MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
June 2000, p. 4036–4048Vol. 20, No. 11
Aberrant Splicing of tau Pre-mRNA Caused by Intronic Mutations
Associated with the Inherited Dementia Frontotemporal Dementia
with Parkinsonism Linked to Chromosome 17
ZHIHONG JIANG,1JOCELYN COTE,1JENNIFER M. KWON,2ALISON M. GOATE,3
AND JANE Y. WU1,4*
Department of Pediatrics,1Department of Neurology,2Departments of Psychiatry & Genetics,3and Department of
Molecular Biology and Pharmacology,4Washington University School of Medicine, St. Louis, Missouri 63110
Received 4 November 1999/Returned for modification 8 December 1999/Accepted 1 March 2000
Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia
with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau
gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative
disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases.
To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have
constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro
biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene
lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the impor-
tance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron
mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase
H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that
sequesters the 5? splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding
and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase
rather than decrease alternative splicing can be an important pathogenic mechanism for the development of
tau is a major microtubule (MT)-binding protein that pro-
motes MT assembly and stabilizes the MT tracks (for reviews,
see references 26, 27, 43, and 44). The tau gene is expressed in
developing and mature neurons and is especially enriched in
the axon. A low level of tau expression is also found in glial
cells. In addition to posttranslational regulation by phosphor-
ylation, expression and function of the tau gene are under
complex regulation by alternative splicing. In the human brain,
alternative splicing produces six isoforms with variations in the
amino-terminal region and in the carboxyl domain, containing
either three or four MT-binding repeats (MT1 to MT4) (1, 19,
35, 40). This expression pattern results from a combination of
alternative inclusion of exon 2 or exons 2 and 3 in the amino-
terminal region and of exon 10 in the MT-binding domain of
the carboxyl-terminal region. Exclusion or inclusion of exon 10
leads to the formation of tau proteins containing either three
or four MT-binding repeats. In the normal human adult cere-
bral cortex, the ratio of four- to three-MT-binding repeat-
containing tau transcripts (Tau4R/Tau3R) is approximately 1
(see Fig. 2) (15, 16, 18, 29, 30).
A large number of studies have suggested that changes in the
tau protein play a critical role in neurodegeneration (reviewed
in references 17, 35, 41, 61, 62). tau-containing neurofibrillary
lesions are found in myotonic dystrophy, Pick’s disease, corti-
cobasal degeneration and progressive supranuclear palsy. In
addition, several other neurodegenerative diseases have intra-
neuronal lesions containing aberrantly processed tau protein,
including Niemann-Pick disease type C, Gerstmann-Straussler-
Scheinker disease with tangles, prion protein amyloid angiop-
athy, amyotrophic lateral sclerosis-parkinsonism-dementia
complex of Guam, Down syndrome, and familial presenile
dementia with tangles (reference 61 and references therein).
Recent genetic studies have placed the tau gene at the center
of pathogenesis of frontotemporal dementia with parkinson-
ism linked to chromosome 17 (FTDP-17), an autosomal dom-
inant condition characterized by prominent atrophy of the
frontal and temporal cortex with later involvement of subcor-
tical structures (14). The brain atrophy is usually accompanied
by neuronal cell death, gliosis, and formation of intraneuronal
deposits containing tau protein. Missense mutations in the tau
gene have been identified in FTDP-17 cases, and several of
these mutations lead to a reduction in the ability of tau to bind
MT and to promote MT assembly (24, 29–31, 50). In addition
to mutations found in the coding region of the tau gene, sev-
eral intronic mutations were identified, accounting for a sig-
nificant fraction of FTDP-17 cases (Fig. 1A) (for reviews, see
references 16 and 61). All of the intronic mutations reported so
far are associated with increases in the ratio of four- to three-
repeat-containing isoforms of tau (Tau4R/Tau3R). This sug-
gests that a proper ratio of Tau4R to Tau3R is essential for
normal function of tau in the human brain and that distur-
bance of this delicate balance may lead to deleterious effects
on neuronal survival and function. A change in the Tau4R/
Tau3R ratio appears to be sufficient for development of the
filamentous tauopathy (16, 32, 61). Discovery of these intronic
mutations in the tau gene in dementia patients highlights the
importance for normal brain function of controlling the bal-
ance of different tau isoforms by alternative splicing.
Based on analyses of the nucleotide sequence of the tau
gene and the observation that intronic mutations increase in-
* Corresponding author. Mailing address: Department of Molecular
Biology and Pharmacology, Washington University School of Medi-
cine, St. Louis, MO 63110. Phone: (314) 454-2081. Fax: (314) 454-2388.
clusion of exon 10 in the tau transcripts, a hypothesis was
proposed that a stem-loop-type secondary structure may form
in the tau pre-mRNA around the exon-intron junction that
could regulate tau exon 10 alternative splicing (6, 30, 63). In
this study, we have established a minigene model system to
examine tau alternative splicing using both cell culture and in
vitro biochemical assays. We have tested the splicing efficiency
of wild-type and mutant tau pre-mRNAs containing either
DDPAC?14 or AusI?16 intronic mutations and demon-
strated that the intronic mutations enhance splicing of exon 10
without affecting RNA stability. Systematic analyses using bio-
chemical approaches indicate that among the different spliceo-
somal U snRNPs, U1 snRNP plays a prominent role in dif-
ferentially recognizing wild-type versus intronic mutant tau
pre-mRNAs and that U6 snRNP also appears to be involved in
this process. A gel mobility shift assay with purified U1 snRNP
preparations demonstrates a significantly higher level of U1
snRNP binding to the mutant tau than to the wild-type tau
pre-mRNA. Our data support the model in which base-pairing
interactions between the intronic sequence downstream of
exon 10 and the splice junction itself prevent maximal recog-
nition of this 5? splice site. Partial disruption of these base-
pairing interactions by intronic mutations leads to enhanced
U1 snRNP interaction with the 5? splice site of exon 10 and
therefore higher splicing efficiency. To our knowledge, this is
the first study with systematic biochemical characterization of
a human disease-causing alternative splicing event in which
intronic mutations increase rather than decrease splicing effi-
MATERIALS AND METHODS
Plasmids. The tau genomic DNA fragments containing exons 9, 10, and 11 as
well as intronic sequences (wild type or mutant) flanking exon 10 were amplified
by PCR from normal adult human or FTDP-17 patient brain genomic DNA. tau
minigene constructs were made by inserting the genomic fragments into mam-
malian expression vector pcDNA3(Invitrogen) between the HindIII and XhoI
sites under the control of the cytomegalovirus promoter. DNA sequence analysis
of tau genomic fragments and different expression plasmids was carried out on an
ABI 373A automatic sequencer using the PRISM Ready Reaction DyeDeoxy
Terminator cycle sequencing kit (Applied Biosystems).
