|
Originally published In Press as doi:10.1074/jbc.M006259200 on August 31, 2000
J. Biol. Chem., Vol. 275, Issue 46, 35914-35919, November 17, 2000
Restoration of Correct Splicing of Thalassemic  -Globin
Pre-mRNA by Modified U1 snRNAs*
Linda
Gorman,
Danielle R.
Mercatante, and
Ryszard
Kole
From the Lineberger Comprehensive Cancer Center and Department of
Pharmacology, University of North Carolina, Chapel Hill, North Carolina
27599
Received for publication, July 14, 2000, and in revised form, August 30, 2000
 |
ABSTRACT |
The T G mutation at nucleotide 705 in the
second intron of the  -globin gene creates an aberrant 5' splice site
and activates a 3' cryptic splice site upstream from the mutation. As a
result, the IVS2-705 pre-mRNA is spliced via the aberrant splice
sites leading to a deficiency of -globin mRNA and protein and to
the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of
-thalassemic IVS2-705 pre-mRNA was permanently corrected by a
modified murine U7 snRNA that incorporated sequences antisense to the
splice sites activated by the mutation. To explore the possibility of
using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5'
splice site recognition sequence with nucleotides complementary to the
aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the
control of the U1 promoter and terminator sequences resulted in the
highest levels of correction (up to 70%) in transiently and stably
transfected target cells.
| |
INTRODUCTION |
The use of antisense oligonucleotides for down-regulation of gene
expression is well documented. The oligonucleotides are most frequently
targeted to mRNA where they block translation or lead to
degradation of the message by RNaseH (1). Alternatively, to inhibit
transcription, oligonucleotides are designed to form triplex structures
with the sequences in promoter regions of DNA. Both strategies result
in down-regulation of the targeted genes and may be useful in the
treatment of cancer (2-4), viral infections (5-7), and other diseases
(8-11).
A variety of antisense RNAs were also used for down-regulation of gene
expression. They were especially effective when the antisense function
of the targeting RNA was combined with ribozyme activity (12-14). In
another approach, antisense RNAs were designed as an external guide
sequence that activated endogenous RNaseP, resulting in degradation of
the targeted message (15-17). To promote stability, the antisense
constructs were usually embedded within larger stable RNA molecules
such as mRNAs, tRNAs, and small nuclear RNAs
(snRNAs)1 (18-22). This
combination not only stabilized the RNAs but also directed them either
to the cytoplasm (mRNA and tRNA) or to the nuclei (snRNAs).
Work in this laboratory showed that in addition to down-regulation of
target genes, antisense oligonucleotides could restore the activity of
genes inactivated by mutations that affect splicing of pre-mRNA and
result in genetic disorders. Restoration of gene activity was
accomplished by targeting the splice sites created or activated by
several mutations in thalassemia (23-26), cystic fibrosis (27), and in
a mouse mdx model of Duchenne muscular dystrophy (28). Thus,
modification of splicing by treatment with antisense oligonucleotides
provides a potential alternative to gene therapy protocols involving
the replacement of defective genes.
Treatment of patients with genetic diseases with antisense
oligonucleotides, which do not remove the mutation but rather repair the defective pre-mRNA, would require lifelong periodic
administrations. This drawback would be alleviated if the patients were
subjected to treatment with vectors stably expressing the appropriate
antisense RNAs. Under these conditions the effects of RNAs should
result in long term, if not permanent, restoration of gene expression.
A group of snRNAs, U snRNAs, which in cells form ribonucleoprotein
particles (snRNPS) and are involved in numerous RNA processing reactions (29-31), appear particularly attractive as carriers for antisense sequences effecting correction of splicing. They are localized in the nucleus (the site of splicing), are stable, are expressed at relatively high levels, and most importantly, interact with their natural RNA targets by base pairing via complementary, i.e. antisense nucleotides. Indeed, we have shown previously
that stable expression of U7 snRNAs, which are normally involved in the
processing of the 3'-ends of histone pre-mRNAs (32-35), but which
were modified to target splice sites in the human -globin gene, led
to permanent restoration of correct splicing of thalassemic -globin
pre-mRNA (19). In this report we have investigated whether U1 snRNA
may be used as an effective modifier of splicing.
The design of U1 snRNA as an antisense agent for modification of
splicing was based on several considerations. There are approximately 1 × 106 copies of U1 per cell as compared with 5 × 103 copies of U7 snRNA molecules (29). Because there are
30 functional U1 genes (36) and only one U7 gene (37, 38), the
expression of U1 snRNA per gene copy is still approximately 6-fold
higher than that of U7, making U1 potentially a more attractive
antisense carrier than the modified U7 snRNA. Experiments reported here show that U1 snRNA targeted to the 3'- but not the 5'-splice site could
be used for antisense repair of aberrantly spliced thalassemic human
-globin pre-mRNA.
| |
EXPERIMENTAL PROCEDURES |
Cell Lines--
The HeLa cell line carrying the thalassemic
IVS2-705 human -globin gene (39) and the cell lines stably
expressing the modified U7 snRNAs (19) were grown in minimum essential
medium modified for suspension cells (S-MEM), 5% fetal calf sera, 5%
horse sera, 50 µg/ml gentamicin, and 200 µg/ml kanamycin.
Cotransfection of the HeLa IVS2-705 cells with a plasmid carrying a
hygromycin-resistance gene and a U1.U7.324 snRNA expressing plasmid
(see below and Fig. 5A) in the presence of LipofectAMINE (8 µg/ml, Life Technologies, Inc., as recommended by the manufacturer),
was used to generate the stable cell line. Stable transfectants were
isolated after selection in media containing 250 µg/ml hygromycin.
U1 snRNA Constructs--
The wild type U1 plasmid (U1.wt) was a
kind gift from Dr. W. Marzluff (University of North Carolina). It
contains the mouse U1 snRNA gene, U1 promoter, and 3' sequences.
