From the Division of Structural Cellular Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Received for publication, September 10, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Spinal muscular atrophy is caused by the
homozygous loss of survival motor neuron 1 (SMN1). SMN2, a nearly
identical copy gene, differs from SMN1 only by a single nonpolymorphic
C to T transition in exon 7, which leads to alteration of exon 7 splicing; SMN2 leads to exon 7 skipping and expression of a
nonfunctional gene product and fails to compensate for the loss of
SMN1. The exclusion of SMN exon 7 is critical for the onset of this
disease. Regulation of SMN exon 7 splicing was determined by analyzing the roles of the cis-acting element in intron 7 (element
2), which we previously identified as a splicing enhancer element of
SMN exon 7 containing the C to T transition. The minimum sequence essential for activation of the splicing was determined to be 24 nucleotides, and RNA structural analyses showed a stem-loop structure.
Deletion of this element or disruption of the stem-loop structure
resulted in a decrease in exon 7 inclusion. A gel shift assay using
element 2 revealed formation of RNA-protein complexes, suggesting that
the binding of the trans-acting proteins to element 2 plays
a crucial role in the splicing of SMN exon 7 containing the C to T transition.
Spinal muscular atrophy
(SMA)1 is a common autosomal
recessive disorder characterized by the loss of motor neurons in the
spinal cord, which presents as proximal, symmetrical limb, and trunk muscle weakness that ultimately leads to death (1). The survival of the
motor neuron (SMN) gene has been identified as the disease-causing gene
of SMA and is present on chromosome 5 at 5q13 (2, 3). Humans contain
two nearly identical copies of the SMN gene, SMN1 and SMN2. These genes
encode an identical protein, a 294-amino acid RNA-binding protein. Only
homozygous deletions or mutations of SMN1 result in the SMA phenotype
(4-15).
SMN1 mRNA expresses a full-length transcript, whereas SMN2 produces
low levels of the full-length transcript and high levels of an isoform
lacking exon 7 (SMN Previously, we tried to determine the critical cis-acting
elements on the SMN pre-mRNA responsible for the aberrant splicing of the SMN exon 7 containing the C to T transition (24). We identified
two cis-acting elements (elements 1 and 2) responsible for
the regulation of SMN exon 7 splicing. The mutation in element 1, which
is composed of 45 bp in intron 6, or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. The ~33-kDa protein was demonstrated to associate with
element 1 in the SMN exon 7 containing the C to T transition, suggesting that the binding of the ~33-kDa protein to element 1 plays
crucial roles in the skipping of the SMN exon 7 containing the C to T
transition. Element 2 was composed of 66 bp in intron 7 and plays roles
in the inclusion of exon 7, but the detailed mechanisms responsible for
the splicing regulation of this element were unclear. In this report,
we investigate the functions of element 2, which we identified
previously as being involved in the regulation of SMN exon 7 splicing.
Cell Cultures--
COS-7 and SK-N-SH cells were used for
in vivo splicing assays. COS-7 cells were grown in 10%
fetal bovine serum/Dulbecco's modified Eagle's medium, and
SK-N-SH cells were cultured in In Vivo Splicing--
Constructs of SMN1 and SMN2 mini-genes
containing exons 6-8 in a pCI mammalian expression vector were gifts
from Drs. Elliot Androphy (Tufts University) and Christian Lorson
(Arizona State University) (21). Mini-genes containing SMN 1 exons 6-8
and the C to T transition in exon 7 cloned into the pCI vector were mutated in element 1 by site-directed mutagenesis. The constructs (1.0 µg) were transfected into cells using LipofectAMINE reagent or
LipofectAMINE ACE reagent (Invitrogen) according to the manufacturer's protocol. Transfected cells were lysed in buffer RLT (Qiagen), and the
total cellular RNA was purified using the RNeasy mini kit (Qiagen).