Cell culture and transient transfection. For transfection experiments, all plas-
mid DNA samples were purified by double cesium chloride centrifugation. HeLa
RB or N2a cells were grown in 3.5-cm dishes in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum. At the time of transfection,
HeLa RB cells were approximately 50% confluent and N2a cells were approxi-
mately 60% confluent. Transfection was performed by the calcium phosphate
precipitation method with 2 to 3 ?g of tau minigene construct DNA. Cells were
harvested 48 h after transfection. Under these conditions, the transfection effi-
ciency in these experiments was approximately 60% for HeLa RB cells and 50%
for N2a cells.
RT-PCR assay. RNA was prepared from different tissues or transfected HeLa
RB or N2a cells with Trizol reagent (Gibco-BRL), and reverse transcription
(RT)-PCR was carried out in the presence of [?-32P]dCTP (Amersham Life
Sciences) as previously described (68). Human or murine and rat tau-specific
primers in exon 9 and exon 11 were used to detect the endogenous tau mRNA.
Alternatively spliced tau products expressed from the transfected minigene were
detected by RT-PCR with primers specific to the transfected plasmid (T7 and
SP6 primers). The PCR cycle number was kept to a minimum (20 cycles) to
maintain linearity. The ratio of tau exon 10?(Tau4R) to tau exon 10?(Tau3R)
was measured with a PhosphorImager (Molecular Dynamics).
Transcription and splicing. Splicing substrates were synthesized using T7
RNA polymerase (Promega) from corresponding linear templates, with catabo-
lite gene activator protein analog (Pharmacia Biotech) and [?-32P]UTP (Amer-
sham Life Sciences). RNA purification was performed as described before (5).
HeLa cell nuclear extracts were prepared as described before (8). Some prepa-
rations yielded very low splicing activity for uncharacterized reasons and allowed
only the first step of the splicing reaction to occur when incubated for 2 h at 30°C.
It was observed previously that these extracts could be rendered fully active by
the addition of 2 U of creatine kinase per 25-?l splicing reaction mixture (7).
Splicing mixtures were set up and processed as described before (45). Splicing
products were separated on 7 or 8% acrylamide (acrylamide-bisacrylamide, 38:
2)–8 M urea gels. The identity of lariat intermediates was confirmed by perform-
ing debranching reactions in S100 extracts followed by gel migration.
Oligonucleotide-targeted RNase H cleavage assay. Minigene constructs
TauEx10?11 and TauEx10?11d5 were linearized with EcoRI (Promega) and
transcribed into RNA with T7 RNA polymerase. [?-32P]UTP-labeled wild-type
and mutant RNAs were incubated at 37°C for 20 min in standard splicing buffer
in the presence of 0.5 U of RNase H (Gibco-BRL) in 12.5-?l reaction mixtures
with various concentrations of an oligonucleotide (5?-GAAGGTACTCACACT
GCC-3?) complementary to the exon 10 splice donor site. When assays were
performed in the presence of nuclear extracts, RNAs were incubated with nu-
clear extract for the indicated times in the presence of RNase H and 0.2 pmol of
oligonucleotide. These concentrations of oligonucleotide and RNase H were
used so that differences in cleavage between the wild-type and mutant tau RNAs
were not masked by excessive amounts of oligonucleotide or RNase H. Cleaved
RNA products were then separated on a 6% polyacrylamide–8 M urea gel. The
cleavage ratios were measured with a PhosphorImager (Molecular Dynamics).
U snRNP inactivation and blocking assays. U1 snRNA was either inactivated
using oligonucleotide-targeted RNase H cleavage as described above or blocked
by incubation with 2?-O-methyl-oligoribonucleotides. Specifically, 2?-O-methyl-
oligonucleotides (U1, 3 to 12 ?M; U2, 0.3 ?M; U5, 12 ?M; U6, 13 ?M) were
added to splicing reaction mixtures and incubated at 30°C for 10 min prior to the
addition of RNA substrates (55). Incubations were then carried on for 1.5 h at
30°C in the presence or absence of creatine kinase (1 U). In the absence of
creatine kinase, splicing reactions with this particular HeLa nuclear extract did
not efficiently proceed through the second step of the splicing reaction. As a
control, a 2?-O-methyl-oligonucleotide against U7 was used (6 ?M). HeLa cell
nuclear extracts depleted of U1 snRNP activity were produced by incubation at
30°C for 1 h in the presence of RNase H and the oligonucleotide CCAGGTA
AGTAT, complementary to the 5? end of U1 snRNA (3). As a control, a
mock-treated nuclear extract was obtained by incubation with RNase H and an
unrelated oligonucleotide. Splicing products were processed as before and re-
solved on 8% denaturing gels. Aliquots of the mock- and U1 oligonucleotide-
treated nuclear extracts were incubated with proteinase K (1 mg/ml) for 30 min
at 37°C, extracted with phenol, and ethanol precipitated. The extracted RNAs
were then run on an 8% polyacrylamide–8 M urea gel and visualized by ethidium
bromide staining to assess the efficiency of RNase H cleavage.
U1 snRNP protection assay. Splicing reactions were carried out with 2 fmol of
32P-labeled-tau pre-mRNA transcripts with U1-depleted or mock-treated nu-
clear extracts at 30°C for 0, 10, or 20 min. RNase H (0.4 U) and 20 pmol (molar
excess) of the oligonucleotide complementary to the 5? splice site of exon 10
(GAAGGTACTCACACTGCC) were then added to 12.5-?l splicing reaction
mixtures (11), and incubation was continued for 15 min at 37°C to completely
cleave the pre-mRNA transcripts that were not protected. U1 snRNP- or mock-
depleted extracts were prepared as described above.
U1 binding assay. The U1 snRNP preparation was a kind gift from A. Kramer,
and its purification and characterization were described previously (37, 38). The
major snRNA species detected in the preparation is U1 snRNA.32P-labeled
pre-mRNA transcripts from constructs TauEx10?11d5wt and -DDPAC were
incubated under splicing conditions with various amounts of purified U1 snRNP
at 30°C for 5 min except that no ATP or creatine phosphate was added. Heparin
was then added to a final concentration of 0.5 mg/ml, and reaction mixtures were
incubated at 30°C for an additional 5 min. The RNA-U1 snRNP complexes were
resolved on 4% nondenaturing polyacrylamide gels (acrylamide-bisacrylamide,
80:1), and electrophoresis was carried out in Tris-glycine buffer (50 mM Tris, 50
mM glycine) (34).