In the U1.309, U1.318, and U1.324 constructs, the natural 9 nucleotide
sequence (nt. 3-11) complementary to pre-mRNA 5' splice sites was
replaced with a 9-, 18-, or 24-nucleotide sequence complementary to the
cryptic 3' splice site activated by the IVS2-705 mutation. In the
U1.Beta and U1.524 construct, the same 9-nucleotide sequence (nt.
3-11) was replaced with 168 nucleotides from the second intron
(IVS2-549-717) or 24 nucleotides complementary to the 5' splice site,
respectively. In the U1.U7.324 construct, nearly the entire U1 gene
(nt. 3-161) was replaced with the U7.324 gene, whereas the
U1 promoter and 3' sequences were retained. Polymerase chain
reaction (PCR)-based mutagenesis methods were used in all the above
constructions (40, 41). Refer to (19) for U7.324 construction.
Transient Expression of Modified U1 and U7 snRNAs--
For all
experiments, HeLa IVS2-705 cells were plated 24 h before treatment
in 24-well plates at 105 cells per 2-cm2 well.
All HeLa cell lines were grown in S-MEM, 5% fetal calf sera, 5% horse
sera, 50 µg/ml gentamicin, and 200 µg/ml kanamycin. The cells were
treated for 10 h with the modified snRNA plasmids (indicated in
the figure legends) complexed with 8 µg/ml LipofectAMINE or 8 µg/ml
DMRIE-C (Life Technologies, Inc.). Unless otherwise indicated, the RNA
was isolated 24-h post-transfection. Note that the variability of
efficiency of transfection between experiments may be responsible for
the differences in the effects of U7.324 seen in Figs. 2B,
3, 4A, and 6A. This variability is much less pronounced within a single experiment, and therefore the comparisons of
the efficiency of correction within an experiment are more accurate.
RT-PCR Analysis--
Total RNA was isolated using TRI-Reagent
(MRC, Cincinnati, OH). RNA (200 ng) was analyzed by reverse
transcription-PCR (RT-PCR) using rTth DNA polymerase as recommended by
the manufacturer (PerkinElmer Life Sciences). To maintain the linear
concentration-dependent response, PCR was carried out for
18 cycles (42) with the addition of 0.2 µCi of
[ -32P]dATP to the PCR reaction mixture. Correction of
human -globin pre-mRNA was detected with forward and reverse
primers spanning positions 21-43 of exon 2 and positions 6-28 of exon
3, respectively, in -globin mRNA. RT-PCR products were separated
on 8% non-denaturing polyacrylamide gels. The gels were dried and
autoradiographed with Kodak BioMax film.
RNase Protection Assay--
The assay was performed using the
RPA II Ribonuclease Protection Assay Kit (Ambion Inc., Austin, TX) with
probes antisense to the regions containing the U1.324, U1.524, or
U1.U7.324 snRNA genes. The probes included 258 nucleotides
complementary to the U1.324 or U1.524 plasmids and 145 nucleotides from
the U1.U7.324 plasmid. Probes generated from U1.524 and U1.324 (mouse
U1a) hybridize with endogenous human U1 snRNA (U1a) with 1-base pair
mismatch resulting in a 152-base pair band after RNase digestion.
U1.524 and U1.324 snRNAs are detected as 176-base pair bands. The
digestion of the U1.U7.324 (mouse U7) probe generates a 62-nucleotide
U7-protected band; this probe does not hybridize with
endogenous human U7 snRNA.
Following transient transfection, 1 µg of total cellular RNA or 1 µg of tRNA was hybridized at 44 °C overnight with 0.5 µl of
radiolabeled probe in the hybridization buffer. Following RNase treatment, samples were separated on 5% non-denaturing polyacrylamide gels. The gels were dried and autoradiographed with Kodak BioMax film.
Protein Analysis--
Transfected cells were treated with hemin
(10 µM, Fluka, Switzerland) in serum free medium for
4 h immediately preceding the isolation of protein. Blots of
proteins separated on a 10% Tricine-SDS-polyacrylamide gel (43) were
incubated with polyclonal affinity purified chicken anti-human
hemoglobin IgG as primary antibody and rabbit anti-chicken horseradish
peroxidase-conjugated IgG as secondary antibody (Accurate Chemicals,
Westbury, NY). The blots were developed with an ECL detection system
(Amersham Pharmacia Biotech).
Image Processing--
All autoradiograms were captured by a
Dage-MTI CCD72 video camera (Michigan City, IN), and the images were
processed using NIH IMAGE 1.61. NIH IMAGE was also used for
quantification of the autoradiograms. Correctly spliced -globin
mRNA was quantified by densitometry of the autoradiograms, and the
results were expressed as the percentage of correct product relative to
the sum of the correct and aberrant products. The results were
corrected to account for the 2-fold higher
[32P]dAMP content of the PCR product derived from
aberrantly spliced mRNA than that from correctly spliced mRNA.
| |
RESULTS |
U1.324 snRNA Targeted to the Aberrant 3' Splice Site in IVS2-705
pre-mRNA--
A TG mutation at nucleotide 705 of intron 2 in
human -globin pre-mRNA (IVS2-705) generates an aberrant donor
(5') splice site and activates a cryptic acceptor (3') splice site 126 nucleotides upstream from the mutation. As a result, IVS2-705
pre-mRNA is spliced incorrectly, and the -globin mRNA
retains a fragment of the intron (Fig.
1A). This fragment prevents
proper translation of -globin leading to reduced levels of
hemoglobin and to -thalassemia (44).
