From the Mayo Clinic Jacksonville, Jacksonville,
Florida 32224, the § Division of Neuroscience, School of
Biological Sciences, University of Manchester, Manchester M13 9PT,
United Kingdom, and the ¶ Nathan Kline Institute,
Orangeburg, New York 10962
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ABSTRACT |
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Missense and splice site mutations in the
microtubule-associated protein tau gene were recently found
associated with fronto-temporal dementia and parkinsonism linked to
chromosome 17 (Poorkaj et al. (1998) Ann.
Neurol. 43, 815-825; Hutton et al. (1998)
Nature 393, 702-705; Spillantini et al. (1998)
Proc. Natl. Acad. Sci. U. S. A. 95, 7737-7741). The
mutations in the 5' splice site of exon 10 were shown to increase the
ratio of tau mRNAs containing exon 10 and thus the
proportion of Tau protein isoforms with 4 microtubule binding repeat
domains, although how this increase leads to neurodegeneration is
presently unclear. The mechanism by which these mutations increase
tau exon 10 splicing was not determined, although the
mutations were predicted to disrupt a potential stem-loop structure
that was likely involved in the regulation of exon 10 alternative
splicing. Here we describe in vitro splicing assays and RNA
structural analysis that demonstrate that the mutations do indeed act
through disruption of the stem-loop structure and that the stability of
this secondary structure feature at least partially determines the
ratio of tau exon 10+/ The microtubule-associated protein Tau plays an important role in
the polymerization and stabilization of neuronal microtubules (for
review, see Ref. 4). Tau is thus crucial to both maintenance of the
neuronal cytoskeleton and axonal transport (4). Abnormal intraneuronal
inclusions, termed neurofibrillary tangles, composed of Tau are a
feature of the pathology in several neurodegenerative conditions (5)
including Alzheimer's disease, Pick's disease, fronto-temporal
dementia (6), progressive supra-nuclear palsy, and the Lytico and Bodig
diseases of Guam.
The tau gene is localized to chromosome 17q21 (7) and
consists of 15 exons (8) of which 11 encode the six major Tau protein
isoforms in human brain. The six different Tau isoforms are generated
by alternative splicing of exons 2, 3, and 10 (9). Exons 9-12 encode
four microtubule-binding domains that are imperfect repeats of 31 or 32 residues (10). Alternative splicing of exon 10 gives rise to Tau
isoforms with 3 (exon 10 FTDP-17 (6) is inherited as an autosomal dominant condition
characterized clinically by behavioral, cognitive, and motor disturbance (many cases of this disease continue to be described clinically as "Pick's disease"). The age of onset is highly
variable but is usually 45-65 years. At autopsy, patients with FTDP-17 display pronounced fronto-temporal atrophy with neuronal cell loss,
gray and white matter gliosis, and superficial cortical spongiform
changes. In addition, virtually all FTDP-17 cases have abnormal
intraneuronal Tau inclusions, with glial Tau inclusions present in some
families (6, 12). The morphology and isoform composition of the Tau
filaments that compose the inclusions also varies in the different
families (12). The identification of different mutations in the
tau gene has largely explained the variability in Tau
pathology observed in FTDP-17 (2, 3, 13). Families with missense (2,
14) (P301L, N279K) or splice site mutations (2, 3) that affect exon 10, and thus 4 repeat Tau isoforms, have intraneuronal and glial
Tau inclusions consisting predominantly of four repeat Tau isoforms.
The Tau filaments in these cases have a longer periodicity than the
paired helical filaments that comprise the neurofibrillary tangles
observed in AD (12, 13). In contrast, families with missense mutations (G272V, V337M, and R406W) outside of exon 10 (1, 2), that affect all
Tau isoforms, have neuronal inclusions (glial inclusions are absent)
that are composed of all six Tau isoforms and are made up of filaments
identical to the paired helical filaments observed in AD (12, 13,
15).
All but one of the reported (1, 2, 14) missense mutations (G272V,
N279K, P301L, and V337M) associated with FTDP-17 occur
within the microtubule binding domains of Tau and four missense mutations (G272V, P301L, V337M, and R406W) have been demonstrated to
disrupt the interaction between Tau and the microtubules in vitro (16, 17). In contrast, the splice site mutations in FTDP-17
affect alternative splicing of exon 10 such that an increased proportion of Tau exon 10+ transcripts are generated; this leads to an
increase in Tau isoforms with four, as opposed to three, binding
repeats (2, 3). The mechanism by which this increase leads to
neurodegeneration and FTDP-17 is presently unclear; however, it
demonstrates that the ratio of four repeat to three repeat isoforms is
crucial to the correct functioning of Tau (2, 3). This is consistent
with the observation that alternative splicing of exon 10 is
developmentally regulated with three repeat Tau alone, in the absence
of four repeat Tau, present in fetal brain tissue in multiple species
(11, 18). The predominance of three repeat Tau during neuronal
development has clear significance for the likely function of these
isoforms in adult brain perhaps implying that human neurons require
three repeat Tau to maintain plasticity (4). In addition, the ratio of
four to three repeat Tau isoforms varies markedly in different species
with a slight predominance of three repeat Tau present in adult human
brain (11), while in adult mouse neurons only four repeat Tau is
observed (18). The ratio of Tau isoforms also varies in different
neuronal populations with the granule cells of the human dentate gyrus being reported to contain only three repeat Tau isoforms (11).