Alternative splicing of tau exon 10. To understand alterna-
tive splicing regulation of genes involved in neuronal function
and neurodegeneration, we have initiated a study of tau pre-
mRNA alternative splicing. Because all of the intronic muta-
tions identified so far in the tau gene are in the intron following
exon 10 and many exonic mutations are also located in exon 10
(6, 25, 30, 63), we have focused our efforts on this region of the
human tau gene (Fig. 1A). Based on our previous experience
(33, 68) we established a specific and sensitive RT-PCR pro-
tocol to examine tau alternative splicing in tissues or cultured
cells. This allowed us to reliably measure the level of inclusion
or exclusion of tau exon 10 in human brain tissues and in
cultured cell lines. A number of human or rodent cell lines of
neural as well as nonneural origin were screened. The tau
alternative splicing pattern detected in these cell lines was
compared with that in adult human and rat brain tissues. Con-
sistent with previous studies (30, 35), endogenous tau expres-
sion was mostly restricted to cell lines derived from neural
tissues. tau expression was also detected in embryonic kidney
cell lines HEK293 and 293T in addition to neural cell lines
VOL. 20, 2000DEMENTIA MUTATIONS INCREASE U1 snRNP BINDING 4037
FIG. 1. (A) Positions of reported mutations in exon 10 or the downstream intron region of the human tau gene. The first nucleotide in the intron is defined as
position ?1. 5? SS, 5? splice site. (B) Schematic of a series of tau minigene constructs. The genomic DNA fragments containing exons 9, 10, and 11 as well as intronic
sequences (wild type [wt], AusI, or DDPAC) flanking exon 10 were inserted in mammalian expression vector pcDNA3under the control of the cytomegalovirus
promoter (PCMV). The chimeric minigene constructs Ad-TauEx10?11wt and -DDPAC were made by replacing the upstream exon 9 and associated intronic sequences
with the first exon region (L1) of the adenovirus (Ad) major late transcription unit. The sizes of the corresponding exons and introns are indicated above the respective
regions (in base pairs).
4038 JIANG ET AL.MOL. CELL. BIOL.
such as SH-SY5Y, N2a, and GT1-7. In the adult human brain,
the ratio of Tau3R to Tau4R is approximately 1 (Fig. 2A, lane
6) (30). However, in the adult rat brain, Tau4R is the predom-
inant tau RNA transcript (Fig. 2A, lane 9). This suggests that
alternative splicing regulation of the tau gene in human brain
is different from that in rat brain.
Intronic mutations affect exon 10 alternative splicing. tau
minigenes were constructed by using genomic DNA fragments
containing intact exons 9, 10, and 11 with shortened flanking
intronic sequences (Fig. 1B, TauEx9-11wt and TauEx9-
11d5wt). These tau minigenes, when introduced into either
HeLa RB or N2a cells, underwent alternative splicing similar
to that seen in the human adult brain, with the formation of
both Tau3R and Tau4R (data not shown) (Fig. 2B, lanes 1 and
2 and 7 and 8). We therefore concluded that the minimal
TauEx9-11d5wt minigene with a total of 800 bp of tau genomic
DNA sequence contains cis elements essential for tau exon 10
alternative splicing. We noticed that transfected tau minigenes
in these cells produced a slightly higher level of Tau4R com-
pared to the endogenous tau expression pattern (Fig. 2), sug-
gesting that overexpression of the tau minigene may titrate
certain limiting factors controlling the ratio of Tau3R to
Derivatives of tau minigenes containing the FTDP-17-asso-
ciated mutations AusI?16 and DDPAC?14 were then con-
structed (Fig. 1B, TauEx9-11AusI and -DDPAC and TauEx9-
11d5AusI and -DDPAC, respectively). These intronic mutant
tau minigenes were introduced into either HeLa RB or N2a
cells. The tau splicing pattern in these transfected cells was
examined by RT-PCR. Both AusI?16 and DDPAC?14 deriv-
atives led to predominantly Tau4R production when tran-
siently expressed in these cell lines (data not shown) (Fig. 2B,
lanes 3 to 6 and lanes 9 to 12), as detected in brain tissues of
FTDP-17 patients (30; Z. Jiang and J. Y. Wu, unpublished
data). This observation suggests that the AusI?16 and
DDPAC?14 intronic mutations in the tau gene most likely
mediate their effect by affecting exon 10 alternative splicing
and that cis elements required to observe the mutant profile
were present in our TauEx9-11d5 minigene. However, these
results, similar to previous studies (6, 8, 23, 30, 63), could not
rule out the possibility that differences in RNA stability con-
tributed to the alternative splicing difference. Therefore, we
tested whether the AusI?16 and DDPAC?14 mutations af-
fected the RNA stability of tau pre-mRNA transcripts both in
transfected cells and in an in vitro biochemical assay. Specifi-
cally, following transfections, cells were treated with actinomy-
cin D to block transcription, and levels of tau pre-mRNA and
mRNA transcripts were then quantified at different time points
by RT-PCR. No significant difference in RNA stability was
detected between wild-type and AusI?16 or DDPAC?14 pre-
mRNAs (Fig. 3). We also compared the stability of tau wild-
type and mutant pre-mRNAs using in vitro-synthesized and
radiolabeled transcripts. Following incubation with HeLa nu-
clear extracts for different periods of time, the tau pre-mRNA
and splicing products were separated by gel electrophoresis.
The levels of the RNA transcripts were then quantified with a
FIG. 2. tau alternative splicing in tissues and cell lines. (A) Expression of
endogenous tau exon 10?(Tau4R) and exon 10?(Tau3R) mRNAs in the adult
human brain, rat brain, and human (HeLa, 293T, HEK293, HeLa RB, and
SH-SY5Y) and murine (N2a and GT1-7) cell lines as determined by RT-PCR
with specific primers in exon 9 and exon 11 of human or rat and mouse tau. (B)
Alternative splicing of exon 10 from transfected wild-type (WT) and mutant tau
minigenes (AusI and DDPAC). The tau minigene constructs (TauEx9?11d5)
with wild-type and mutant intronic sequences were transfected into HeLa RB
and N2a cells. Alternatively spliced tau products expressed from the transfected
minigene were detected by RT-PCR with primers specific to the transfected
plasmids (T7 and SP6 primers). The percentage of Tau4R (exon 10 inclusion) in
the total splicing products was measured using a PhosphorImager.
FIG. 3. Intronic mutations do not affect the RNA stability of tau transcripts.
Wild-type (WT) and mutant (AusI?16 and DDPAC?14) tau minigenes were
transfected into N2a cells. Following transfections, cells were treated with acti-
nomycin D to block transcription, and tau pre-mRNA and mRNA transcripts
were then detected at different time points by RT-PCR (A). The tau transcripts
were quantified using a PhosphorImager. RNA stability was expressed as the
ratio of total tau transcripts at different time points to that at time zero (B).
VOL. 20, 2000DEMENTIA MUTATIONS INCREASE U1 snRNP BINDING 4039
PhosphorImager. Again, no difference was detected in the
stability of wild-type and mutant tau transcripts (Fig. 4 and 5).