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
A, correction of aberrant
splicing of IVS2-705 pre-mRNA by modified U1snRNA. Box,
exons; line, introns. The dotted lines represent
correct and aberrant splicing pathways. Modified U1 snRNA (heavy
bar) is targeted to the cryptic 3' splice site. B,
structure of U1 snRNA constructs. Wild-type U1 snRNA includes two stem
loops, the Sm sequence (open box) and a sequence antisense
to the 5' splice sites of pre-mRNA (shaded box). In
anti-705 U1 snRNAs, this sequence is replaced with antisense sequences
to the aberrant 3' or 5' splice sites in the -globin gene. The U1
promoter and 3'-end forming regions flank the gene.
|
|
Success in utilizing modified U7 constructs that had the 18 nucleotide
anti-histone pre-mRNA sequence replaced with the anti-IVS2-705 sequence, as well as contained a modified Sm sequence (SmOPT), prompted the modification of U1 snRNA as an antisense carrier. To block
aberrant splicing and induce correct splicing of the IVS2-705
-globin pre-mRNA, a series of modified U1 snRNAs, containing sequences antisense to the aberrant 3' splice site were generated (Fig.
1B). The length of the antisense sequences in the constructs ranged from 9 to 168 nucleotides.
Transfection of HeLa cells stably expressing IVS-705 -globin
pre-mRNA with U1.309 resulted in barely detectable correction of
-globin splicing (Fig. 2A,
lane 2). In the U1.309 construct, the 9 nucleotides, which
in native U1 snRNA are antisense to the 5' splice site, were replaced
with nucleotides complementary to the cryptic 3' splice site in the
-globin intron. This modification does not change the overall length
of U1 snRNA and therefore should not destabilize it. Thus, the negative
result suggests that the antisense sequence was not able to compete
with the splicing factors that assembled at the 3' splice site during
aberrant splicing of IVS2-705 pre-mRNA. This assumption was further
supported by the findings that the levels of splicing correction by
U1.318 and U1.324, containing, respectively, 18 and 24 nucleotide
antisense sequences (Fig. 2A, lanes 3 and
4) were increased in a manner commensurate with the length
of the inserted sequence. Further elongation of the antisense sequence
to 168 nucleotides (U1.Beta, Fig. 2A, lane 5),
abolished rather than enhanced correction of splicing. The antisense sequence, which extends through the putative branch points
and the aberrant 3'and 5' splice sites in the IVS2-705 -globin
intron, was expected to interfere with aberrant splicing on the basis
of its effect when placed in the modified U7 gene (U7.Beta, Fig.
2B, lane 3). Unlike in U1 snRNA, the U7.Beta and
U7.324 corrected splicing to a similar degree (Fig. 2B,
lanes 2 and 3). This suggests that the long
antisense sequence affects the structure, stability, and/or interaction of the U1 and U7 particles with the target sequences in a very different manner.
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of the length of antisense sequences
in U1.3 snRNA on correction of IVS2-705 pre-mRNA splicing.
A, RT-PCR assay of total cellular RNA from IVS2-705 HeLa
cells transfected with U1 constructs. Lane 1, mock
transfection. Lanes 2-4, U1.309, U1.318, and U1.324 snRNAs
directed against the 3' cryptic splice site with respectively, a 9-, 18-, or 24-nucleotide antisense sequence. Lane 5, U1.Beta,
with a 168-nucleotide antisense sequence spanning two putative branch
sites, the 3' cryptic splice site and the aberrant 5' splice site.
Lane 6, RNA from human blood (Hb). B,
effect of antisense length in modified U7 snRNAs on correction of
IVS2-705 pre-mRNA splicing. Lane 1, mock transfection;
lane 2, U7.324, with a 24-nucleotide sequence antisense to
the 3' cryptic splice site; lane 3, U7.Beta; lane
4, RNA from human blood (Hb). The sizes (in
nucleotides) of PCR bands representing aberrantly and correctly spliced
mRNAs are indicated on the right. The same designations
are used in RT-PCR assays shown in Figs. 3, 4, 5, and 6. Quantitation
of these results took into account the approximately 2-fold higher
[32P]dAp content of the aberrant PCR product than that of
the correct one.
|
|
We have compared in more detail the ability of the two similar U7 and
U1 snRNAs, i.e. those with 24 nucleotide antisense
sequences, U7.324 and U1.324, to correct IVS2-705 pre-mRNA
splicing. For both snRNAs, transient transfection of IVS2-705 HeLa
cells resulted in dose-dependent correction of splicing
(Fig. 3, lanes 6-11). Quantitation of the results by densitometry (see "Experimental Procedures") showed that at 1 µg of DNA plasmids per
105 cells, the level of correction was as high as 56 and
70% for the U1.324 and U7.324 constructs, respectively. The effect of U1.324 was dependent on the antisense sequence because the transfection of the cells with a plasmid expressing wild-type U1 snRNA did not
restore correct splicing of  globin IVS2-705 pre-mRNA (Fig. 3,
lanes 3-5).
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3.
Correction of aberrant splicing by U1.324 and
U7.324 snRNA. IVS2-705 cells transiently transfected with 0.25, 0.5, and 1 µg of U1 or U7 snRNA constructs (top).
Lane 1, RNA from human blood (Hb); lane
2, mock transfection; lanes 3-5, cells transfected
with wild type U1 snRNA gene (U1.WT); lanes 6-8,
and 9-11, cells transfected with U1.324 and U7.324
constructs, respectively. All other designations are as described in
the legend to Fig. 2.
|
|
U1.524 snRNA Targeted to the Aberrant 5' Splice Site--
The fact
that transfection with U1.324 and U7.324 plasmids led to efficient
correction of splicing of IVS2-705 pre-mRNA indicated that the
24-nucleotide antisense sequence did not destabilize the snRNPs, and
that it bound to the 3' splice site strongly enough to inhibit aberrant
splicing. Because modified U7.524 snRNA targeted to the aberrant
IVS2-705 5' splice site was effective in correction of splicing (Fig.