We previously employed RT-PCR analysis of FTDP-17 brains to demonstrate
that the 5' splice site mutations are associated in vivo
with a 2-6-fold increase in the ratio of exon 10+ to exon 10 In this study we again utilize in vitro exon trapping
(splicing) assays (2, 19) to test the hypothesis that the stability of
the potential stem-loop structure in the 5' splice site of tau exon 10 at least partially determines the ratio of
tau exon 10+/ Generation of Exon Trapping Constructs--
Mutant and wild-type
versions of tau exon 10 were amplified from the DNA of the
patients with the FTDP-17 +13, +14, and +16 splice site mutations
(residues numbered from the exon 10 5' splice site) and from an
unaffected individual. PCR products contained exon 10 and flanking
intron sequence at either end (Fig. 1).
PCR products were cloned into the splicing vector pSPL3b using
XhoI and PstI sites incorporated into the
amplification products. Mutant and wild-type constructs were identified
by sequence analysis. Site-directed mutagenesis was performed on these
constructs using the Transformer site-directed mutagenesis kit
(CLONTECH). Mutagenic primers for introducing the
+17(G/T)/+18(T/G) "extended stem" mutations were generated for the
wild-type construct and for each of the three FTDP-17 splice site
mutant (+13, +14, and +16) constructs. Also generated were mutagenic
primers for the mutations for the complementary rescue analysis (see
Figs. 2-5 for list of mutations). A selection primer was used in each
mutagenesis reaction to remove a single HpaI site from the
pSPL3b vector. For each reaction vector DNA was denatured at 100 °C,
mismatch and selection primers were annealed and T4 DNA polymerase, and
T4 DNA ligase were used to synthesize the second strand. Each mutant
was transformed into BMH 71-18 mutS Escherichia coli and the
plasmid DNA isolated using the Wizard miniprep kit (Promega). The DNA
was treated with HpaI as a primary selection process and
then re-transformed into DH10B. Colonies were screened by PCR and
restriction enzyme digestion, and the identity of each mutant was
confirmed by sequencing.
Wild-type and FTDP-17 mutant (+13, +14, and +16) exon trapping
constructs containing additional flanking intronic sequence (1 kb) on
either side of tau exon 10 were also generated in pSPL3b to
ensure that the short intronic sequences used in the original constructs (Fig. 1) were not artifactually affecting splicing assay
results. These constructs were generated by performing PCR on control
genomic DNA with primers (5'-CACCCTCGAGGGAAGACGTTCTCACTGATCTG-3' and
5'-GTGGCGGATCCATGGCTCCTTGCAACTTC-3') designed to more distant 5' and 3'
intronic sequences (GenBankTM). Amplification was performed
with the high fidelity PCR system (Boehringer Mannheim). The 2082-bp
PCR products were cloned into pSPL3b using XhoI and
BamHI sites incorporated into the amplification products and
sequenced. Mutagenesis was then performed, as described above, on the
wild-type extended splicing construct to introduce the FTDP-17 exon 10 5' splice site mutations (+13, +14, and +16). These constructs were
analyzed by exon trapping in identical fashion to the original wild
type and mutant pSPL3b constructs (see below).
Exon Trapping--
For splicing assays, the "exon trapping
system" from Life Technologies, Inc. was used. Briefly, COS-7 cells
were transfected in triplicate with 1 µg of each construct using
LipofectACE (Life Technologies, Inc.). Cells were collected 24 h
post-transfection and RNA prepared using Trizol reagent (Life
Technologies, Inc.). First-strand synthesis was performed using
reagents supplied with the system, using the conditions described in
the manufacturer's instructions. PCR was performed using the primers
SD2 and SA4 (the sequence for these primers was taken from the exon
trapping system) and Taq Gold (Perkin-Elmer). To enable
quantification of PCR products on an automated sequencer, the SD2
primer was labeled with a TET (Applied Biosystems) fluorescent dye tag.
An initial denaturation stage of 5 min at 94 °C was followed by six stage 1 cycles (94 °C for 1 min, 60 °C for 1 min, 72 °C for 5 min), 24 stage 2 cycles (94 °C for 1 min, 60 °C for 1 min,
72 °C for 4 min), and a final extension phase of 72 °C for 10 min. Performing PCR in a single stage for 30 cycles with shorter
extension times (45 s at 72 °C) did not significantly alter the
results. To verify that the RT-PCR was quantitative, the total number
of amplification cycles was varied (24-39 cycles, data not shown). PCR
products were visualized on 2% agarose gels; exon 10+ products were
246 bp, while exon 10 Sequencing of the 5' Splice Site in tau Exon 10 from Different
Species--
Analysis was performed on genomic DNA from rhesus,
marmoset, bovine, rabbit, rat, and mouse. PCR was performed between
tau exon 10 to exon 11 using the Expand Long Template PCR
system (Roche Molecular Biochemicals) with primers (10F,
5'-GATCTTAGCAACGTCCAGTCCAAGTG-3' and 11R,
5'-GTTGCCTAATGAGCCACACTTGGAGGTC-3') generated from conserved exonic
sequence. For the PCR, an initial denaturation stage of 92.5 °C for
2 min was followed by 10 stage 1 cycles (92.5 °C for 10 s,
54 °C for 30 s, 68 °C for 12 min), 20 stage 2 cycles
(92.5 °C for 10 s, 54 °C for 30 s, 68 °C for 12 min
increasing by 20 s each cycle), and a final extension phase of 10 min at 72 °C. Sequencing of the PCR product was performed after
agarose gel purification using the Big Dye terminator cycle sequencing
kit (Perkin-Elmer). Sequencing reactions were analyzed on an Applied Biosystems 377 automated sequencer running Factura software
(Perkin-Elmer). Sequences of the tau exon 10 5' splice site
were aligned by the Sequence Navigator Package (Perkin-Elmer).