These results demonstrated that the effects of these FTDP-17
intronic mutations on tau alternative splicing were not due to
differential RNA stability.
To investigate the biochemical mechanism by which the in-
tronic mutations affected tau exon 10 alternative splicing, we
set up an in vitro splicing system using tau minigenes. When
incubated under splicing conditions in HeLa nuclear extracts,
the TauEx9-11d5wt and mutant substrates both yielded only
the Tau3R (exon 10 skipping) mRNAs, with no detectable
Tau4R (exon 10 inclusion) mRNAs (data not shown). This
could be explained by the fact that the 3? splice site upstream
of exon 10 as well as the 5? splice site of exon 9 are both
divergent from consensus sequences, and therefore these splic-
ing signals are too weak to be recognized by the splicing ma-
chinery in vitro. Therefore, we replaced the upstream exon 9
and associated intronic sequences for the first exon region (L1)
of the adenovirus (Ad) major late transcription unit (Fig. 1B,
Ad-TauEx10?11wt and -DDPAC). Even with this chimeric
substrate, we were able to detect only a low level of exon 10
inclusion in the wild-type tau substrate and a slight enhance-
ment of exon 10 inclusion with the DDPAC?14 derivative
(data not shown). We then used single-intron substrates to
determine which tau splicing unit (9-10 or 10-11) was specifi-
cally affected by these intronic mutations (Fig. 1B, TauEx9-
10d5 and TauEx10?11d5). Neither DDPAC?14 nor AusI?16
intronic mutations affected splicing between exon 9 and exon
10 (data not shown). However, splicing between exons 10 and
11 was significantly enhanced by AusI?16 and DDPAC?14
mutations, because significantly higher levels of splicing inter-
mediates or products can be detected at various time points
(Fig. 4, compare lanes 3 and 5 with lane 1 or lanes 4 and 6 with
lane 2). This result suggests that AusI?16 and DDPAC?14
intronic mutations increase specific recognition of the se-
quence around the 5? splice site of exon 10 by the splicing
Oligonucleotide-directed RNase H cleavage experiments
support the presence of a secondary structure around the 5?
splice site of exon 10. It has been proposed in previous studies
that the tau exon 10 splice donor region could form a stem-
loop structure with downstream intronic sequences. This stem-
loop model was first proposed based on sequence analysis and
exon-trapping experiments where exon 10 was inserted with
minimal intronic sequence into a heterologous splicing cassette
(6, 23, 30, 63). In this model (Fig. 5A), there are base-pairing
interactions between the nucleotide residues surrounding the
5? splice site of exon 10 in the region that extends from posi-
tions ?2 to ?16 (with the first G nucleotide in the intron being
?1). In wild-type tau pre-mRNA, this structure would contain
at least six uninterrupted base pairs, whereas in either
AusI?16 or DDPAC?14 mutant tau pre-mRNA, a G-C in-
teraction is disrupted in the stem region, presumably leading to
a less stable structure. More recently, nuclear magnetic reso-
nance spectroscopy was employed to demonstrate that short
RNA oligonucleotides corresponding to this region of tau pre-
mRNA could form a stable, folded stem-loop structure in the
absence of protein factors (65). However, it is not clear
whether such a secondary structure exists in the longer tau
pre-mRNA transcripts under splicing conditions or whether
the splicing machinery indeed differentially recognizes wild-
type versus mutant tau pre-mRNA transcripts.
We probed the potential secondary structure of the wild-
type and mutant tau gene in the region around the exon 10 5?
splice site using an oligonucleotide-directed RNase H cleavage
assay with a specific DNA oligonucleotide (illustrated in Fig.
5A) complementary to the 5? splice site region. To avoid com-
plication of splicing products (when the assay was performed in
the presence of nuclear extract) and nonspecific cleavage, we
used shorter RNA transcripts. With tau transcripts containing
exon 10 and 261 nucleotides of downstream intronic se-
quences, both DDPAC?14 and AusI?16 mutant tau consis-
tently showed significantly more cleavage products than wild-
type tau. Consistently, RNase H cleavage at a cryptic splice site
downstream of the authentic 5? splice site occurred at a lower
level in the mutant than in the wild-type transcripts (Fig. 5B
and F). These data are consistent with an increased accessibil-
ity of the sequence around the 5? splice site to the oligode-
oxynucleotide-mediated RNase H cleavage in the mutant tau
transcripts (Fig. 5B). These results support the idea that a
secondary structure forms around the 5? splice site of exon 10,
reducing the binding of the oligonucleotide and, therefore, the
efficiency of RNase H cleavage. Destabilization of this second-
ary structure by the DDPAC?14 or AusI?16 mutations in-
creases cleavage by RNase H. To further define this potential
secondary structure, we made additional truncations in the
intron. Shortening this intronic sequence downstream of the 5?
splice site to 53 nucleotides did not affect the differential
RNase H cleavage of wild-type and mutant tau transcripts (Fig.
5C and G), consistent with the observation that these short-
ened tau minigene constructs behave similarly to those with
longer intron sequences (data not shown). Finally, the RNase
H cleavage assay was carried out in the presence of HeLa
nuclear extract. Again, as was observed in the absence of nu-
clear extract, there were significantly more cleavage products
in the reactions with DDPAC?14 or AusI?16 tau transcripts
than in those with the wild-type tau transcript (Fig. 5D and H).
Quantification of the cleavage products at different oligonu-
cleotide concentrations and at different time points clearly
indicates that wild-type and intronic mutant (DDPAC?14 as
well as AusI?16) tau RNAs have distinct susceptibilities to the
FIG. 4. Intronic mutations affect exon 10 alternative splicing. In vitro splicing
of TauEx10?11d5wt and mutant substrates. Labeled pre-mRNA substrates were
incubated in HeLa nuclear extracts (approximately 6 ?g/?l) under splicing con-
ditions for the times indicated.
4040JIANG ET AL.MOL. CELL. BIOL.
RNase H cleavage. Because the oligonucleotide used in the
RNase H cleavage assay does not extend to the position of
these mutations (?14 for DDPAC and ?16 for AusI; Fig. 5A)
and because the difference in cleavage efficiency between the
wild-type and mutant RNAs was detectable in the absence of
other protein factors or spliceosomal components, these data
are best explained by the difference in the RNA secondary
structure. Taken together, these results support the hypothesis
that at the U1 snRNP binding site around the 5? splice site
downstream of exon 10 there exists a secondary structure in-
volving 53 or fewer intronic nucleotides. This secondary struc-
ture could form on naked tau pre-mRNA transcripts as well as
with tau transcripts in the presence of splicing-competent nu-
clear extracts. This structure is altered in the DDPAC?14 and
AusI?16 mutants so that base-pairing interaction in the stem
is weakened and the accessibility of the oligonucleotide is in-
creased, leading to enhanced RNase H cleavage in the reaction
mixtures containing the mutant tau transcripts.