First strand cDNA was synthesized in a 20-µl reaction volume
using a random primer (TaKaRa) and Moloney murine leukemia virus
reverse transcriptase (Invitrogen). PCR amplification analysis of the
plasmid-derived cDNAs was performed using the primer set, pCI
forward (5'-GCT AAC GCA GTC AGT GCT TC-3') and pCI reverse (5'-GTA TCT
TAT CAT GTC TGC TCG-3'). PCR was performed in a total volume of 50 µl
that contained 1.2 µg of first strand cDNA, 0.4 µM
of each primer, 0.2 µM dNTPs supplemented with trace
amounts of [ Exon Trapping Systems--
Various deletion mutants of SMN1
containing intron 6, exon 7, and intron 7 were generated by PCR using
the following primer sets: 600Fwd (5'-AAG CTT GGC ATG AGC CAC TGC AAG
AAA AC-3') and OR (5'-GGA TCC GAG AAT TCT AGT AGG GAT GTA G-3') for
I7DM1, 600Fwd and OR-IR (5'-AAG CTT GTT TTA CAT TAA CCT TTC AAC T-3')
for I7DM2, 600Fwd and DR-11R (5'-GGA TCC GAA CTT TTT AAA TGT TCA AAA
AC-3') for I7DM3, and 600Fwd and IR (5'-GGA TCC CAC AAA CCA TAA AGT TTT AC-3') for I7DM4 (see Fig. 1B). These PCR products were
digested with NotI and BamHI and inserted into
the exon trapping vector pSPL3 (Invitrogen) (25). The mutation of the
stem-loop structure was generated by PCR using the template as an
I7DM1-T exon trapping vector with mutagenized oligonucleotides (5'-CCC
CTG AAC ATT TAA AAA GTT CAG ATG-3' and 5'-CAT TTG TTT TCC ACA AAC CAT
AAA GTT TTA-3'). The constructs of tandemly repeated element 2 were
generated by PCR using the I7DM-1T vector with mutagenized
oligonucleotides (5'-GTG GAA AAC AAA TGT TTT TGA ACA GTG GAA AAC AAA
TGT TTT TGA ACA GTG GAA AAC AAA TGT TTT TGA ACA TTT AAA AAG TTC-3' and
5'-AAA CCA TAA AGT TTT ACA AAA GTA AGA TTC AC-3'). These PCR products were ligated by a BKL kit (Takara, Japan). All of the constructs were
sequenced before use in the experiments. Total cellular RNA was
isolated from transfected COS-7 cells 24 h after the transfection using the RNeasy mini kit (Qiagen). Aliquots of 3.0 µg of RNA were
reverse-transcribed using the SA2 primer (5'-ATC TCA GTG GTA TTT GTG
AGC-3') and Moloney murine leukemia virus reverse transcriptase
(Invitrogen). Splicing products were detected by PCR using a pSPL3
vector-specific primer set, SD6 (5'-TCT GAG TCA CCT GGA CAA CC-3') and
SA2. PCR was performed in a total volume of 40 µl. Amplification was
conducted as follows: 1 min at 94 °C, 1 min at 60 °C, and 1 min
at 72 °C for 30 cycles followed by 72 °C for 5 min. The PCR
products were electrophoresed in a 5% acrylamide gel.
Gel Mobility Shift Assay--
For the collection of nuclear
extracts, SK-N-SH cells were homogenized in 50 volumes of 10 mM HEPES-NaOH (pH 7.9) containing 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 5 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride at
4 °C. Buffers and any other solutions used in this study were
sterilized before each use by filtration through a Steritop (Millipore
Corp.) with a pore size of 220 nm. Following the addition of 10%
Nonidet P-40 to a final concentration of 0.6%, the homogenates were
centrifuged at 15,000 rpm for 5 min. The pellets were resuspended in 10 volumes of 20 mM Tris-HCl (pH 7.5) containing 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride, followed by centrifugation at 15,000 rpm
for 5 min. The supernatants thus obtained were stored at
For gel mobility shift assays, the reactions were carried out in a
final volume of 20 µl (binding buffer: 20 mM HEPES, pH 7.9, 72 mM KCl, 1.5 mM MgCl2, 0.78 mM magnesium acetate, 0.52 mM dithiothreitol,
3.8% glycerol, 0.75 mM ATP, and 1 mM GTP) by mixing various concentrations of nuclear extracts with
radioisotope-labeled RNA oligonucleotide (present at a final
concentration of 6 nM). The oligonucleotide sequences were
5'-GUG GAA AAC AAA UGU UUU UGA ACA-3' (oligo-element 2), and 5'-GUG GAA
AAC AAA UGC CCC UGA ACA-3' (oligo-mutant element 2). The samples were
incubated at room temperature for 25 min and were then loaded on a 4%
native acrylamide gel in a loading buffer (37.5 mM
Tris-HCl, 315 mM glycine, and 1.5 mM EDTA),
which was run at a constant 100 V for 2 h at 4 °C. The gel was
then dried and exposed to autoradiographic film.