RT-PCR Analysis of Exon 10 Alternative Splicing in Different
Species--
RNA was isolated from human frontal lobe and from rabbit,
rat, and mouse whole brain using the Trizol reagent and protocol (Life
Technologies, Inc.). Reverse transcription was performed using the
Superscript pre-amplification system (Life Technologies, Inc.) on 1-4
µg of brain RNA with an oligo(dT) primer. PCR was performed using
primers designed to exonic sequence that was conserved in each species
from exon 9 (forward, 5'-CTGAAGCACCAGCCAGGAGG-3') and exon 13 (reverse,
5'-TGGTCTGTCTTGGCTTTGGC-3'). Amplification involved 30 cycles of
94 °C for 30 s, 65-50 °C for 30 s, and 72 °C for
45 s with a final 72 °C extension phase for 10 min. We demonstrated previously that these conditions were quantitative by
performing this amplification using a range of PCR cycles (18-37) (2).
PCR products were visualized on 2% agarose gels with exon 10+ RT-PCR
products being 367 bp, while exon 10 Human Genomic tau Transgenic Mouse Generation and
Analysis--
Transgenic mice expressing the entire normal human
tau gene under the control of the human tau
promoter were generated through micro-injection of a tau
containing PAC (P1-derived artificial chromosome) (24i13, ~200 kb)
into mouse embryos. RNA was isolated from hemi-brains from transgenic
and littermate control animals, from two different lines, using the
Trizol reagent and protocol (Life Technologies, Inc.). For analysis of
exon 10 alternative splicing in mouse endogenous and human
transgene-derived tau mRNA, RT-PCR was performed between
exon 9 and exon 11 with mouse-specific (9F, 5'-CACCAAAATCCGGAGAACGA-3'
and 11R, 5'-CTTTGCTCAGGTCCACCGGC-3') and human specific primers (9F,
5'-CTCCAAAATCAGGGGATCGC-3' and 11R, 5'-CCTTGCTCAGGTCAACTGGT-3'). PCR
was performed (30 cycles of 94 °C for 30 s, 62 °C for
30 s and 72 °C for 45 s with a final 72 °C extension
phase for 10 min). Mouse-specific and human-specific PCRs were analyzed
on agarose gels; products corresponding to exon 10+ tau
mRNA gave a band at 390 bp, while products corresponding to exon
10 RNA Structural Mapping and Gel Migration Analysis--
Wild-type
and mutant (+8, +13, +14, +16, and 10-bp stem) constructs containing
tau exon 10 and flanking intronic sequence (Fig. 1) were
cloned into pBluescript(KS
To further demonstrate the effect of the FTDP-17 splice site mutants on
RNA secondary structure, the variable migration of in vitro
transcribed wild-type and mutant (+13, +14, +16, and 10-bp extended
stem) RNA transcripts was analyzed on both nondenaturing 4% Metaphor
agarose (FMC) gels and on denaturing 4% agarose/formaldehyde gels.
In vitro transcription was performed as described above. 1 µg of each RNA was loaded onto each gel. Nondenaturing 4% Metaphor agarose gels were run at 100 V in TAE buffer (Tris/acetic acid/EDTA). Denaturing 4% agarose/formaldehyde gels were run at 100 V in 1 × MOPS buffer, pH 7.0 (20 mM MOPS, 5 mM sodium
acetate, 1 mM EDTA), samples were preheated to 55 °C for
10 min in 50% formamide, 6.5% formaldehyde 1 × MOPS loading
buffer prior to loading.
Mutations That Disrupt the Predicted Stem-Loop Increase the
Incorporation tau Exon 10--
To demonstrate that mutations that
disrupt the predicted stem-loop in the 5' splice site increase splicing
of tau exon 10 in vitro a series of mutations
were introduced into exon trapping constructs. Mutations were made at
positions
The results of these experiments (Fig. 2) demonstrated that each of the
FTDP-17-associated splice mutants (+3, +13, +14, and +16) significantly
increased the incorporation of tau exon 10 into the
artificial transcripts generated by the exon trapping assay, compared
with the wild-type constructs consistent with previous reports of the
effects of these mutants in this in vitro system (2). In
addition artificial mutants at
In contrast, mutations at +8 and +9 had little impact on exon 10 splicing in this assay relative to the wild-type construct (Fig. 2).
This was expected since these mutants occur within the loop region and
therefore are not predicted to affect the stability of the stem-loop
structure. The small increase in splicing observed with the +8 mutation
probably reflects the small increase in U1 snRNP binding stability to
the 5' splice site that is predicted to be generated by this change.
Taken together the results from this series of experiments are entirely
consistent with the hypothesis that the predicted stem-loop structure
at the 5' splice site plays a major role in regulating the alternative
splicing of tau exon 10 in vitro. However the
precise ratio of exon 10+ to 10 Increasing the Stability of the Predicted Stem-Loop Reduces the
Incorporation tau Exon 10--
To further test the hypothesis that the
stability of the predicted stem-loop at least partially determines the
ratio of exon 10+ to exon 10
As before, each of the FTDP-17-associated splice mutants significantly
increased the incorporation of tau exon 10 into the artificial transcripts generated by the exon trapping assay, compared with the wild-type constructs. This increase was present regardless of
whether comparisons were between wild-type and mutant constructs with
the normal 6 bp or with the extended 10 bp stem-loop structure (Fig.
3). The mutation with the greatest effect on splicing in both the 6- and 10-bp constructs was the +14 mutation (observed in the
disinhibition dementia parkinsonism amyotrophy complex family (25)),
which also causes the greatest loss in stem-loop stability (2) of the
three mutations tested.