U1 and U6 snRNPs are important players in differential
recognition of tau wild-type and mutant pre-mRNAs. To dis-
sect the mechanism by which intronic mutations affect tau exon
10 splicing, we employed the in vitro splicing assay using tau
pre-mRNA substrates containing exon 10 and exon 11 with the
shortened intron in between. Because the effect of the
DDPAC?14 mutation was consistently stronger than that of
the AusI?16 mutation in enhancing exon 10 splicing both in
vitro and in transfected cells, we used the DDPAC?14 mutant
for further characterization.
We first carried out titration experiments to examine
whether certain splicing factors were limiting for tau exon 10
splicing. Serial dilution of the HeLa nuclear extract led to a
general reduction in splicing efficiency for both wild-type and
FIG. 5. Oligonucleotide-targeted RNase H cleavage assays support a potential secondary structure around the 5? splice site of exon 10. (A) The stem-loop structure
formed with exon 10 downstream intronic sequences. The mutations AusI?16 and DDPAC?14 are indicated. (B, C, and D). Oligonucleotide-targeted RNase H
cleavage assays. Minigene constructs of TauEx10?11 (B) and TauEx10?11d5 (C and D) were linearized with EcoRI and transcribed into RNA with T7 RNA
polymerase. Labeled wild-type (WT) and mutant (AusI and DDPAC) tau RNAs were incubated with RNase H at 37°C for 20 min under splicing conditions in the
presence of various concentrations of the oligonucleotide complementary to the 5? splice site. The line with an arrowhead over the stem-loop shows the position of the
oligonucleotide used (A). TauEx10?11 constructs contain a cryptic 5? splice site, as indicated by the asterisk (B). (D) RNase H cleavage assay carried out under splicing
conditions with 0.2 pmol of the oligonucleotide in the presence of HeLa nuclear extract (1.6 ?g/?l). In this experiment, tau transcripts, the oligonucleotide, the nuclear
extract, and RNase H were mixed together prior to incubation at 37°C for the time indicated in minutes). The uncut RNA and products of cleavage at the exon 10 splice
donor site are shown on the right, while the products resulting from the cryptic site are marked on the left (B). Quantification of the RNA cleavage products in panel
B is shown in panels E (cleavage at the authentic site) and F (cleavage at the cryptic site). Panels G and H show the quantification of RNA cleavage products in panels
C and D, respectively. The efficiency of cleavage was expressed as the ratio of total cleavage products to the corresponding input transcript as measured using a
VOL. 20, 2000 DEMENTIA MUTATIONS INCREASE U1 snRNP BINDING4041
DDPAC?14 mutant tau pre-mRNAs (Fig. 6A). However, at
medium to low concentrations of HeLa nuclear extracts, the
difference in splicing efficiency between the DDPAC?14 and
wild-type tau became even more obvious (Fig. 6A, compare
lanes 2 with 5 and 3 with 6, respectively). This suggested the
involvement of trans-acting factors in differential recognition
of wild-type and mutant splicing substrates. We then investi-
gated which spliceosomal U snRNPs were involved in differ-
entially recognizing wild-type versus mutant tau pre-mRNAs.
U1, U2, U5, or U6 snRNPs were partially blocked by using
specific individual 2?-O-methyl-oligoribonucleotides at appro-
priate concentrations to treat HeLa nuclear extracts prior to
the splicing reactions (55). In the mock-treated or control (U7)
oligoribonucleotide-treated nuclear extracts, splicing of either
wild-type or DDPAC mutant tau pre-mRNAs was the same as
that in the untreated nuclear extract, with an approximately
twofold increase in the splicing products in the reactions with
DDPAC tau compared with wild-type tau constructs (Fig. 6B,
lanes 1 and 2 and lanes 11 and 12). When either U2 snRNP or
U5 snRNP was partially blocked, both wild-type tau splicing
and DDPAC?14 tau splicing were partially inhibited to a
similar extent. The ratio of DDPAC?14 splicing products to
wild-type tau splicing products remained approximately the
same (Fig. 6B, lanes 5 and 6 and lanes 7 and 8). With U6
snRNP partially blocked, splicing of wild-type tau was more
severely affected than that of DDPAC?14 tau (Fig. 6B, lanes
9 and 10). However, when U1 snRNP was partially blocked,
splicing of wild-type tau was drastically decreased, whereas
DDPAC?14 tau splicing was only slightly affected. Thus, the
ratio of DDPAC?14 splicing products to wild-type tau splicing
products was increased from 2-fold in mock-treated nuclear
extract to approximately 10-fold when U1 snRNP was partially
blocked. This is consistent with the idea that the secondary
structure on the wild-type tau pre-mRNA renders the 5? splice
site of exon 10 less accessible for recognition by U1 snRNP at
the early stage and by other factors, such as U6, at a later stage
during spliceosome assembly.
To further demonstrate that the involvement of U1 snRNP
in differentially recognizing wild-type and DDPAC?14 tau
substrates was an early event, we made use of HeLa nuclear
extracts that would not proceed to the second step of splicing
without the addition of exogenous creatine kinase (see Mate-
rials and Methods). In the presence of 8 ?M 2?-O-methyl U1
oligonucleotide and creatine kinase, wild-type tau splicing was
remarkably reduced but DDPAC?14 tau splicing was only
slightly decreased (Fig. 7A, lanes 1 to 4). When exogenous
creatine kinase was omitted from the splicing reaction and the
splicing reaction was blocked at the first step, wild-type tau
FIG. 6. (A) Presence of a limiting factor(s) for tau splicing in HeLa nuclear extracts. Splicing reactions were set up as previously described except that decreasing
amounts of HeLa nuclear extract (NE) were used, specifically: lanes 1 and 5, 6 ?g/?l; lanes 2 and 6, 4 ?g/?l; lanes 3 and 7, 2 ?g/?l; and lanes 4 and 8, 0.8 ?g/?l. Positions
of pre-mRNA, splicing intermediates, and products are indicated. (B) U snRNP inactivation differentially affects wild-type (WT) and mutant tau splicing. 2?-O-Methyl-
oligoribonucleotides (2?O-MeU) complementary to U1, U2, U5, U6, and U7 snRNAs were added individually to HeLa nuclear extracts, and the splicing reaction
mixtures were preincubated at 30°C for 10 min. The concentration of individual 2?-O-methyl-oligonucleotides was titrated to give partial inhibition of splicing (U1, 8
?M; U2, 0.3 ?M; U5, 12 ?M; and U6, 13 ?M). The U7 2?-O-methyl-oligonucleotide (6 ?M), which has been shown not to affect splicing, was used as a control.
TauEx10?11d5wt and the DDPAC?14 mutant pre-mRNAs (WT and DD, respectively) were then added, and the incubation was continued for 2 h. Splicing reaction
products were analyzed by gel electrophoresis.
