
La Jolla, CA). The resultant two PCR fragments were gel-purified and
used in a subsequent fusion reaction under the same PCR condition except for the extension time increased to 2 min. The final products purified from the agarose gel were digested with EcoRI and
BamHI and then ligated into the pcDNA3.1 vector. The
presence of the introduced intron mutations was confirmed by dideoxy
sequencing.
Oligonucleotide primers used to create the desired intron mutations
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
19 to 25 bases upstream of the 3'-splice site. For
convenience, we designated the nucleotide at 19 relative to the
3'-splice site as the first position of the branchpoint sequence. Based on the wild type pcDNA3.1-LCAT minigene, we made a series of point mutations in the branch site using the synthetic oligonucleotides as
described under "Experimental Procedures."
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Fig. 1.
Schematic presentation of the
pcDNA3.1-LCAT minigene construct. A, the
pcDNA3.1 contains the human cytomegalovirus immediate early
promoter (CMV), a multiple cloning site region
(MCS, shaded box), and the 3' portion of the
bovine growth hormone gene, including the polyadenylation site. The
human LCAT minigene insert encompasses full length LCAT cDNA as
well as the wild type sequence of intron 4. The sizes of the exons 1-4
and 5-6 as well as the intron, in base pairs, are indicated above and
below the elements, respectively. B, the sequence of intron
4 of the human LCAT gene. The branchpoint consensus sequence is
underlined, in which T is mutated to C in the patients with
fish-eye disease. The asterisk indicates the adenosine of
the putative branchpoint nucleotide.
The effect of mutations in the branch site adenine on the activity
of LCAT from medium of HEK-293 cells
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Fig. 2.
The effect of the branchpoint mutations on
the LCAT pre-mRNA splicing as analyzed by RT-PCR. Total
cytoplasmic RNA isolated from HEK-293 cells that were transiently
transfected with the branchpoint mutants were analyzed by RT-PCR using
primers complementary to sequences of exons 2 and 5, respectively.
Correct splicing results in the excision of intron 4 giving a fragment
of 389 bp corresponding to the same size of the LCAT cDNA. The
unspliced product has a size of 472 bp containing the 83-bp sequence of
intron 4. Lane 1, pcDNA 3.1 vector without insert;
lane 2, LCAT cDNA; lane 3, IVS4-WT;
lane 4, IVS4-MUT; lane 5, IVS4-MUT-1; lane
6, IVS4-MUT-2; lane 7, IVS4-20c; lane 8,
IVS4-20g; lane 9, IVS4-20t; and M, molecular
weight marker.
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Fig. 3.
Insertion of a natural branch site into the
sequences of mutated intron 4 of the human LCAT genes. Both the
inserted and mutated branch sites are underlined. *, normal
branchpoint; (*), inserted branchpoint. A, the insertion of
a natural branch site into the wild type sequence of intron 4 of the
LCAT gene. B, the insertion of a natural branch site into
the naturally mutated intron 4 of the LCAT gene. The highly conserved T
was mutated to C in the patients with FED. C, the insertion
of a natural branch site into the branchpoint mutation of intron 4 of
the LCAT gene. The normal branchpoint A was substituted to T in the
branch site.
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Fig. 4.
The effect of the introduction of a normal
branch site into the intron sequences on the splicing of the LCAT
mRNA precursors containing the natural (IVS4-MUT) and the
branchpoint (IVS4-20t) mutations. A, enzymatic activity
secreted by transiently transfected HEK-293 cells. Bar 1,
IVS4-WT; bar 2, IVS4-BPS-WT; bar 3, IVS4-MUT;
bar 4, IVS4-BPS-MUT, bar 5, IVS4-20t; bar
6, IVS4-BPS-20t. ***p < 0.0001 compared with the
intron wild type. B, RT-PCR analysis of total cytoplasmic
RNA from HEK-293 cells transfected with the LCAT intron 4 mutants
containing the branchpoint sequence insertion. Lane 1,
pcDNA 3.1 vector without insert; lane 2, LCAT cDNA;
lane 3, IVS4-WT; lane 4, IVS4-BPS-WT; lane
5, IVS4-MUT; lane 6, IVS4-BPS-MUT, lane 7,
IVS4-20t; lane 8, IVS4-BPS-20t; and M, molecular
weight marker. BPS represents the inserted normal branchpoint
sequence.