4A, lane 3), it
seemed possible that U1.524 might be similarly effective. However, as
shown in Fig. 4A, lane 5, U1.524 did not correct
-globin splicing; other constructs, U1.324, U7.524, and U7.324, were
active as expected (Fig. 4A, lanes 2-4).
View larger version (52K):
[in this window]
[in a new window]
|
Fig. 4.
RNase protection assay of U1 snRNA
expression. A, correction of aberrant splicing.
Lane 1, mock transfection; lanes 2 and
3, cells transfected with 1 µg of U7.324 and U7.524,
respectively; lanes 4 and 5, cells transfected
with 1 µg of U1.324 and U1.524, respectively. B, RNase
protection assay using RNAs from A. Lanes 1 and
4, tRNA (t) mixed with probe; lanes 2 and 5, RNA from mock transfected cells mixed with probe
(M); lane 3, cells transfected with U1.324;
lane 6, cells transfected with U1.524. Bands from
modified U1 (U1.mod, 176 nucleotides) and wild-type U1
(U1.wt, 152 nucleotides) are indicated. In lanes
1-3 and 4-6, the probes were transcribed
from U1.324 and U1.524 plasmids, respectively, as described under
"Experimental Procedures."
|
|
To confirm that the inability of the U1.524 construct to correct
splicing was not because of lack of its expression, an RNase protection
assay was performed (Fig. 4B). Densitometry analysis of the
autoradiogram indicated that both U1.324 and U1.524 were expressed in
transient transfections at comparable levels (Fig. 4B,
lanes 3 and 6). Comparison of the intensity of
the bands representing anti-thalassemic and endogenous U1 snRNAs (176 and 152 nucleotides, respectively) indicated that these RNAs were
expressed at, approximately, a 1:5 ratio. Thus, assuming that the
wild-type U1 snRNA is present at 106 copies per cell (29),
the amount of modified U1.324 and U1.524 was estimated to equal
2-3 × 105 copies per cell.
U7.324 snRNA Expressed from the U1 Promoter--
In an attempt to
further improve the effectiveness of snRNA-based vectors, we combined
the strong U1 promoter and terminator sequences with the coding
sequence of the modified U7.324 gene. In transient transfections with 1 µg of plasmid, this construct (U1.U7.324, Fig.
5A) was effective, resulting
in approximately 70% correction of -globin splicing (Fig.
5B, lane 2), whereas the U7.324 gene controlled
by the U7 promoter (Fig. 5B, lane 3) corrected
splicing to approximately 60%. This small increase in correction
efficiency was consistent with the higher level of U1.U7.324 expression
over that of the U7.324 construct (Fig. 5C, lanes
3 and 4).
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
A, structure of U1.U7.324 snRNA
construct. U7.324 snRNA includes a stem loop structure, the SmOPT
sequence, and a 24-nucleotide sequence antisense to the 3' cryptic
splice and is flanked by the U1 promoter and terminator. B,
correction of aberrant splicing by U1.U7.324 snRNA. IVS2-705 cells were
mock transfected (lane 1) or transfected with 1 µg of
U1.U7.324 (lane 2) and U7.324 (lane 3) plasmid
DNA. C, RNase protection assay. Lane 1, tRNA;
lanes 2-4 RNAs from B mixed with probe.
Bands from modified U7 (62 nucleotides) and undigested probe
(145 nucleotides) are indicated.
|
|
The effectiveness of the U7.324 and U1.U7.324 constructs was compared
in a time-course experiment in transiently transfected cells. For both
constructs, correctly spliced globin mRNA became detectable
12-h post-transfection (Fig.
6A, lanes 4 and
5), and its level increased in a time-dependent
fashion at the 24- and 48-h time points (Fig. 6A,
lanes 6-9). There was no further increase 72-h
post-transfection (Fig. 6A, lanes 10-11). At
every time point, the level of correction effected by U1.U7.324
snRNA was higher than that by U7.324 snRNA. The average of the results
from this figure and Fig. 5 indicates that the U1.U7.324 construct is
approximately 30% more effective in splicing correction than its
U7-only counterpart.
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 6.
Time course of splicing correction by
U1.U7.324 snRNA in transiently transfected cells. A,
RT-PCR. Total RNA from cells transfected with U7.324 (lanes 2, 4, 6, 8, and 10) and U1.U7.324 (lanes 3, 5, 7, 9, and 11) constructs was isolated at 6-, 12-, 24-, 48-, and 72-h post-transfection (top). Lane 1,
untreated cells; lane 12, human blood (Hb).
Percent correction at every time point is shown below the
panel. B, expression of U7 snRNA. The RNA from
transfected cells was analyzed by RNase protection assay. Lane
1, tRNA; lane 2, RNA from untreated cells; lanes
3-12, the same RNA samples as analyzed in A,
lanes 2-11, respectively. C, immunoblot. Total
protein from transfected cells was isolated 12-, 24-, 48-, and 72-h
post-transfection as indicated in lanes 2-9. Lane
10, human globin as size marker.
|
|
The RNase protection assay shows that the time course of splicing
correction is consistent with the levels of expression of the two
constructs (Fig. 6B). The U7.324 snRNA is not yet generated 6-h post-transfection but is clearly detectable at 12-72-h time points, and its levels are higher upon transcription from the U1
promoter (Fig. 6B, lanes 5, 7, 9 and
11 versus lanes 6, 8, 10, and 12,
respectively). Interestingly, translation of globin protein was
delayed relative to transcription and splicing because it was not
detectable for 24-h post-transfection (Fig. 6C, lanes 2-5); globin was translated 24 h later (Fig.