4042 JIANG ET AL.MOL. CELL. BIOL.
splicing was almost completely blocked in the presence of 3 to
16 ?M 2?-O-methyl U1 oligonucleotide (Fig. 7A, lanes 7 and
8). However, under the same conditions, DDPAC tau splicing
was hardly affected (Fig. 7A, compare lanes 10 to 12 with lanes
6 to 8). This observation clearly indicates that U1 snRNP is
crucial for wild-type tau splicing, especially during the earliest
step of the splicing reaction, and that the DDPAC?14 intronic
mutation allows tau splicing to occur even when functional U1
snRNP is at a very low level. To rule out the possibility of
artifacts related to the use of 2?-O-methyl-oligonucleotides, we
depleted the extract of U1 snRNAs by using a DNA oligonu-
cleotide and RNase H (Fig. 7B and C). Under these condi-
tions, more than 90% of U1 snRNA in the nuclear extract was
cleaved (Fig. 7C, lane 2). When splicing reactions were carried
out using this U1-inactivated nuclear extract, wild-type tau
splicing was completely blocked (Fig. 7B, lane 2), whereas
significant amounts of splicing intermediates were still de-
tected in the reactions with DDPAC?14 tau pre-mRNA (Fig.
7B, lane 4). This demonstrates that a low level of U1 snRNP
(less than 10% of that in the untreated nuclear extract) was
sufficient to support splicing of DDPAC?14 but not wild-type
tau pre-mRNA. This result also suggests that the DDPAC?14
mutation may affect tau splicing through increasing initial rec-
ognition of the 5? splice site of exon 10 by U1 snRNP.
DDPAC?14 intronic mutation promotes more efficient as-
sembly of U1-dependent complexes on the 5? splice site of exon
10. To examine whether the DDPAC?14 intronic mutation
could influence the formation of U1 snRNP-dependent early
complexes on the 5? splice site, a specific RNase H protection
assay (Fig. 8) was performed as described before (11). Wild-
type or DDPAC?14 tau splicing substrates were first incu-
bated with HeLa nuclear extract under splicing conditions for
0 to 20 min. Then, 20 pmol (in molar excess to tau pre-mRNA
transcripts) of the oligodeoxynucleotide complementary to the
5? splice site and corresponding to the U1 snRNP binding
region was added together with RNase H, and the incubation
was continued for another 15 min. The RNase H-cleaved and
-protected fragments were then resolved by denaturing poly-
acrylamide gel electrophoresis and quantified. An increasing
level of protection was observed for both substrates upon in-
cubation at 30°C (Fig. 8A, lanes 2 to 4 and 6 to 8), which likely
reflects early 5? splice site recognition by the spliceosomal
commitment complexes. This is consistent with the previous
observation with other pre-mRNAs that U1 snRNP binding to
the 5? splice site is detectable at 0°C but requires incubation
with nuclear extract at 30°C to be stabilized (11). The
DDPAC?14 tau mutant transcript reproducibly gave rise to
twofold more protected product than wild-type tau (Fig. 8A,
lanes 2 to 4 and lanes 6 to 8, and Fig. 7B, 10- and 20-min time
points). Finally, when the nuclear extract was depleted of U1
snRNP by oligonucleotide-targeted RNase H cleavage, no pro-
tected tau pre-mRNA was detectable in the reactions with
either wild-type or DDPAC?14 tau transcripts (Fig. 8A, lanes
1 and 5). The requirement for preincubation with nuclear
FIG. 7. Wild-type (WT) tau pre-mRNA is more sensitive to U1 snRNP inactivation than DDPAC mutant pre-mRNA. (A) Effect of U1 snRNP 2?-O-methyl-
oligonucleotide inactivation on TauEx10?11d5 splicing. The same experiment was done using a different preparation of HeLa nuclear extract in the presence (lanes
1 to 4) and absence (lanes 5 to 12) of creatine kinase. This extract specifically needs the addition of creatine kinase to proceed through the second step of the splicing
reaction. Incubation was for 1.5 h at 30°C. Addition of the 2?-O-methyl U1 (2?O-MeU1) oligonucleotide is shown above each lane. The concentration used was 8 ?M
for lanes 2 and 4 and 0, 4, 8, or 16 ?M for lanes 5 and 9, 6 and 10, 7 and 11, and 8 and 12, respectively. (B) Inactivation of U1 snRNA by RNase H cleavage affects
wild-type (WT) and DDPAC tau splicing. Splicing reactions with tau transcripts were performed with either mock-treated HeLa nuclear extract (lanes 1 and 3) or
nuclear extract after treatment of oligonucleotide-targeted RNase H cleavage of U1 snRNA (lanes 2 and 4). Splicing reactions were incubated at 30°C for 1.5 h. An
asterisk marks the RNA species produced by a cleavage induced by nonspecific hybridization of the U1-specific oligonucleotide to the tau pre-mRNA transcripts. (C)
Efficiency of oligonucleotide-targeted RNase H cleavage of U1 snRNA. An aliquot of mock-treated (lane 1) or U1 snRNA-depleted (lane 2) nuclear extracts as
described for panel B was analyzed by gel electrophoresis for the presence of different snRNA species (as indicated on the right). U1?, U1 snRNA molecules containing
a shortened 5? end after RNase H cleavage. Under these conditions, more than 90% of U1 snRNA in the nuclear extract was cleaved.
VOL. 20, 2000DEMENTIA MUTATIONS INCREASE U1 snRNP BINDING 4043
extract to observe formation of the U1 snRNP protected band
also suggests that certain trans-acting factors in the nuclear
extract may play a role in making the 5? splice site accessible
for U1 snRNP binding. These results demonstrate that the
DDPAC?14 intronic mutation enhances the formation of U1
snRNP-dependent complexes on the 5? splice site of exon 10 in
a dynamic fashion during splicing. Supporting this result, we
also observed greater accumulation of spliceosomal complexes
A and B at these same time points, as monitored by native gel
electrophoresis (data not shown).
To test directly whether DDPAC?14 intronic mutation pro-
motes U1 snRNP binding, we used a gel mobility shift assay
with purified U1 snRNP as described in Materials and Meth-
ods. As shown in Fig. 9, incubation of the purified U1 snRNP
preparation with32P-labeled tau pre-mRNA transcripts led to
the formation of a complex that migrated more slowly than the
free RNA, with significantly more complex detected in the
DDPAC?14 tau reaction than in the wild-type tau reaction.