A at the Third Position of the Branchpoint
Sequence--
We have previously proposed (25, 26) that the natural
mutation T C at the fourth position of the branch site of intron 4 of the LCAT gene might not only destroy the base pairing itself with
the fourth position of AUGAUG sequence in human U2 snRNA but also disrupt the immediate downstream weak G-U base pairing interaction between the branchpoint sequence and U2 snRNA (Fig. 5A, scheme b). This
disruption would result in the inappropriate bulging of the adenosine
residue in the branchpoint region and thus lead to the defective
splicing. Therefore, if the G at the third position of the branchpoint
sequence is altered to A, the Watson-Crick A-U base pair between the
branch site and the AUGAUG sequence in U2 snRNA might compensate, at
least in part, for the natural mutation in the branchpoint sequence
(Fig. 5A, scheme c). As shown in Fig.
5B, the alteration from the sequence CCCCGAC to CCCCAAC did
indeed increase the LCAT activity, although it was still very low as
compared with the wild type intron. However, a correctly spliced
transcript could be clearly identified (Fig. 5C, lane
5). By contrast, no band corresponding to the mature mRNA
transcript was observed in the cells that expressed the natural mutant
(Fig. 5C, lane 4). To exclude the possibility
that the effect of the double mutant (IVS4-21a, -22c) on the splicing
of LCAT pre-mRNA resulted from the differences in the efficiency of
transient transfection rather than specific base-pairing interaction, we assayed the relative abundance of each expressed construct by RT-PCR
with a set of primers that amplify 502-bp fragment of the neomycin
resistance gene (NeoR), which is under the control of the
SV40 early promoter in the pcDNA3.1 vector. As shown in Fig.
5C, the amount of amplified NeoR gene product
was similar in the cells transfected with each plasmid construct
(lanes 6-10), suggesting that there were no differences in
the transient transfection efficiency.
View larger version (28K):
[in a new window]
Fig. 5.
The effect of the change of the branchpoint
sequence from CCCCGAC to CCCCAAC on the splicing of LCAT
pre-mRNA. A, proposed base pairing interaction
between the branchpoint sequence and U2 snRNA. a, U2 snRNA,
wild type intron 4 of LCAT pre-mRNA; b, U2 snRNA, the
natural mutation of intron 4 of LCAT pre-mRNA; c, U2
snRNA, the doubly mutated intron 4 of LCAT pre-mRNA. B,
LCAT activity from the media of HEK-293 cells transfected with the
natural and the IVS4-21a, -22c mutants. **p < 0.001 compared with the natural mutant. C, total cytoplasmic RNA
from the natural and the IVS4-21a, -22c mutants for either LCAT
fragment (lanes 1-5) or NeoR (lanes
6-10) was amplified by RT-PCR with the primer pairs as described
under "Experimental Procedures." Lanes 1 and
6, pcDNA 3.1 vector without insert; lanes 2 and 7, LCAT cDNA; lanes 3 and 8,
IVS4-WT; lanes 4 and 9, IVS4-MUT; lanes
5 and 10, IVS4-21a, -22c; and M, molecular
weight marker.
A transversion or C T transition at the sixth position (IVS4-24a or IVS4-24t) of the consensus sequence had only a
mild decreased enzyme activity (Fig. 6A, bars 9 and 10) as compared with wild type control. Interestingly,
in contrast to these downstream mutations, the change at the last
position of the branchpoint sequence, i.e. IVS4-25a or
-25g, resulted in approximately a 2.7-fold increase in LCAT activity
compared with the intron 4 wild type (Fig. 6A, bars
11 and 12).
View larger version (43K):
[in a new window]
Fig. 6.