Introduction of Antisense Oligonucleotides--
All of the
antisense oligonucleotides containing 2'-O-methyl and
phosphorothioate backbone modifications were synthesized by Japan Bio
Service Inc. The sequences of the antisense oligonucleotides were
5'-GAU AGC UAU AUA UAG AU-3' (As-con), 5'-GAU AGC UAU AUA UAG AU-3'
(As-pyr), and 5'-UGU UCA AAA ACA UUU GUU UUC-3' (As-element2). Antisense oligonucleotides and exon trapping vectors were
co-transfected into COS-7 by lipofection. The final concentrations of
antisense oligonucleotides in the culture medium ranged from 5 to 25 nM.
RNA Structure Analyses--
The RNA secondary structures and
free energies were predicted using the GCG version of the MFOLD program
(26). The most stable structures of element 2 are presented in Fig.
1D.
Identification of the Unique Stem-Loop Structure in the SMN Intron
7, Which Enhances the Exon 7 Splicing--
It is well known that SMN1
mRNA expresses a full-length transcript, whereas SMN2 produces low
levels of the full-length transcript and high levels of an isoform
lacking exon 7 (SMN
For the deletion mutants in the flanking intron 7, the deletion mutant
I7DM4-T, which contained a deletion of 66 bp at the 3'-flanking regions
from I7DM1-T, increased the exclusion of SMN exon 7 (23% inclusion),
whereas the splicing pattern of deletion of the mutant I7DM3-T, which
contained a deletion of 52 bp from I7DM1-T, was similar to that of
I7DM1-T (Fig. 1C). Therefore, the 14-bp region from +59 to
+72 of the flanking intron 7 may be a critical element for the
inclusion of SMN exon 7 containing the C to T transition. The position
of this element within intron 7 is presented in Fig. 1D.
Analysis of the higher order structure by the MFOLD program (26) showed
that element 2, composed of 24 nucleotides, possessed a unique
stem-loop structure (Fig. 1D). The sequence showed a
complete matching between SMN1 and SMN2, suggesting that element
2 may be important for the regulation of the splicing of the SMN
1 exon 7 containing the C to T transition and wild type SMN2.
Functional Analyses of the Intronic Splicing Enhancer
Element--
To determine whether the stem-loop structure in element 2 is important for regulation of the splicing of the SMN exon 7 containing C to T transition, we variously mutated the stem-loop
structure in element 2 of I7DM1-T (Fig.
2A). Mutation or deletion of
the loop region did not affect the splicing of exon 7 (constructs A and B in Fig. 2A), suggesting that the
loop structure of element 2 is not important for the splicing
regulation (Fig. 2B). In contrast, disruption of the stem
structure caused a significant decrease of exon 7 inclusion compared
with the original structure of I7DM1-T (constructs C and
D in Fig. 2A), and the effects on the decrease in
exon 7 inclusion were similar to those of the deletion mutant I7DM4-T
(Fig. 2B), indicating that the stem structure is important for the activities of element 2. Furthermore, a C-G base pair in the
stem structure is also essential for splicing activity of element 2 because one point mutation of G to C in the stem caused a dramatic
decrease in exon 7 inclusion (construct E in Fig.