Increasing the length of the stem from 6 to 10 bp, and thereby the
stability of the stem-loop structures, consistently resulted in a
dramatic reduction in the ratio of exon 10+ to exon 10 Rescue Analysis of Stem-Loop Mutations by Alteration of
Complementary Bases in the 5' Splice Site of Exon 10--
Analysis of
the stem-loop structure was also performed by mutating a residue on one
side of the stem and then attempting to rescue the observed increase in
splicing by altering the corresponding residue on the opposite side of
the stem such that base pairing is restored (Fig.
4). However this analysis is complicated
by the short length of the stem-loop (6 bp) as all of the residues on
one side of the stem occur within the U1 snRNP binding site. As a
result several of the residues could not be mutated without causing the
complete loss of exon 10 splicing (+1, +2, +3 (G to C), and +4, Fig.
5). In addition, other mutations are
predicted to cause an increase in U1 snRNP binding, which in turn leads to an increase in splicing independent of stem-loop stability (
However, rescue analysis was performed using constructs with artificial
mutations at positions
As predicted the artificial mutations at
Exon trapping analysis with the Alteration of Complementary Bases Does Not Rescue the Affects of
FTDP-17 Splice Mutants at Positions +16 and +3--
Rescue analysis
was also attempted with constructs designed to restabilize FTDP-17
splice site mutants at +16 (C to T) (2) and +3 (G to A) (3). Similar
analysis could not be performed for FTDP-17 splice mutations at +13 (2)
and +14 (2), since altering the residue on the opposite side of the
stem-loop, to each mutant, would result in the loss of the +1/+2 GT
sequence that is minimally required in a 5' splice site. This in turn
would have resulted in an uninterpretable loss of exon 10 splicing.
Rescue constructs for the +16 (C to T) and +3 (G to A) FTDP-17 mutants
were generated by altering the equivalent residue ( Analysis of the tau Exon 10 5' Splice Site in Other Species
Confirms the Role of Pre-mRNA Secondary Structure in the Regulation
of Alternative Splicing of This Exon--
The results from the exon
trapping analysis demonstrate that the effect of the FTDP-17-associated
5' splice site mutations on the alternative splicing of tau
exon 10 is mediated through destabilization of the predicted stem-loop
structure. However exon trapping is an artificial system that does not
fully replicate the alternative splicing of tau exon 10 in vivo. Therefore to determine if the predicted stem-loop
structure was also likely to play a role in the regulation of
tau exon 10 alternative splicing in vivo, we
examined the sequence of the 5' splice site in a range of other
mammalian species (human, rhesus, marmoset, bovine, rabbit, rat, and mouse).
The sequence of the 5' splice site in primates (human, rhesus, and
marmoset) and bovine had an identical predicted stem-loop structure
with a 6-bp stem and a 6-base loop region (Fig.
6). In contrast in each of the rodent
species (rabbit, rat, and mouse) some part of the sequence that makes
up this structure was absent (Fig. 6), resulting in a predicted
stem-loop of reduced stability. In the rabbit the +11 residue was not
conserved (C in rabbit, U in human), resulting in a predicted 5-bp stem
and 7-base loop, which mimics the artificial +11 (T to C) mutation
employed in exon trapping studies. In the rat residue +13 is not
conserved (G in rat and A in human), which mimics the +13 (A to G)
FTDP-17 mutation, and results in a 6-bp stem with an internal mismatch (G-U). In the mouse both the +13 and the +16 residues are not conserved. Thus the order of predicted stem-loop stability in the
different mammalian species analyzed is primates/bovine > rabbit > rat > mouse (Fig. 6). In order to relate the
stability of the stem-loop in the 5' splice site to the alternative
splicing of exon 10, we performed RT-PCR analysis on RNA isolated from the brains of the different species (human frontal lobe, rabbit, rat,
and mouse). The results of this analysis demonstrated clearly (Fig. 6)
that there was an inverse relationship between the predicted stability
of the stem-loop in the sequence of the splice site in each species and
the ratio of tau exon 10+/
In the mouse, tau exon 10 Transgenic Mice Expressing the Human tau Gene Demonstrate
Alternative Splicing of Exon 10 Consistent with the Involvement of
Cis-acting Elements in Regulation of This Splice
Event--
Alternative splicing of exon 10 was investigated in the
brains of transgenic mice expressing a human PAC (P1-derived artificial chromosome) (~200 kb) transgene containing the entire tau
gene. RT-PCR analysis with human and mouse tau-specific
primers was performed on RNA isolated from the brains of adult
transgenic and littermate control animals. This analysis (Fig.
6C) demonstrated that splicing of tau exon 10 in
the endogenous mouse gene was unaffected in the transgenic animals
(only exon 10+ mouse tau RNA was observed). In contrast,
with human-specific primers products corresponding to both exon 10+ and
exon 10
The fact that the human tau transgene generates pre-mRNA
that undergoes alternative splicing of exon 10 similar to that seen in
the human brain (although the exon 10+/ RNA Structural Mapping Confirms the Presence of a Stem-Loop
Structure in the 5' Splice Site of tau Exon 10 That Is Disrupted by the
FTDP-17 Splice Site Mutations--
RNA secondary structure prediction
analysis with the program RNAFOLD (28) suggested two possible
structures of similar stability for the region around the 5' splice
site of exon 10 (Fig. 7). In both
structures the minimal 6-bp stem-loop is maintained, however, beyond
this region it is possible for either a short second stem to form
(structure 1) or a lateral stem-loop (structure 2). The structures are
of similar stability, because they are both formed with an identical
9-base sequence (ACACGUCCC) that is repeated at virtually equidistant
positions from the 5' splice site (
Initial characterization of the effects of the FTDP-17 splice site
mutants on the secondary structure of tau pre-mRNA was performed by examining the migration of in vitro transcribed
RNAs containing exon 10 and flanking intronic sequences (247 bases) on
denaturing and nondenaturing agarose gels (Fig.