The effects of the mutations in the
branchpoint sequence on the efficiency of LACT pre-mRNA
splicing. A, LCAT activities of HEK-293 cell media for
intron 4 wild type and the various intron mutants. The enzymatic
activities (nanomoles/ml/h) are expressed as a percentage of wild type
LCAT intron 4 activity. Bar 1, IVS4-WT; bar 2,
IVS4-yeast; bar 3, IVS4-19a; bar 4, IVS4-19g;
bar 5, IVS4-21c; bar 6, IVS4-21t; bar
7, IVS4-23a; bar 8, IVS4-23g; bar 9,
IVS4-24a; bar 10, IVS4-24t; bar 11, IVS4-25a;
bar 12, IVS4-25g. ***p < 0.0001, **p < 0.001, *p < 0.05 compared with
the wild type intron. B, RT-PCR on total cytoplasmic RNA
from HEK-293 cells that were transiently transfected with LCAT minigene
constructs carrying a series of mutations in the branchpoint sequence.
Lane 1, pcDNA 3.1; vector without insert. Lane 2,
LCAT cDNA; lane 3, IVS4-WT; lane 4,
IVS4-yeast; lane 5, IVS4-19a; lane 6, IVS4-19g,
lane 7, IVS4-21c; lane 8, IVS4-21t; lane
9, IVS4-23a; lane 10, IVS4-23g; lane 11,
IVS4-24a; lane 12, IVS4-24t; lane 13, IVS4-25a;
lane 14, IVS4-25g; and M, molecular weight
marker.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A). This alteration would produce a stronger Watson-Crick base pair
between the branchpoint region and the AUGAUG sequence in U2 snRNA
compared with the natural mutation (Fig. 5A), which might,
at least in part, suppress the effect of the natural mutation on the
LCAT pre-mRNA splicing by increasing the ability to tolerate a
mismatch at the adjacent position. As expected, the base change just
one nucleotide upstream of the branchpoint adenosine residue did
recover some of the LCAT activity in the medium and produce a clearly
spliced RNA band, although these changes were relatively small compared
with the wild type control (Fig. 5, B and C). The
results reported here indicate that the strength of the base pairs
between the pre-mRNA and U2 snRNA may be one of the critical
factors in determining the efficiency of nuclear pre-mRNA splicing.
A or G transversion significantly enhanced the efficiency of LCAT
pre-mRNA splicing up to 2.7-fold as compared with the wild type
control, whereas the substitution of the sixth position in the
branchpoint sequence appeared to have less effect on the splicing,
which is consistent with the consensus sequence in which the nucleotide
could be either a purine or a pyrimidine. These observations suggest
that the potential base pairing between the first four nucleotides in
the branchpoint region and the U2 snRNA are probably one of the major
determinants in the splicing of nuclear pre-mRNA, and this might
also explain why the branch site in mammalian introns could be more
degenerate. The reasons for the markedly increased splicing efficiency
in the two mutants, IVS4-25a and -25g, are not clear. However, these findings raise the possibility that a DNA polymorphism involving the
branchpoint sequence might affect the efficiency of mRNA splicing and thus have significant clinical implications. A common C T
polymorphism that resides in the last position of the branchpoint consensus sequence of intron 9 of the low density lipoprotein receptor
gene has been reported (10). However, this polymorphism has been
observed to have no effect either on splicing in vitro or on
plasma lipid concentrations in the population. This is not surprising,
because the last position of the consensus sequence for the mammalian
branchpoint sequence could be either C or T.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr. Ross T. A. MacGillivray and Dr. John S. Hill for suggestions during the experiments and critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by a grant from British Columbia and the Yukon Heart and Stroke Foundation of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Atherosclerosis
Specialty Laboratory, Healthy Heart Program, St. Paul's Hospital, 1081 Burrard St., Vancouver, British Columbia, V6Z 1Y6 Canada. Tel.:
604-806-8609; Fax: 604-806-8590; E-mail: haydn@unixg.ubc.ca.
Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.M910197199
| |
ABBREVIATIONS |
|---|
The abbreviations used are: snRNP, small nuclear ribonucleoprotein particle; pre-mRNA, message RNA precursor; IVS, intervening sequence; LCAT, lecithin:cholesterol acyltransferase; FED, fish-eye disease; HEK, human embryonic kidney; PCR, polymerase chain reaction; RT, reverse transcription; bp, base pair(s); BPS, branchpoint sequence.
| |
REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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