2A). When A-U base pairs in the stem were substituted to G-C
base pairs, inclusion of exon 7 was slightly decreased (construct F in Fig. 2A). This finding suggests that the higher
ordered stem structure in element 2 is important, but the sequences of
the A-U base pairs are not very important.
To test whether element 2 can activate the splicing of the SMN exon 7 containing the C to T transition, we constructed exon trap vectors
containing three or four repeats of element 2 that were tandemly
inserted into the I7DM1-T vector, as shown in Fig. 3A. The insertion of multiple
element 2 in the intron 7 of SMN increased the inclusion of SMN exon 7 in an inserted number-dependent manner (Fig.
3B). Altogether, it is demonstrated that element 2 is an
intronic splicing enhancer of SMN exon 7 containing the C to T
transition.
Data base analysis showed there were several genes containing similar
sequences to element 2 in its intron. Some genes that reveal complete
matching to the stem-loop structure within element 2 are listed in the
Table I. As shown in the table, these
elements listed are positioned relatively close to the 5' splice sites of each intron similar to element 2 of the SMN gene. Furthermore, we
found expressed sequence tag clones of MRPS35 (mitochondrial ribosomal
protein S35) and RDGBB (retinal degeneration B A trans-Acting Factor Specifically Binds to Element 2--
Because
a disruption of element 2 revealed dynamic changes in the splicing
patterns of SMN exon 7, it is possible that trans-acting factors specifically bind to element 2 and regulate its splicing through direct binding. Therefore, we performed gel shift assay using
32P-labeled oligo-element 2 and nuclear extracts of
neuroblastoma SK-N-SH cells. Efficient binding was observed, and the
binding activity was increased corresponding to the amounts of nuclear extracts (Fig. 4A). To confirm
that the binding was specific, competition studies with unlabeled
oligo-element 2 were performed. Preincubation of nuclear extracts with
25-fold molar excesses of unlabeled oligo-RNAs resulted in a nearly
complete elimination of the activity, whereas preincubation of the
extracts with equivalent amounts of unlabeled mutated oligonucleotides
of element 2 (oligo-mutant element 2), which were mutated by
substitution of T to C (construct C), as shown in Fig. 2A,
did not inhibit the binding of the RNA-nuclear proteins (Fig.
4B). These data demonstrate that a trans-acting factor specifically binds to element 2 in the intron 7 of SMN pre-mRNA and that binding to the cis-acting element 2 may enhance the splicing of the SMN exon 7 containing the C to T
transition.
We performed UV cross-linking experiments using SMN pre-mRNA, but
we could not detect any difference in proteins binding to SMN
pre-mRNA between wild type and mutant SMN pre-mRNA, which deleted element 2. Because many bands were detected in both binding assays and bands of interest may be hidden in the many bands, we could
not obtain further information concerning putative
trans-acting factor(s) bound to this enhancer element, such
as the molecular weight(s) of the factor(s).
Effects of Treatment with Antisense Oligonucleotides--
Having
demonstrated that element 2 in intron 7 of the SMN gene plays a crucial
role in the regulation of exon 7 splicing and that
trans-acting proteins could directly bind to this element, it was of interest to examine whether the treatment of cells
transfected with I7DM1-T exon trapping vectors with antisense
oligonucleotides was sufficient to lead to a decrease in the inclusion
of SMN exon 7 containing the C to T transition. We thus synthesized
antisense oligonucleotides directed toward element 2 (Fig.
5A, As-element2). We further synthesized antisense oligonucleotides that were directed toward the polypyrimidine tract on intron 6 of SMN1 (Fig.