6C, lanes 6-9). However, although an increasing
time-dependent correction of splicing is confirmed by the
Western blot, a quantitative difference between the U7 and U1.U7
constructs is not evident. This may be because of low sensitivity of
the Western blot or loading error. Note that this error is eliminated
in RT-PCR assays by comparing the ratio of the spliced products.
Because the main advantage of vector-transcribed antisense RNA over
synthetic antisense oligonucleotides is the possibility of stable
intracellular expression, a U1.U7.324-expressing IVS2-705 HeLa cell
line was generated. As expected, correct splicing of IVS2-705
pre-mRNA was restored very efficiently (77%) and persisted at this
level throughout the time of culture (Fig.
7). The growth rate of the cell line was
comparable with that of the wild-type HeLa cells (data not shown),
suggesting that the modified U7 snRNA is not toxic to the cells.
View larger version (74K):
[in this window]
[in a new window]
|
Fig. 7.
Stable correction of IVS2-705 pre-mRNA
splicing in cell line 705U1.U7.324.F. Lane 1, RNA from
IVS2-705 cell line; lane 2, RNA from cell line
705U1.U7.324F; lane 3, RNA from human blood
(Hb).
|
|
| |
DISCUSSION |
Approximately 15% of all point mutations that cause genetic
diseases affect splicing of pre-mRNA (45). Moreover, a great majority of genes in higher eukaryotes are transcribed into
pre-mRNAs that undergo alternative splicing (46-49). Thus,
manipulation of splicing with antisense oligonucleotides and RNAs
offers a promising method of modification or repair of spliced
mRNAs. On the other hand, splice sites and other sequences involved
in pre-mRNA splicing appear to be unlikely targets for the
antisense approach because they interact with numerous protein splicing
factors and several snRNP particles within the spliceosome (31, 50).
Nevertheless, work from this laboratory showed that blocking of
aberrant splice sites by antisense agents leads to restoration of
correct splicing in pre-mRNAs for -globin, cystic fibrosis
transmembrane conductance receptor (CFTR) and
dystrophin genes in systems modeling thalassemia, cystic fibrosis, and
Duchenne muscular dystrophy, respectively (19, 23, 27, 28, 39).
Previous work showed that antisense oligonucleotides targeted to splice
site junctions in immediate early genes led to inhibition of
replication of herpes simplex virus (52-54). Recently it has been
found that modification of alternative splicing of bcl-x pre-mRNA
by antisense oligonucleotides sensitized the cells to apoptotic stimuli
(55).2 Clearly, splice sites
are accessible to antisense agents, most likely because the spliceosome
is formed de novo for every splicing event and its
interaction with pre-mRNA is very dynamic (31).
Our previous work showed that the accessibility of the aberrant 3'
splice site in IVS2-705 pre-mRNA to oligonucleotides was ~8 times
lower than that of the 5' splice site (26). The difference in the
accessibilities of the 3' and 5' aberrant splice sites in IVS2-705
pre-mRNA to modified U7 snRNPs, particles much larger than the
oligonucleotides (Ref. 19 and Fig. 4A, lanes 2 and 3), was not as pronounced. This difference may be
because of the fact that the modified U7 snRNP particle, carrying the
appropriate Sm proteins, may mimic other snRNPs in the spliceosome
assembling on the splice sites. However, because of inappropriate
antisense sequences and/or inappropriate structure of modified U7, the
spliceosome is dysfunctional, resulting in inhibition of aberrant splicing.
In view of the observations regarding U7 snRNA it was surprising that
the modified U1 snRNA, U1.524, targeted to the aberrant 5' splice site,
was unable to inhibit aberrant splicing. Lack of expression or
instability of U1.524 was excluded by the RNase protection assay, which
confirmed that the inactive U1.524, and the active U1.324 were produced
at similar levels. One possibility is that U1.524 could not compete
with wild type U1 for binding to the 5' splice site because it was
expressed at one-fifth of the level of endogenous U1. Because
endogenous U1 does not initially interact with the 3' splice site, it
would not compete with U1.324 for binding to its target.
U1.524 ineffectiveness in inhibiting splicing may also be because of
the fact that, despite its modification, the construct is still able to
support the proper spliceosome assembly at the 5' splice site. This
hypothesis is supported by several reports, which have shown that
modified U1 could, albeit inefficiently, restore splicing at defective
splice sites or redirect splicing to sequences adjacent to canonical
splice sites (56, 57). Of particular interest is the observation that
U1 snRNA-targeted 14 nucleotides downstream from the 5' splice site
still promoted correct splicing presumably by forming the so-called
commitment complex, which then evolved into a functional spliceosome
(56, 58). This interpretation is consistent with the fact the U7 snRNAs
targeted to the 3' and 5' splice sites were both effective in splicing
correction (Fig. 4A, lanes 2 and 3 and
Ref. 19) even though their level of expression is likely to be lower
than that of U1 snRNA (59).
Several reports showed that modified U1 snRNA could be used for
down-regulation of targeted mRNAs (14, 18, 60-62). They were
particularly effective in inhibiting HIV replication when a hammerhead
ribozyme sequence was incorporated into the U1 snRNA molecule (14, 60,
61). It is notable however, that the most effective constructs were
targeted either to the coding sequences or to regions that included the
3' splice site sequences (14). Recently, an effective anti-HIV
U1-ribozyme targeted to the 5' splice site of REV pre-mRNA was
constructed. In this construct, the antisense/ribozyme sequence was
located within the body of the U1 molecule whereas the regular 5'
antisense sequence was retained. Thus, the resulting RNA bound
simultaneously to the 5' splice site and to the adjacent sequences,
forming a double-target antisense molecule (14). Interestingly,
double-target U7 snRNAs were also found to be more effective than the
single target ones in correction of splicing of several thalassemic
-globin pre-mRNAs (44). It appears that the arrangement of
closely spaced antisense sequences may loop out and deform the targeted
pre-mRNA, preventing the formation of the spliceosome and
inhibiting aberrant splicing.