8A). RNAs containing wild-type
exon 10, the +8, +13, +14, +16 splice site mutants and the extended (10 bp) stem-loop (Fig. 3) were analyzed. All five in vitro
transcribed RNAs ran as single bands with identical migration on
denaturing (formaldehyde/agarose) gels consistent with each RNA being
the same size (247 bases). In contrast, migration on nondenaturing
agarose gels differed significantly between different RNAs (Fig.
8A). The wild-type and +8 mutant RNAs gave essentially identical migration patterns consistent with the +8 mutation occurring in the predicted loop region and therefore not significantly affecting the stability of the stem-loop. The wild-type and +8 RNAs migrated as a
doublet band corresponding to one major product and to one slower minor
product, suggesting that these RNAs exist in at least two
confirmations. This is consistent with the prediction of the RNAFOLD
analysis that there are two possible secondary structures for this
region with similar stability (Fig. 7). In contrast, the FTDP-17 splice
mutant (+13, +14, and +16) RNAs, which are predicted to disrupt the
stem-loop, were observed as a single major band with faster migration
than the wild-type RNA. This is consistent with these mutations
altering RNA secondary structure presumably resulting in a more
flexible molecule. Weaker minor products in the FTDP-17 mutant RNAs
(+13, +14, and +16) are also visible that appear to co-migrate with the
wild-type RNA major product, suggesting that a proportion of these
mutant RNA molecules maintain the wild-type secondary structure (Fig.
8A). Interestingly these minor products are strongest with
the FTDP-17 +16 mutation, which also produces the smallest increase in
exon 10+ to 10
To further investigate the likely pre-RNA secondary structure around
tau exon 10, we performed mapping analysis with RNase enzymes that recognize double-stranded (V1 RNase) and single stranded (T1 RNase) RNA (20). The sites of cleavage were mapped by primer extension with an oligonucleotide complementary to a sequence downstream of the region to be studied (see "Experimental
Procedures"). In vitro transcribed RNAs containing exon 10 (wild-type, +14 mutant, 10-bp stem-loop mutant) were subject to this
analysis (Fig. 8B).
The extended 10-bp stem RNA gave the strongest signals for V1
(double-stranded) digestion in regions predicted to be double-stranded in the extended 10-bp stem-loop (Fig. 8B). The V1 digestion
was strongest at residues (
The results from the secondary structure mapping analysis are not
sufficiently clear to enable a prediction of the precise structure of
the stem-loop. Indeed results from RNAFOLD predictions and gel
migration analyses would suggest that there may be multiple (at least
two) confirmations formed in this region. However the mapping results
from different RNAs, wild-type and mutant (+14 and 10-bp stem), are
clearly consistent with the presence of a stem-loop, which is disrupted
by the FTDP-17 +14 mutation. This conclusion is further supported by
the gel migration studies of different tau exon 10 wild-type
and mutant RNAs (described above).
Concluding Remarks--
Our results demonstrate that alternative
splicing of tau exon 10 (in humans and other mammals) is at
least partially regulated by a stem-loop structure that forms in the
pre-mRNA at the 5' splice site. In addition, the ratio of exon
10+/
The 5' splice site mutations that are associated with FTDP-17 (2, 3)
act by disrupting this stem-loop, which leads to increased
incorporation of exon 10 in tau mRNA. This in turn leads to an increase in the proportion of Tau isoforms with four
microtubule-binding domains, although how this leads to
neurodegeneration is yet to be determined. The identification of splice
site mutations in tau associated with FTDP-17 has
demonstrated the significance of Tau isoform composition to neuronal
function. In addition, these mutations in tau are the first
shown to cause human disease through disruption of pre-mRNA
secondary structure that regulates alternative splicing.
transcripts. In addition, we
provide evidence that the stability of the stem-loop structure
underlies the alternative splicing of this exon in other species.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
) or 4 (exon 10+) microtubule binding domains
(11). The recent identification of four missense (1, 2) and four splice
site mutations (2, 3) in the tau gene, associated with
fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17)1 (6), has
demonstrated that Tau dysfunction can lead to neuronal cell death and
is not simply a secondary consequence of neurodegenerative disease.
tau mRNA (2). In addition, we utilized an exon trapping protocol (2, 19) as a splicing assay to demonstrate in vitro that the 5' splice site mutations were also capable of increasing the
incorporation of tau exon 10 into artificial transcripts. However while both RT-PCR analysis of FTDP-17 brains and splicing assays demonstrated that the 5' splice site mutations act by increasing the incorporation of exon 10 into tau mRNAs, neither
method indicated the mechanism by which the mutations affected splicing
(2). Examination of the intronic sequence downstream of exon 10 revealed that each of the mutations was predicted to disrupt a
potential stem-loop structure that was likely involved in the
regulation of exon 10 alternative splicing by competing with the U1
snRNP for binding to the 5' splice site (2). Stem loop structures have
previously been implicated in regulating the selection of alternative
5' splice sites (20) and distant branch points (21) and also in the
tissue-specific splicing of the chicken
-tropomyosin exons 6A and 6B
(22, 23).
transcripts and that the splice site
mutations act by disrupting this structure. We also examined the
sequence of the exon 10 5' splice site in the tau gene from
bovine, rabbit, rat, mouse, and two other primates to determine if this
pre-mRNA structure might regulate alternative splicing in other
mammals. Finally, we utilize RNA secondary structure analysis to
demonstrate that a stem-loop is present in the normal 5' splice site of
tau exon 10 and that this structure is disrupted by the
FTDP-17 splice site mutations.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Exon trapping assay constructs.