5A, As-pyr) and another sequence in the intron 6 (Fig. 5A, As-con) as controls. These antisense
oligonucleotides were co-transfected with an exon trapping vector
cloned I7DM1-T into COS-7 cells, and total RNA was extracted 24 h
after the transfection. RT-PCR was performed to examine the effects of
these antisense oligonucleotides on the in vivo splicing of
SMN exon 7. Treatment with As-element2 led to a decrease in the
inclusion of SMN exon 7, which was dependent on the amount of antisense
oligonucleotides (Fig. 5B). Treatment with the As-con showed
no change in the splicing of SMN exon 7 (Fig. 5C). In
contrast, treatment with As-pyr showed a slight decrease in the
inclusion (data not shown). The polypyrimidine tract is essential for
the pre-mRNA splicing. Therefore, the As-pyr was expected to work
effectively as the antisense oligonucleotides that blocked its
cis-acting element. Similarly, the As-element 2 caused an
inhibition of the splicing of SMN exon 7, suggesting that the results
supported the above findings that element 2 is an important
cis-acting for the splicing of SMN exon 7 containing the C
to T transition.
SMN1 mRNA expresses a full-length transcript, whereas SMN2
produces a low level of full-length transcript predominantly as an
isoform lacking exon 7 (2, 16, 17). The critical difference between
SMN1 and SMN2 is a silent nucleotide transition in SMN exon 7. That is,
SMN1 contains a C located six nucleotides inside exon 7, whereas SMN2
contains a T at this position. This transition leads to an alteration
in the recognition of exon 7 by components of the splicing
machinery (20, 21). A previous report demonstrated the presence of an
ESE within exon 7 and that human Tra2- Deletion analysis of SMN1 pre-mRNA sequences showed that the
regions from +59 to +72 of the flanking intron 7 are significant elements for the inclusion of the SMN1 exon 7 containing the C to T
transition. However, deletion of these elements from wild type SMN1
pre-mRNA did not affect the splicing of SMN exon 7. Therefore,
although element 2 does not play a role in exon 7 splicing of the wild
type SMN, it is necessary for enhancing the exon 7 splicing of the
mutant type SMN. It is not known why element 2 affects only the
splicing of SMN exon7 containing the mutation. A possible mechanism is
that the alteration of the splicing patterns of the SMN exon 7 may
result from the binding of a specific regulatory factor, as shown in
Fig. 4A, to element 2 caused by changes in the higher order
structure of the pre-mRNA containing the C to T transition in exon
7, and the binding of the regulatory factor may activate the
recognition or usage of 3' or 5' splice sites by splicing machineries
such as small nuclear ribonucleoproteins or
serine-arginine-related proteins (27, 28).
It has previously been reported that the binding of the polypyrimidine
tract-binding protein to the enhancer pyrimidine tract is functional in
that the exon inclusion increases when in vivo levels of
polypyrimidine tract-binding protein increase (29). Members of the CELF
family of RNA-binding proteins, including CUG-BP, have also been known
to bind to a conserved intronic splicing element (MSE containing CUG
motif) and positively regulate alternative splicing (30, 31). Element
2, identified in the present study, is considered to act for an
intronic splicing enhancer because of the decrease in SMN splicing by
deletion or mutation of this element. However, element 2 does not
contain the consensus sequences to which the polypyrimidine
tract-binding protein or the CELF family bind, although it contains the
unique stem-loop structure. Therefore, element 2 could not be regulated
by these proteins. However, a novel molecule may associate with the
element to enhance the splicing of the SMN exon 7 containing the C to T
transition. Indeed, we found specific interactions of element 2 oligonucleotide and nuclear extracts. The element that we identified in
the present study has not been shown to be critical for splicing or for
the specific binding site of splicing factors. Therefore,
the mechanisms responsible for the regulated splicing of SMN exon 7 by
the cis-acting element remain unknown. Identification and
characterization of the trans-acting factors that bind to
the element are needed to elucidate the mechanisms. Data base analysis
showed a complete matching of the stem-loop structure of the element 2 nucleotide sequence to the intron sequences of several genes. Although
a detailed analysis of the splicing of these genes is needed, it raises
the possibility that these genes may also be regulated by an
alternative splicing by this cis-acting element and its trans-acting proteins.