Because U6 snRNA has been used as an antisense vector by several groups
(20, 63-65) and because it differs from U7 and U1 snRNA in important
aspects of metabolism and structure (29), we built a series of U6
constructs carrying either 24 or 168 nucleotide sequences antisense to
the aberrant splice sites of IVS2-705 pre-mRNA. Surprisingly, when
transfected into HeLa IVS2-705 cells, all of the constructs failed to
correct splicing of IVS2-705 pre-mRNA (data not shown). Notably, in
previous work, the effective U6 constructs were not targeted to splice
sites. It therefore seems plausible that in our system U6 was not able
to compete with the splicing factors for splice site targets. This
could be because of the fact that U6 snRNA is incorporated into snRNP
particles only in conjunction with U4 snRNA (66-67 and 51), and in its
absence U6 snRNA may be unable to access the splice site.
The combination of the promoter and termination sequences from the U1
gene with the modified U7 snRNA sequence increased expression of U7
snRNA and concomitant correction of globin pre-mRNA splicing, providing an improvement over the U7.324 vector. Thus, the ability to
modify splicing by two types of snRNA molecules, U1 and U7, generated
from three different vectors U1, U7 and U1.U7 broadens the
possibilities of using the antisense approach as a form of gene therapy
and may offer certain advantages in different cell types or tissues.
The obvious next step for this work is to deliver the antisense U1 or
U7 constructs in vectors that are stably expressed in stem cells and/or
erythroid progenitor cells. If efficient correction of splicing by
antisense RNAs were achieved in these cells in a thalassemic patient, a
more balanced synthesis of - and -globin would have been restored
and the clinical symptoms of thalassemia ameliorated. Note that the
correction would have occurred in the -globin pre-mRNA, which
was properly transcribed from the gene that remained in its natural
chromosomal environment. In consequence, the possibility of
overexpression or inappropriate expression of -globin mRNA, an
important consideration in treatment of hemoglobinopathies, would have
been precluded. One concludes that repair of defective pre-mRNAs by
antisense agents offers an attractive alternative to gene replacement therapy.
| |
ACKNOWLEDGEMENT |
We thank Elizabeth Smith for excellent
technical assistance.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL-51940 (to R. K.).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: University of North
Carolina,Lineberger Comprehensive Cancer Center, CB no. 7295, Chapel
Hill, NC 27599-7295. Tel.: 919-966-1143, Fax: 919-966-3015; E-mail:
kole@med.unc.edu.
Published, JBC Papers in Press, August 31, 2000, DOI 10.1074/jbc.M006259200
2
D. R. Mercatante and R. Kole, manuscript in preparation.
| |
ABBREVIATIONS |
The abbreviations used are:
snRNA, small nuclear
RNA;
snRNP, small nuclear ribonucleoprotein;
nt, nucleotide;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
HIV, human immunodeficiency virus;
PCR, polymerase chain reaction;
RT-PCR, reverse transcription-PCR.
| |
REFERENCES |
| 1.
|
Crooke, S. T.
(1998)
Antisense Nucleic Acid Drug Dev.
8,
133-134
|
| 2.
|
Rubenstein, M.,
Mirochnik, Y.,
Chou, P.,
and Guinan, P.
(1998)
Methods Find Exp. Clin. Pharmacol.
20,
825-831
|
| 3.
|
Roh, H.,
Pippin, J.,
Boswell, C.,
and Drebin, J. A.
(1998)
J. Surg. Res.
77,
85-90
|
| 4.
|
Tzai, T. S.,
Lin, C. I.,
Shiau, A. L.,
and Wu, C. L.
(1998)
Anticancer Res.
18,
1585-1589
|
| 5.
|
Offensperger, W. B.,
Offensperger, S.,
and Blum, H. E.
(1998)
Mol. Biotechnol.
9,
161-170
|
| 6.
|
Kenney, J. L.,
Guinness, M. E.,
Curiel, T.,
and Lacy, J.
(1998)
Blood
92,
1721-1727
|
| 7.
|
Zhang, H.,
Hanecak, R.,
Brown-Driver, V.,
Azad, R.,
Conklin, B.,
Fox, M. C.,
and Anderson, K. P.
(1999)
Antimicrob. Agents Chemother.
43,
347-353
|
| 8.
|
Hashiramoto, A.,
Sano, H.,
Maekawa, T.,
Kawahito, Y.,
Kimura, S.,
Kusaka, Y.,
Wilder, R. L.,
Kato, H.,
Kondo, M.,
and Nakajima, H.
(1999)
Arthritis Rheum.
42,
954-962
|
| 9.
|
Yacyshyn, B. R.,
Bowen-Yacyshyn, M. B.,
Jewell, L.,
Tami, J. A.,
Bennett, C. F.,
Kisner, D. L.,
and Shanahan, W. R., Jr.
(1998)
Gastroenterology
114,
1133-1142
|
| 10.
|
Li, B.,
Hughes, J. A.,
and Phillips, M. I.
(1997)
Neurochem. Int.
31,
393-403
|
| 11.
|
Bennett, C. F.,
Kornbrust, D.,
Henry, S.,
Stecker, K.,
Howard, R.,
Cooper, S.,
Dutson, S.,
Hall, W.,
and Jacoby, H. I.
(1997)
J. Pharmacol. Exp. Ther.
280,
988-1000
|
| 12.
|
Cantor, G. H.,
Stone, D. M.,
McElwain, T. F.,
and Palmer, G. H.
(1996)
Antisense Nucleic Acid Drug Dev.
6,
301-304
|
| 13.
|
Perez-Ruiz, M.,
Sievers, D.,
Garcia-Lopez, P. A.,
and Berzal-Herranz, A.