A, design of the basic exon trapping construct is
shown with tau exon 10 and flanking intronic sequences
cloned into the XhoI and PstI sites in the pSPL3b
splicing vector. The cloned tau sequence is flanked by
artificial introns (311 and 2039 bp) from the human immunodeficiency
virus tat gene. RT-PCR primers SD2 and SA4 were designed to
the human immunodeficiency virus tat exons that are
incorporated into the artificial mRNAs. Mutagenesis was performed
on the basic constructs as required for each experiment. Extended exon
10 constructs (not shown) containing ~1 kilobase pair of flanking 5'
and 3' intronic sequence were also generated in pSPL3b to ensure that
similar exon trapping results were obtained when additional intronic
sequence was included. B, schematic splicing diagrams
displaying the RT-PCR product sizes produced by mRNAs containing
tau exon 10 (246 bp) and mRNAs with tau exon
10 skipped (153 bp).
products were 153 bp. Quantification of
products and estimation of the molar ratio of exon 10+ and exon 10
mRNA was performed using an Applied Biosystems 377 automated sequencer running Genescan software. Triplicate independent
transfections were used to determine the mean exon 10+/
ratio and
S.D. for each experiment. The identity of RT-PCR products (exon 10+/
) was confirmed by sequencing.
products were 274 bp.
mRNA gave a band at 297 bp). A complete description of the
generation and neuropathological analysis of these human tau
transgenic mice will be published
separately.2
) using XhoI and
PstI. The plasmid DNA was linearized with BamHI
to generate a template for in vitro transcription with T3
RNA polymerase. In vitro transcription was performed using
the Ribomax system (Promega) according to manufacturer's instructions.
RNase digests were performed using 4 µg of RNA and 0.08 unit of V1
double strand-specific RNase (Amersham Pharmacia Biotech) or 0.5 unit
of T1 single strand-specific RNase (Ambion) for 15 min at 25 °C in a
25-µl reaction, using the manufacturers' recommended buffers.
Control samples for each RNA were treated identically, to the RNase
digests, but enzyme was omitted. Samples were then immediately purified
by phenol/chloroform extraction and resuspended in 15 µl of diethyl
pyrocarbonate-treated water. RNA recovery was assessed UV absorption at
260 nM. Primer extension was performed on 1 pmol of
digested RNA using 50,000 cpm of 32P-labeled
oligonucleotide (5'-CCGGGCTGCAGACACCACTTCC-3') and Superscript II
reverse transcriptase (Life Technologies, Inc.) in a 20-µl reaction
according to manufacturer's instructions. The RNA template was
digested with RNase H (Life Technologies, Inc.), and the resultant primer extension products were isopropyl alcohol precipitated, washed
in 70% ethanol, and resuspended in 5:1 formamide/blue dye loading mix.
Sequencing reactions of the wild-type construct were performed using
33P-labeled terminator sequencing system (Amersham
Pharmacia Biotech) and the same, unlabeled, oligonucleotide as was used
for the primer extension reactions. Primer extension samples were run
on a 6% polyacrylamide sequencing gel alongside the sequencing
reactions and the results visualized by autoradiography. Approximate
equal gel loading was verified by comparison of bands generated from full-length cDNA products from control reactions.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2,
1, +3, +11, +13, +14, and +16 (Fig.
2) relative to the 5' splice site that are predicted to lie within the stem, while mutations at +8 and +9
affected loop residues that were not expected to affect the stability
of the stem-loop. Mutations at +3 (G to A), +13 (A to G), +14 (C to T),
and +16 (C to T) were those that had been shown previously to increase
splicing of exon 10 in vivo and to be pathogenic in FTDP-17
(2, 3). Exon trapping transfections were performed in triplicate on
each of the mutant constructs as well as on the wild-type
construct.
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Fig. 2.
Exon trapping analysis of mutations in the
stem and loop regions of the 5' splice site.
A, proposed stem-loop structure shown with the
location of mutations used in exon trapping analysis (+3, +13, +14, +16
mutations were observed in FTDP-17 families (2, 3)).
B, the results of exon trapping analysis shows
that mutations which are predicted to disrupt the stem-loop structure
( 2,
1, +3, +11, +13, +14, +16) cause increased exon 10+ (246-bp
product) to exon 10
(153 bp) RNA ratios. In contrast, mutations in
the loop region (+8 and +9) are not predicted to affect the stability
of the stem-loop and do not alter exon 10 splicing. Molar ratios and
S.D. values (shown beneath gel lanes) were calculated from three
independent transfections. Qualitatively identical results were
obtained with wild-type and mutant (+13, +14, +16) tau exon
10 constructs containing ~1 kb of 5' and 3' flanking intronic
sequence (not shown).
2 (G to A),
1 (T to C), and +11 (T
to C), which are predicted to disrupt the stem-loop, also increased
splicing of exon 10 relative to the wild-type construct. Mutations at
2 and +3 are also predicted to increase the stability of U1 snRNP
binding to the 5' splice site (Fig. 5), which has also been shown to
increase incorporation of an alternatively spliced exon (24). The
result of this double effect on the splicing mechanism is that these
mutations (+2 and +3) cause the greatest increase in exon 10 incorporation of the mutations tested (Fig. 2, ratios >10). The +3 (G
to A) FTDP-17 splice site mutation (3) is thus predicted to increase U1
snRNP binding as well as disrupt the stem-loop structure, suggesting that both factors are likely to play a role in the mechanism of this
pathogenic mutation.
RNA is likely to be affected by other
factors such as the stability of U1 snRNP binding to the 5' splice
site. Indeed it is significant that mutations that were predicted to
reduce U1 snRNP base pairing at positions +3 (G to C), +4 (A to G), and
+5 (G to C) resulted in the total loss of exon 10 splicing (Fig. 5).