In summary, we found a stem-loop structure within element 2 in the
intron 7 of SMN that enhances the splicing of the SMN exon 7 containing
the C to T transition. In addition to element 1, which has been
demonstrated to be a negative regulator of the splicing, it is
important for an understanding of the mechanisms of splicing of SMN
exon 7 containing the C to T transition to identify the
trans-acting RNA-binding proteins that specifically interact
with these elements. Experimental manipulation to modify the function
of the cis-acting elements or the trans-acting
factors might allow the development of therapeutic strategies for
SMA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7) (2, 16, 17). The SMN
7 protein is
presumed to be less stable (18) and has a reduced ability to
oligomerize, explaining why SMN2 cannot prevent SMA (2, 19, 20). The
critical difference between SMN1 and SMN2 is a silent nucleotide
transition in SMN exon 7. SMN1 contains a C located six nucleotides
inside exon 7, whereas SMN2 contains a T at this position. This
transition is believed to inhibit one of the splicing regulatory
elements, called exonic splicing enhancers (ESE), within exon 7 (21). A
previous report demonstrated the presence of an ESE within exon 7 and
that human Tra2-
1, a member of the serine-arginine-related proteins
of splicing factors, binds to the elements and stimulates an ESE (22).
Recently, it was discovered that a single nucleotide change occurs
within a heptamer motif of the ESE, which in SMN1 is recognized
directly by SF2/ASF (23). The abrogation of the
SF2/ASF-dependent ESE is considered to be the basis for the
inefficient inclusion of exon 7 in SMN2. However, it is unclear whether
Tra2-
1 and SF2/ASF functionally cooperate to promote the inclusion
of the exon and whether other factors are involved in the regulation of
the splicing of SMN exon 7.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum essential medium with 10%
fetal bovine serum. Prior to transfection, the cells were plated at a
density of 60-80% confluency on 3.5-cm dishes.
-32P]dCTP, 5 units of rTaq DNA polymerase,
and 10× PCR buffer (TaKaRa). The amplification conditions
were as follows: an initial denaturation step (94 °C for 2 min), 30 cycles (94 °C for 30 s, 56 °C for 1.5 min, and 72 °C for
1 min), and a final extension step (72 °C for 10 min). The reaction
products were resolved by electrophoresis through a 5% acrylamide gel.
PCR products were cloned into the pGEM-T vector (Promega) and
sequenced. Quantification of the density of each band was carried out
using a densitography program (ATTO). The ratios of inclusion of exon 7 were quantified and expressed as percentages of inclusion relative to
the total intensities.
80 °C as
nuclear extracts for gel mobility shift assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7). Indeed, the phenomena were confirmed by the
transient transfection of SMN1 and SMN2 mini-gene constructs, including
the genomic exons 6-8 in a pCI mammalian expression vector (Fig.
1A). Furthermore, the
substitution of C to T located six nucleotides inside exon 7 of SMN1
led to the exclusion of exon 7, and the splicing patterns of exon 7 were similar to those of wild type SMN2. In contrast, the mutant SMN2
(substitution of T to C in SMN2) mini-gene produced full-length SMN
transcripts (Fig. 1A). To determine the
cis-acting elements responsible for the skipping of SMN exon
7, we constructed various deletion mutants of both wild type SMN1 and
mutant SMN1 (C to T transition in exon 7) mini-genes that contain exon
7 with flanking introns 6 and 7, and they were cloned into the exon
trapping vector, pSPL3 (Fig. 1B). Previously, we identified
a cis-acting element for the regulation of SMN exon 7 splicing in the intron 6 (element 1) (24). Element 1 has been
demonstrated to be composed of 45 bp, and the core was a
pyrimidine-rich sequence responsible for the negative regulation of SMN
exon 7 splicing. Further, we also found element 2, which was composed
of 66 bp in the intron 7 and plays roles in the inclusion of exon 7, but the detailed mechanisms responsible for the splicing regulation of
this element remain unclear. In the present study, we first tried to
identify the minimum sequence of element 2 essential for the activation of the splicing of SMN exon 7. When the various deletion mutants of
wild type SMN1 (I7DM1~4-C) were transfected into COS-7 cells, all of
the constructs expressed the full-length type of mRNA including SMN
exon 7 (Fig. 1C, +Exon7) but never expressed
isoforms lacking exon 7 (Fig. 1C,
Exon7). In
contrast, the longest construct of mutant SMN1 (I7DM1-T), which
contained a C to T transition in the SMN1 exon 7 with 235 bp of the
flanking intron 6 and 124 bp of the flanking intron 7, expressed
mRNAs with both the inclusion and exclusion of exon 7 (51%
inclusion) (Fig. 1C). The ratios of exon inclusion using
mini-genes containing the C to T transition in exon 7 cloned into exon
trap vectors were higher than those using the SMN mini-genes containing
exons 6-8 cloned into the pCI vector. The differences indicate that
there may be some cis-acting elements that regulate the
splicing of SMN exon 7 in regions that are different from the mini-gene
sequences (about 400 bp) cloned into the exon trap vector. However,
because the mini-genes containing the C to T transition in SMN1 exon 7 cloned into the exon trap vector (pSPL3) showed a significant increase
in the exclusion of exon 7, these constructs are thought to be useful
for determining the cis-elements responsible for the
exclusion of exon 7 in the 400-bp mini-gene.