(1999)
Antisense Nucleic Acid Drug Dev.
9,
33-42
|
| 14.
|
Michienzi, A.,
Conti, L.,
Varano, B.,
Prislei, S.,
Gessani, S.,
and Bozzoni, I.
(1998)
Hum. Gene Ther.
9,
621-628
|
| 15.
|
Werner, M.,
Rosa, E.,
Al Emran, O.,
Goldberg, A. R.,
and George, S. T.
(1999)
Antisense Nucleic Acid Drug Dev.
9,
81-88
|
| 16.
|
Ma, M. Y.,
Jacob-Samuel, B.,
Dignam, J. C.,
Pace, U.,
Goldberg, A. R.,
and George, S. T.
(1998)
Antisense Nucleic Acid Drug Dev.
8,
415-426
|
| 17.
|
Werner, M.,
Rosa, E.,
Nordstrom, J. L.,
Goldberg, A. R.,
and George, S. T.
(1998)
RNA
4,
847-855
|
| 18.
|
Bertrand, E.,
Castanotto, D.,
Zhou, C.,
Carbonnelle, C.,
Lee, N. S.,
Good, P.,
Chatterjee, S.,
Grange, T.,
Pictet, R.,
Kohn, D.,
Engelke, D.,
and Rossi, J. J.
(1997)
RNA
3,
75-88
|
| 19.
|
Gorman, L.,
Suter, D.,
Emerick, V.,
Schumperli, D.,
and Kole, R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4929-4934
|
| 20.
|
Good, P. D.,
Krikos, A. J.,
Li, S. X.,
Bertrand, E.,
Lee, N. S.,
Giver, L.,
Ellington, A.,
Zaia, J. A.,
Rossi, J. J.,
and Engelke, D. R.
(1997)
Gene Ther.
4,
45-54
|
| 21.
|
Biasolo, M. A.,
Radaelli, A.,
Del Pup, L.,
Franchin, E.,
De Giuli-Morghen, C.,
and Palu, G.
(1996)
J. Virol.
70,
2154-2161
|
| 22.
|
Mirochnitchenko, O.,
and Inouye, M.
(1993)
Antisense Res. Dev.
3,
171-179
|
| 23.
|
Sierakowska, H.,
Sambade, M. J.,
Agrawal, S.,
and Kole, R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12840-12844
|
| 24.
|
Dominski, Z.,
and Kole, R.
(1993)
Proc. Natl. Acad. Sci.
90,
8673-1677
|
| 25.
|
Schmajuk, G.,
Sierakowska, H.,
and Kole, R.
(1999)
J. Biol. Chem.
274,
21783-21789
|
| 26.
|
Sierakowska, H.,
Sambade, M. J.,
Schumperli, D.,
and Kole, R.
(1999)
RNA
5,
369-377
|
| 27.
|
Friedman, K. J.,
Kole, J.,
Cohn, J. A.,
Knowles, M. R.,
Silverman, L. M.,
and Kole, R.
(1999)
J. Biol. Chem.
274,
36193-36199
|
| 28.
|
Wilton, S. D.,
Lloyd, F.,
Carville, K.,
Fletcher, S.,
Honeyman, K.,
Agrawal, S.,
and Kole, R.
(1999)
Neuromusc. Dis.
9,
330-338
|
| 29.
|
Baserga, S. J.,
and Steitz, J. A.
(1993)
in
The RNA World
(Gesteland, R. F.
, and Atkins, J. F., eds)
, pp. 359-381, Cold Spring Harbor Press, Plainview, NY
|
| 30.
|
Guthrie, C.,
and Patterson, B.
(1988)
Annu. Rev. Genet.
22,
387-419
|
| 31.
|
Moore, M. J.,
Query, C. C.,
and Sharp, P. A.
(1993)
in
The RNA World
(Gesteland, R. F.
, and Atkins, J. F., eds)
, pp. 303-357, Cold Spring Harbor Laboratory Press, Plainview, NY
|
| 32.
|
Spycher, C.,
Streit, A.,
Stefanovic, B.,
Albrecht, D.,
Koning, T. H.,
and Schumperli, D.
(1994)
Nucleic Acids Res.
22,
4023-4030
|
| 33.
|
Bond, U. M.,
Yario, T. A.,
and Steitz, J. A.
(1991)
Genes Dev.
5,
1709-1722
|
| 34.
|
Strub, K.,
Galli, G.,
Busslinger, M.,
and Birnstiel, M. L.
(1984)
EMBO J.
3,
2801-2807
|
| 35.
|
Schaufele, F.,
Gilmartin, G. M.,
Bannwarth, W.,
and Birnstiel, M. L.
(1986)
Nature
323,
777-781
|
| 36.
|
Dahlberg, J. E.,
and Lund, E.
(1988)
in
Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles
(Birnstiel, M. L., ed)
, pp. 38-70, Springer, Berlin
|
| 37.
|
Gruber, A.,
Soldati, D.,
Burri, M.,
and Schumperli, D.
(1991)
Biochim. Biophys. Acta
1088,
151-154
|
| 38.
|
Grimm, C.,
Stefanovic, B.,
and Schumperli, D.
(1993)
EMBO J.
12,
1229-1238
|
| 39.
|
Sierakowska, H.,
Montague, M.,
Agrawal, S.,
and Kole, R.
(1997)
Nucleosides Nucleotides
16,
1173-1182
|
| 40.
|
Jones, D. H.,
and Howard, B. H.
(1991)
BioTechniques
10,
62-66
|
| 41.
|
Jones, D. H.,
and Winistorfer, S. C.
(1992)
BioTechniques
12,
528-530, 532, 534-535
|
| 42.
|
Chen, I. T.,
and Chasin, L. A.
(1993)
Mol. Cell. Biol.