The difference in the amount of complex formed with the
DDPAC?14 and wild-type tau pre-mRNAs is more obvious at
the lower concentration of U1 snRNP used (Fig. 9, compare
lanes 7 and 8 with lanes 2 and 3). Quantification with the
PhosphorImager revealed that there is an approximately a
seven- to ninefold increase in the level of the complex detected
with the DDPAC?14 mutant compared with the wild-type tau
transcript. The detected complex was U1 snRNP dependent,
because inactivation of U1 snRNP in the preparation with
RNase H in the presence of the U1-specific oligonucleotide
abolished the formation of this complex (Fig. 9, lanes 5 and
10). These results demonstrate that the DDPAC?14 intronic
mutation enhances the formation of U1 snRNP-dependent
complexes on the 5? splice site of exon 10.
We have established a minigene system to dissect the mo-
lecular mechanism underlying alternative splicing of human
tau pre-mRNA exon 10, an event important for pathogenesis
of neurodegenerative disorder FTDP-17. Our systematic bio-
chemical analyses of wild-type and intronic mutant tau pre-
mRNAs demonstrated that single-nucleotide mutations in an
evolutionarily nonconserved intronic region enhance splicing
between exon 10 and exon 11. Experiments using RNase H and
an oligonucleotide complementary to positions ?6 to ?12 at
the splice junction suggest that the intronic mutant tau tran-
scripts harbor a more “open” RNA structure in this region
than wild-type tau in the presence or absence of HeLa nuclear
extracts. This is consistent with the presence of an RNA stem-
loop structure forming around the exon 10 5? splice site in
wild-type tau pre-mRNA. Comparison of the splicing efficiency
of wild-type and mutant tau transcripts when various spliceo-
somal U snRNPs were made limiting by specific 2?-O-methyl
oligonucleotides demonstrated that wild-type tau splicing was
most sensitive to a reduction in the level of functional U1
snRNP. Finally, the U1 snRNP protection assay and the gel
mobility shift experiment with purified U1 snRNP revealed an
increase in the binding of U1 snRNP to the 5? splice site of
exon 10 in the DDPAC?14 mutant, correlating well with the
increase in splicing efficiency observed in our in vitro splicing
assay. These results strongly support the model depicted in Fig.
10B, where the stem-loop structure in the wild-type tau pre-
mRNA is destabilized by FTDP-17 intronic mutations, leading
to enhanced recognition of this 5? splice site by the U1 snRNP-
containing early splicing complex and increased formation of
FIG. 8. Disruption of the putative secondary structure by the DDPAC?14
intronic mutation promotes the assembly of U1-dependent complexes on the 5?
splice site of exon 10. (A) TauEx10?11d5wt and DDPAC?14 RNAs were
incubated with nuclear extract at 0°C (lanes 2 and 6) or 30°C for 10 min (lanes
3 and 7) or 20 min (lanes 4 and 8) or with U1 snRNP-depleted (U1?) HeLa
nuclear extracts for 20 min (lanes 1 and 5). Following incubation, the oligonu-
cleotide complementary to the 5? splice site of exon 10 (Fig. 1A) was added along
with RNase H (0.4 U), and the incubation was continued for another 15 min at
37°C. The RNA cleavage products were then analyzed by gel electrophoresis.
Positions of the U1-protected pre-mRNA and cleavage products are indicated on
the right. The asterisk indicates the position of an artifactual cleavage product
generated by hybridization of the U1 snRNA-specific oligonucleotide on the tau
pre-mRNAs. After a longer exposure, the U1 snRNP-protected band was visible
in lanes 2 and 6 but not detectable in the lanes 1 and 5. (B) Histogram repre-
sentation of the ratio of protected tau pre-mRNA to digested products for
wild-type (WT) and mutant substrates. Error bars are derived from five inde-
FIG. 9. Tau DDPAC?14 intronic mutation increases U1 snRNP binding to
tau pre-mRNA. TauEx10?11d5wt (lanes 1 to 5) and -DDPAC?14 (lanes 6 to
10) RNAs were incubated with purified U1 snRNP, 0 ?l for lanes 1 and 6, 0.35
?l for lanes 2 and 7, 0.7 ?l for lanes 3 and 8, and 1.4 ?l for lanes 4 and 9, or with
depleted U1 snRNP (U1?) (lanes 5 and 10, 1.4 ?l of protein used). The RNA-U1
snRNP complexes were resolved in a nondenaturing 4% polyacrylamide gel. In
lanes 5 and 10, the U1 snRNP preparation was treated with oligonucleotide-
targeted RNase H cleavage as described above to inactivate U1 snRNA.
4044 JIANG ET AL.MOL. CELL. BIOL.
This stem-loop structure model could also explain the be-
havior of several other mutations found in a number of pa-
tients with tau exon 10 aberrant splicing, including ?3 and ?13
mutations (6, 23, 30, 63) in addition to the DDPAC?14 and
AusI?16 mutations (Fig. 10A). It is also consistent with the
observation that in the rat (or mouse), the predominant iso-
form of tau is the exon 10-containing isoform. Exon 10 splicing
may be enhanced in these species because of the destabiliza-
tion of the stem structure caused by the naturally occurring G
at position ?13 in the rat tau gene (G at both ?13 and ?16
positions in mouse; see Fig. 2); (23; Jiang and Wu, unpublished
data). Thus, a single-nucleotide change in this nonconserved
intronic region can have a significant impact on alternative
splicing of exon 10.
While this paper was in preparation, two studies were pub-
lished in which tau exon 10 alternative splicing was examined
more extensively using exon-trapping assays (9, 23). The study
by Grover and colleagues lends further support for the stem-
loop model. On the other hand, D’Souza and colleagues sug-
gested that the stem-loop structure was not supported by a
mutation at ?12 that should restore base-pairing in the stem
structure (9). However, results from compensatory-mutation
analyses have to be carefully interpreted. First, A-U base pair-
ing (in the ?12 compensatory mutant) is expected to be
weaker than G-C base-pairing (in the wild-type tau). Second,
multiple RNA-RNA and RNA-protein interactions are known
to influence splice site selection and splicing efficiency. The
stem-loop structure has to be viewed in the context of these
multiple interactions. It is likely that events other than the U1
snRNA-pre-mRNA interaction also play important roles in
regulating tau exon 10 splicing. In fact, we have found that
certain non-U1 snRNP splicing regulators affect tau exon 10
splicing, and we are currently characterizing the differential
recognition of the wild-type versus intronic mutant pre-
mRNAs by these splicing regulators (Jiang and Wu, unpub-
lished). The single-nucleotide changes around the 5? splice site
may have multiple effects on the RNA-RNA interactions (in-
tramolecular and intermolecular) as well as on RNA-protein
interactions. These effects may not be necessarily in the same
direction. For example, “compensatory mutations” for the
DDPAC?14 and AusI?16 mutants (at positions ?1 and ?2,
respectively) that “restore” the base-pairing interactions in the
stem will also affect U1 snRNP binding at the same time.