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Fig. 1.
in vivo splicing of SMN and the
structure of element 2. A, splicing patterns of SMN1
and SMN2 exon 7. RT-PCR analysis of total RNA isolated from SK-N-SH
cells 24 h after transfection of pCI vectors cloned SMN mini-gene
containing exons 6-8. Upper bands, full-length transcripts
including exons 6-8; lower bands, transcripts lacking exon
7. WT SMN1(C), wild type SMN1 mini-gene; Mutant
SMN1(T), SMN1 mini-gene containing the C to T transition located
six nucleotides inside exon 7; WT SMN2(T), wild type SMN2
mini-gene; Mutant SMN2(C), SMN2 mini-gene containing
substitution of T to C located six nucleotides inside exon 7. B, constructions of the exon trapping vectors. Various
deletion mutants (I7DM1-4) of both wild type SMN1 and mutant SMN1 (C
to T transition in exon 7) mini-genes that contain the exon 7 with
flanking introns 6 and 7 were cloned into pSPL3. BP,
branching point; PPT, polypyrimidine tract. Element 1 ( 112
to
68), negative regulatory element of exon 7 splicing was determined
by previous study (24). C, RT-PCR products from in
vivo splicing assays. +Exon7 and
Exon7
transcripts yield 317- and 263-bp fragments, respectively. Each PCR
product was excised from gels, and the DNA sequence was determined. In
each case, the splicing occurred at the expected sites. All of the
deletion mutants of wild type SMN1 express +Exon 7 transcripts (upper panel), whereas each deletion mutant of
SMN1 containing the C to T transition produced both +Exon7
and
Exon7 (lower panel). The scores shown in
the lower panel are percentages of exon 7 inclusion relative
to total transcripts. The values represent the means of four analyses.
Note that the splicing patterns are changed in the cases where I7DM4-T
was transfected compared with the full-length constructs, I7DM1-T.
D, upper panel, position of the element 2 within
the intron 7. Lower panel, predicted stem-loop structure of
element 2. The 24-nucleotide structure was predicted by the MFOLD
program (26).
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Fig. 2.
Mutations in the stem-loop of element 2 of
mutant SMN1 containing the C to T transition alter the amount of
transcripts containing exon 7. A, the various mutations
(constructs A-F) in the I7DM1-T mini-gene cloned in the
exon trapping vector, pSPL3. Circled letters indicate
substituted nucleotides. B, RT-PCR products from in
vivo splicing analysis. I7DM1-T, mini-gene cloned into
the exon trapping vector; I7DM4-T, deletion mutation, which
is the deleted 66 bp containing the element 2 from I7DM1-T (Fig. 1,
construct B). Mutation in the loop did not affect SMN
splicing, but all of the mutations in the stem structure led to
decreases of the inclusion of SMN exon 7. C, quantitative
analyses of the splicing patterns of each construct. Each band
intensity was determined using the densitography program described
under "Experimental Procedures," and the percentages of exon 7 inclusion relative to the total transcripts are represented (means ± S.D. of four analyses).