These results, while uninterpretable in terms of demonstrating the role
of the stem-loop, do indicate the minimal U1 snRNP binding stability required for this splice site to remain functional in
vitro.
tau mRNAs, we generated
wild-type and mutant (+13, +14, and +16) constructs in which residues
+17/+18 downstream of the exon 10 5' splice site were converted from GT
to TG by site-directed mutagenesis (Fig.
3). This had the effect of increasing the
length of the "stem" from 6 to 10 bp (although the exact structure of the stem-loop has yet to be determined), since residues +19 and +20
also pair with
5 and
6 (Fig. 3), significantly increasing the
stability of the potential stem-loop structure in the 5' splice site
sequence. Exon trapping was performed in triplicate on each of the 4 constructs (wild-type and +13, +14, and +16 splice site mutants) with
the extended (10 bp) stem-loop sequence as well as on wild-type and
mutant constructs with the normal (6 bp) stem-loop (Fig. 3).
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Fig. 3.
Tau exon 10 alternative splicing in
vitro is regulated by the stability of the stem-loop.
A, normal (6 bp) and extended (10 bp) stem-loops
are shown with the location of FTDP-17 splice site mutations (+13, +14,
and +16). The 10-bp stem-loop was generated by mutagenesis of residues
+17/+18 (shaded box). B, the results
of exon trapping analysis with the 6- and 10-bp stem-loop constructs
demonstrated that increasing the stability of the stem-loop resulted in
a reduction in the ratio of exon 10+ (246-bp product) to exon 10
(153-bp product) mRNAs in wild-type (WT) and mutants
(+13, +14, and +16). Note that the mutants (+13, +14, and +16)
consistently increased the proportion of exon 10+ mRNA compared
with wild-type (WT) regardless of whether comparisons were
made among 6- or 10-bp stem-loop constructs. The +14 mutant gives the
largest increase in exon 10+/
ratio consistent with this mutation
producing the greatest reduction in stem-loop stability (2). Molar
ratios and S.D. values (shown beneath gel lanes) were calculated from
three independent transfections.
transcripts, resulting from increased skipping of exon 10. This effect was observed
in both the wild-type construct and in each of the three FTDP-17 mutant
constructs (Fig. 3). Thus increasing the stability of the stem-loop in
this manner reduced the incorporation of exon 10 consistent with the
stem-loop structure regulating alternative splicing of this exon. The
observed reduction in the incorporation of exon 10 with the extended
stem (10 bp) constructs effectively rescues the effect of the FTDP-17
mutations by restabilizing the disrupted stem-loop. However as
mentioned earlier the FTDP-17 mutants even with the 10 bp stem
continued to have a higher exon 10+/
mRNA ratio than observed in
the equivalent 10-bp stem wild-type construct (Fig. 3). Again this
result is consistent with the hypothesis that the overall stability of
the stem-loop determines the proportion of transcripts into which exon
10 is incorporated.
2 (G
to A), +3 (A to G), Fig. 5).
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Fig. 4.
Exon trapping analysis of artificial splice
site mutants ( 1, +16) rescued through alteration of the opposing
residue (+15,
2). A, stem-loop showing
artificial splice site mutants (
1, +16) and opposing bases (+15,
2)
that are altered to generate rescue constructs. Note the generation of
three consecutive G-C pairs in the
1/+15 rescue construct stem-loop.
B, exon trapping analysis (exon 10+ = 246 bp,
exon 10
= 153 bp). Artificial
1 and +16 mutations increase exon 10 splicing compared with wild-type (WT), and this effect is
rescued when the stem-loop is restabilized by the opposing +15 and
2
mutations, respectively (
1/+15, +16/
2). The
1/+15 rescue
construct gives the lowest observed exon 10+/
ratio (0.44), which
presumably reflects the generation of a more stable stem-loop (compared
with wild-type in the
1/+15 construct that contains three consecutive
G-C pairs over the 5' splice site. Molar ratios and S.D. values (shown
beneath gel lanes) were calculated from three independent
transfections.
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Fig. 5.
Rescue analysis of FTDP-17 splice mutants
(+16 and +3) and effects of altering U1 snRNP binding site on
splicing. A, stem-loop showing FTDP-17
splice site mutants (+3 (G to A), +16) and opposing bases (+12, 2)
that are altered to generate rescue constructs.
B, predicted U1 snRNP binding to the
tau exon 10 5' splice site and the effect of
2, +3 (G to
A), +3 (G to C), +4, +5, +8 mutations. Note that mutations at +3 (G to
C), +4 (A to G), and +5 (G to C), which are predicted to reduce U1
snRNP base pairing to the 5' splice site, resulted in the complete loss
of exon 10 splicing (data not shown) C, exon
trapping analysis (exon 10+ = 246 bp, exon 10
= 153 bp). The FTDP-17
mutations +16 and +3 (G to A) increase exon 10 splicing compared with
wild-type (WT); however, splicing is not reduced by
alteration of the corresponding residue
2 and +12, respectively, that
restores base pairing at each site. This likely reflects incomplete
restabilization of the stem-loop in the rescue constructs (+16/
2 and
+3/+12) as a G-C pair is replaced with A-T and increased U1 snRNP base
pairing in the +3 and +16/
2 constructs. Molar ratios and standard
deviations (shown below gel lanes) were calculated from three
independent transfections.
1 (U to C) and +16 (C to G) with rescue
constructs where the opposing residues were altered, +15 (A to G),
2
(G to C), such that the stem-loop in each case is restabilized (Fig.