13,
289-300
|
| 43.
|
Schagger, H.,
and von Jagov, G.
(1987)
Anal. Biochem.
166,
368-379
|
| 44.
|
Schwartz, E.,
and Benz, E. J.
(1991)
in
Hematology: Basic Principles and Practices
(Hoffman, R.
, Benz, E. J.
, Shattil, S. J.
, Furie, B.
, and Cohen, H. J., eds)
, pp. 586-610, Churchill Livingstone, New York
|
| 45.
|
Krawczak, M.,
Reiss, J.,
and Cooper, D. N.
(1992)
Hum. Genet.
90,
41-54
|
| 46.
|
Jiang, Z. H.,
and Wu, J. Y.
(1999)
Proc. Soc. Exp. Biol. Med.
220,
64-72
|
| 47.
|
Edwalds-Gilbert, G.,
Veraldi, K. L.,
and Milcarek, C.
(1997)
Nucleic Acids Res.
25,
2547-2561
|
| 48.
|
Benz, E. J., Jr.,
and Huang, S. C.
(1996)
Trans. Am. Clin. Climatol. Assoc.
108,
78-95
|
| 49.
|
Schiaffino, S.,
and Reggiani, C.
(1996)
Physiol. Rev.
76,
371-423
|
| 50.
|
Cheng, S. C.,
and Abelson, J.
(1987)
Genes Dev.
1,
1014-1027
|
| 51.
|
Bringmann, P.,
Appel, B.,
Rinke, J.,
Reuter, R.,
Theissen, H.,
and Luhrmann, R.
(1984)
EMBO J.
3,
1357-1363
|
| 52.
|
Kean, J. M.,
Kipp, S. A.,
Miller, P. S.,
Kulka, M.,
and Aurelian, L.
(1995)
Biochemistry
34,
14617-14620
|
| 53.
|
Smith, C. C.,
Aurelian, L.,
Reddy, M. P.,
Miller, P. S.,
and Ts'o, P. O.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
2787-2791
|
| 54.
|
Jacob, A.,
Duval-Valentin, G.,
Ingrand, D.,
Thuong, N. T.,
and Helene, C.
(1993)
Eur. J. Biochem.
216,
19-24
|
| 55.
|
Taylor, J. K.,
Zhang, Q. Q.,
Wyatt, J. R.,
and Dean, N. M.
(1999)
Nat. Biotechnol.
17,
1097-1100
|
| 56.
|
Cohen, J. B.,
Broz, S. D.,
and Levinson, A. D.
(1993)
Mol. Cell. Biol.
13,
2666-2676
|
| 57.
|
Zhuang, Y.,
and Weiner, A. M.
(1986)
Cell
46,
827-835
|
| 58.
|
Cohen, J. B.,
Snow, J. E.,
Spencer, S. D.,
and Levinson, A. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10470-11044
|
| 59.
|
Suter, D.,
Tomasini, R.,
Reber, U.,
Gorman, L.,
Kole, R.,
and Schümperli, D.
(1999)
Hum. Mol. Genet.
8,
2415-2423
|
| 60.
|
Liu, D.,
Donegan, J.,
Nuovo, G.,
Mitra, D.,
and Laurence, J.
(1997)
J. Virol.
71,
4079-4085
|
| 61.
|
Michienzi, A.,
Prislei, S.,
and Bozzoni, I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7219-7224
|
| 62.
|
Montgomery, R. A.,
and Dietz, H. C.
(1997)
Hum. Mol. Genet.
6,
519-525
|
| 63.
|
Noonberg, S. B.,
Scott, G. K.,
Garovoy, M. R.,
Benz, C. C.,
and Hunt, C. A.
(1994)
Nucleic Acids Res.
22,
2830-2836
|
| 64.
|
He, Y.,
Zeng, Q.,
Drenning, S. D.,
Melhem, M. F.,
Tweardy, D. J.,
Huang, L.,
and Grandis, J. R.
(1998)
J. Natl. Cancer Inst. (Bethesda)
90,
1080-1087
|
| 65.
|
He, Y.,
and Huang, L.
(1997)
Cancer Res.
57,
3993-3999
|
| 66.
|
Hashimoto, C.,
and Steitz, J. A.
(1984)
Nucleic Acids Res.
12,
3283-3293
|
| 67.
|
Rinke, J.,
Appel, B.,
Digweed, M.,
and Luhrmann, R.
(1985)
J. Mol. Biol.
185,
721-731
|
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S.-N. Grellscheid and C. W. J. Smith
An Apparent Pseudo-Exon Acts both as an Alternative Exon That Leads to Nonsense-Mediated Decay and as a Zero-Length Exon.
Mol. Cell. Biol.,
March 1, 2006;
26(6):
2237 - 2246.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Vacek, H. Ma, F. Gemignani, G. Lacerra, T. Kafri, and R. Kole
High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA
Blood,
January 1, 2003;
101(1):
104 - 111.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. G. De Angelis, O. Sthandier, B. Berarducci, S. Toso, G. Galluzzi, E. Ricci, G. Cossu, and I. Bozzoni
Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells
PNAS,
July 9, 2002;
99(14):
9456 - 9461.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. A. Patel, C. E. Chalfant, J. E. Watson, J. R. Wyatt, N. M. Dean, D. C. Eichler, and D. R. Cooper
Insulin Regulates Alternative Splicing of Protein Kinase C beta II through a Phosphatidylinositol 3-Kinase-dependent Pathway Involving the Nuclear Serine/Arginine-rich Splicing Factor, SRp40, in Skeletal Muscle Cells
J. Biol. Chem.,
June 15, 2001;
276(25):
22648 - 22654.
[Abstract]
[Full Text]
[PDF]
|
|
|
Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
|
Advertisement
Advertisement
|
TOP
ABSTRACT
INTRODUCTION