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Fig. 3.
Tandem repeat of element 2 enhances the
splicing of SMN exon 7. A, the constructs of tandem
repeats of element 2 in the intron 7 of SMN. I7DM1-T, the
original construct containing one copy of element 2; Element
2 × 3, contains three copies of element 2; Element
2 × 4, contains four copies of element 2. B, the
effects of tandem repeats of element 2 in the intron 7 of SMN. RT-PCR
analyses showed that the insertion of element 2 increases the inclusion
of SMN exon 7. The percentages of exon 7 inclusion relative to the
total transcripts are represented (means ± S.D. of four
analyses).
) that have
alternative splicing variants both including and excluding its exon
close to the stem-loop structure, suggesting that the stem-loop
structure within element 2 could play a role in the alternative
splicings of several genes.
Genes containing the stem-loop structure similar to that of
element 2 in SMN
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Fig. 4.
Identification of element 2-nuclear protein
complexes. A, gel mobility shift assay of SK-N-SH cell
nuclear extracts. 32P-Labeled element 2 RNA was incubated
with increased nuclear extracts for 25 min at room temperature. The
binding mixtures were then analyzed on a 5% nondenaturing
polyacrylamide gel. Specific binding was detected, and the activities
increased corresponding to the amounts of nuclear extracts.
B, competition assay was done by adding unlabeled RNA
oligonucleotides (1, 5, and 25 molar excess) of wild type element 2 or
mutant element 2. Excessive amounts (25 molar excess) of unlabeled RNA
oligonucleotides of wild type element 2 inhibited the complex
formation, whereas mutant element 2 did not affect the specific binding
at the same concentrations. Aliquots of 5 µg of nuclear extracts were
used in this experiment.
View larger version (19K):
[in a new window]
Fig. 5.
Treatment with antisense oligonucleotides
directed toward element 2 decreases the expression of transcripts
containing SMN exon 7. A, the regions where the three
types of antisense oligonucleotides (As-element2,
As-con, and As-pyr) can hybridize. B,
RT-PCR of in vivo splicing using the exon trapping system.
Treatment with As-element2 led to a decrease in the ratio of exon
7 inclusion relative to total transcripts. The effects observed were
dependent on increasing concentrations of As-element2 (lower
panel). C, treatment with As-con did not affect the
splicing of SMN exon 7. The percentages of exon 7 inclusions are shown
below each lane, and represent the means for the four
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, a member of the
serine-arginine-related proteins of splicing factors, binds to the
elements and stimulates an ESE (22). It was recently reported that a
single nucleotide change occurs within a heptamer motif of the ESE,
which in SMN1 is recognized directly by SF2/ASF (23). The abrogation of
the SF2/ASF-dependent ESE is considered to be the basis for
the inefficient inclusion of exon 7 in SMN2. However, it is unclear
whether Tra2-
1 and SF2/ASF functionally cooperate to promote the
inclusion of the exon and whether other factors are involved in the
regulation of the splicing of SMN exon 7. Therefore, we have examined
one of the critical cis-acting elements that we identified
in a previous study of the SMN pre-mRNA responsible for the
skipping of the SMN exon 7 containing the C to T transition.
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ACKNOWLEDGEMENTS |
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We thank K. Otori for technical support in this study. We are grateful to Drs. E. Androphy and C. Lorson for the constructs of SMN1 and SMN2 mini-genes in pCI vector.
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FOOTNOTES |
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* This work was supported in part by the Toray Sciences Foundation, grants from Research for Comprehensive Promotion of Study of the Brain of the Ministry of Education, Culture, Sports, Science and Technology, and a Grant-in-Aid for Scientific Research (A).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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 81-743-72-5411; Fax: 81-743-72-5419; E-mail: imaizumi@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M209271200
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ABBREVIATIONS |
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The abbreviations used are: SMA, spinal muscular atrophy; SMN, survival motor neuron; ESE, exonic splicing enhancer; RT, reverse transcriptase.
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REFERENCES |
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