Therefore, the compensatory mutation strategy that we and
FIG. 10. Model explaining enhanced splicing of exon 10 in DDPAC?14 and AusI?16 intronic tau mutations via increased U1 snRNA interaction. In the wild-type
tau, a putative stem-loop structure forms around the 5? splice site of exon 10, as depicted in panel A. The base-pairing interaction between U1 snRNA and the 5? splice
site is shown in panel B. The stem-loop structure prevents efficient interaction of U1 snRNA with the 5? splice site and leads to partial skipping of exon 10. Such a
stem-loop structure is less stable in the presence of the DDPAC?14 and AusI?16 mutations (or other likely mutations, including those at positions ?2, ?3, and ?13)
because of reduced base-pairing interactions, resulting in increased recognition by U1 snRNP and therefore increased exon 10 splicing. The Watson-Crick base-pairing
interactions are depicted as thick black bars. The G-to-A mutations at the ?3 (MSTD) and ?2 (S305N) positions not only disrupt the base-pairing interactions in the
stem (A) but also lead to the formation of additional base-pairing interactions between the tau pre-mRNA and U1 snRNA (●●). Thus, it is possible that such mutations
at ?3 or ?2 positions have a more severe effect on exon 10 splicing.
VOL. 20, 2000 DEMENTIA MUTATIONS INCREASE U1 snRNP BINDING4045
other groups used may not be optimal for testing the stem-loop
model in the presence of the spliceosome.
The role of U1 snRNP in mammalian pre-mRNA splicing
has been well established (for reviews, see references 2, 36, 46,
and 52). It has been demonstrated that differential binding of
U1 snRNP could affect 5? splice site recognition in both yeast
and mammalian cells (10, 20–22, 39, 49, 57, 58, 66). It is worth
noting that these previous studies have all focused on either
the upstream exonic region or the intronic region at the U1
snRNP binding site less than 10 nucleotides from the splice
junction. Sequences further downstream in the intron have
been found to be less conserved (47, 56). Our results demon-
strate that a nonconserved intronic region outside the U1
snRNP binding site can also influence U1 snRNP binding via
formation of a secondary structure(s) that masks the U1
snRNP binding site. The formation of secondary structures
that potentially sequester 5? splice sites has been proposed as
a mechanism to regulate alternative 5? splice site selection (12,
13, 59, 60). However, there has been little direct biochemical
evidence demonstrating differential U1 snRNP binding in the
pre-mRNA with proposed secondary structures (4, 58). In
yeast cells, a systematic analysis to examine the effects of sec-
ondary structures on U1 snRNP binding using artificial hair-
pins to sequester the 5? splice site of the yeast RP51A intron
has been carried out (22). Pre-mRNAs containing hairpin
structures with longer than 9 consecutive base pairs began to
show a reduction in splicing in vivo, whereas structures with up
to 6 consecutive base pairs had little effect on splicing efficiency
(22). Our study demonstrates that even a single-nucleotide
change at position ?14 or ?16, which potentially disrupts one
base-pairing in a 6-bp stem involving the 5? splice site, leads to
a significant increase in splicing of tau exon 10 both in vitro and
in vivo. In addition, these mutations are located in a noncon-
served region of the intron. This is significant and prompts
revised strategies for identifying potentially important genes
for human diseases, since many have focused only on evolu-
tionarily conserved regions.
It should be pointed out that this secondary-structure model
is not inconsistent with the potential involvement of factors
other than U1 snRNP. It is possible that the intronic mutations
also disrupt the interaction of certain splicing-repressing fac-
tors (either protein or RNA). The potential involvement of
splicing factors other than U1 snRNP in differentiating wild-
type and intronic mutant tau pre-mRNAs was suggested by
several observations. In human brain tissues and in cells trans-
fected with tau minigenes, the increase in exon 10 splicing in
both AusI?16 and DDPAC?14 mutant tau pre-mRNAs, com-
pared with wild-type tau, appeared more dramatic than the
difference observed in the RNase H cleavage assay in the
absence of nuclear extract. In this study (Fig. 2) and in a
previous study (30), the ratio of Tau4R to Tau3R was in-
creased from 1 in wild-type tau to 3 or higher in AusI?16 tau
and 4 or higher in DDPAC?14 tau. SR proteins have been
shown to bind directly to 5? splice sites (69) and recruit and/or
stabilize the binding of the U1 snRNP to pre-mRNA (11, 34).
In our previous studies, we have demonstrated interaction
between SR proteins and U1 70K, a U1 snRNP protein (67),
and distinct functional activities of SR proteins in alternative 5?
splice site selection (68) as well as in alternative exon inclusion
(33). Recently, a yeast U1 snRNP protein, Nam8p, was shown
to interact with nonconserved intronic sequences and affect 5?
splice site selection (51). U5 PRP8 yeast and human homologs
have been shown to interact with 5? and 3? splice sites (53, 54,
64), although their role in regulating alternative splicing is not
yet clear. It is possible that some of these proteins, or other
novel or known alternative splicing regulators, also play a role
in tau alternative splicing.
Abnormal pre-mRNA splicing has been implicated in the
pathogenesis of a large number of human diseases, including
neurodegenerative disorders such as amyotrophic lateral scle-
rosis (42). Almost all splicing mutations reported in human
diseases either weaken recognition by spliceosomal snRNPs or
cause activation of cryptic splice sites, leading to exon skipping,
intron retention, or usage of cryptic splice sites (28, 48). The
FTDP-17-associated intronic mutations analyzed in this study
represent the first case in which single-nucleotide mutations
cause increased rather than decreased splicing of an alterna-
tively spliced exon, thereby altering the balance between dif-
ferent isoforms of normal gene products and leading to neu-
rodegeneration. Our study provides strong evidence that
enhanced U1 snRNP binding to a normal alternative splice
site, as a result of single-nucleotide mutations in the noncon-
served intronic region outside of the U1 snRNP binding site,
can be a pathogenic mechanism. Such aberrant splicing can
cause alteration in the delicate balance of different alternative
splicing products. Considering the size of introns compared
with exons and the complexity of alternative splicing regulation
in mammalian genes, it is likely that simple alterations in the
balance of different isoforms of critical genes as a result of
aberrant splicing could be a more important mechanism for
pathogenesis of human diseases than previously appreciated.
We thank A. Kramer, W.-Y. Tarn, and M. McNally for generous
gifts of purified U1 snRNP preparation and 2?-O-methyl-oligonucleo-
tides and Y. Rao, A. Strauss, and members of the Wu laboratory for
critical reading of the manuscript.
This work is supported by grants from the National Institute of
Health (RO1 GM53945/AG17518 to J.Y.W. and P50 AG05681 to
A.M.G.), by the Leukemia Society of America Scholarship to J.Y.W.,
by a postdoctoral fellowship from Natural Sciences and Engineering
Research Council of Canada to J.C., by NSADA to J.M.K., and by an
NIH career development award to A.M.G. (AG000634).
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