4). The
1 and +16 mutations are predicted to reduce the stability of
the stem-loop while the
1/+15 and +16/
2 rescue constructs generate
G-C base pairs that restore stem-loop stability. The
1/+15 stem-loop
is more stable than in the wild-type construct, since an A-T pair is
replaced with a G-C pair; this results in a group of three consecutive
G-C pairs.
1 and +16 resulted in an
increase in the proportion of exon 10+ mRNA (ratios 3.16 and 1.55, respectively), consistent with destabilization of the stem-loop (Fig.
4). The
1/+15 rescue construct reduced exon 10 splicing to levels
significantly lower (ratio 0.44) than those observed with wild-type
constructs (ratio 0.82). This reflects restabilization of the stem-loop
by the opposing +15 mutation, which gives a stem-loop that is more
stable than with the wild-type sequence (Fig. 4). The exceptionally low
exon 10+/
ratio observed with the
1/+15 rescue (it was the lowest
ratio observed where splicing was not abolished) probably reflected the
generation of three consecutive G-C base pairs, over the splice site,
which would be expected to significantly increase stem-loop stability. The +16/
2 rescue construct also reduced splicing significantly (ratio
1.32) relative to the +16 mutant construct (ratio 1.55) consistent with
restabilization of the stem-loop. However the rescue construct still
gave a higher exon 10+/
ratio than wild-type (0.82). The reason for
this difference is unclear although it may reflect a subtle effect on
either stem-loop stability, U1 snRNP binding or another part of the
splicing process produced by replacing a purine residue with a
pyrimidine (G to C) at position
2.
1, +16 mutants, and
1/+15, +16/
2
rescues (Fig. 4) yielded results that are consistent with data from the
extended (10 bp) stem-loop constructs (Fig. 3). Together these studies
demonstrate that the stability of the predicted stem-loop in the 5'
splice site has a major influence on the alternative splicing of
tau exon 10 in vitro.
2 and +12,
respectively) on the opposite side of the stem-loop (
2, G to A; +12,
C to U) to restore the base pairing at this position (Fig. 5). However
in both rescue constructs the predicted stem-loop structure is
significantly less stable than in the wild-type splice site, since a
G-C pair is replaced with A-U with the result that in both rescues
consecutive A-U base pairs are created in the stem-loop. The
anticipated result was that the two rescue constructs would show
reduced incorporation of exon 10 relative to the +16 and +3 mutants
alone. However, neither rescue construct significantly reduced the
splicing of exon 10, compared with the +3 and +16 mutant constructs,
and indeed the
2/+16 construct displayed a small (1.8-fold), but
significant, increase in the proportion of exon 10+ mRNA (Fig. 5).
The explanation for these results is likely to be a combination of two
factors: first both of the complementary "rescue" constructs have
stem-loops that are significantly less stable than in the wild-type
construct, with a G-C pair replaced with A-T; second the +3 FTDP-17
mutant and the
2/+16 rescue (to the FTDP-17 +16 mutant) are both
predicted to increase the base pairing of the 5' splice site with the
U1 snRNP (Fig. 5). Increased binding of the U1 snRNP to the 5' splice
site would be expected to increase splicing of exon 10 (24),
independent of stem-loop stability. In both cases, the overall result
is that the restoration of base pairing in the rescue constructs
(+3/+12 and
2/+16) is insufficient to significantly reduce exon 10 splicing compared with the FTDP-17 mutants (+3 and +16).
mRNAs, thus the order of
different species in exon 10+/
ratio was: mouse > rat > rabbit > human frontal lobe (bovine and other primates not
tested). The exon 10+/
ratios observed in the human, rabbit, and rat
brain RNA by RT-PCR are also highly similar to the ratios obtained
through in vitro exon trapping studies performed with
equivalent tau exon 10 constructs: wild-type, +11 (T to C)
and +13 (A to G), respectively (Fig. 2). The results are consistent
with the predicted stem-loop structure in the 5' splice site of exon 10 regulating the alternative splicing of this exon in multiple species
and that the stability of this structure at least partially determines
the proportion of exon 10+ mRNA. It should be noted, however, that
other factors in addition to the stem-loop must also be involved in the
regulation of exon 10 alternative splicing, since only three repeat Tau
(exon 10
) is observed in the mammalian fetal brain (18). One possible explanation of this phenomenon is that an inhibitory splicing factor is
present in fetal brain that is absent in the majority of adult neurons;
previous studies have also suggested that thyroid hormone
expression regulates the generation of four repeat Tau during brain
development (26).
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Fig. 6.
Analysis of tau exon 10 alternative splicing in other mammals and transgenic mice.
A, aligned 5' splice site sequences.
Boxes denote the location of the stem-loop with nonconserved
residues, in the rodents, excluded. B, RT-PCR
analysis of tau exon 10 alternative splicing in human
frontal lobe and rabbit, rat, and mouse whole brain RNA. PCR was
between exon 9 and exon 11 (exon 10+ products are 367 bp and exon 10
products are 274 bp), and the identity of PCR products was confirmed by
sequencing. The results demonstrate an inverse relationship between
stem-loop stability and tau exon 10+/
mRNA ratio. In
Humans (most stable stem-loop) the lowest tau
exon 10+/
RNA ratio is observed while in mouse (least
stable stem-loop) exon 10
mRNA could not be detected.
C, RT-PCR analysis of tau exon 10 alternative splicing in transgenic mice expressing the entire human
tau gene (TG) and in littermate controls
(Non-TG). PCR was performed on RNA isolated from adult mouse
brains between exon 9 and exon 11. Primers were designed to be human-
or mouse-specific. Mouse tau-specific primers gave only exon
10+ products (390 bp), demonstrating that splicing of the endogenous
mouse gene is unaffected by the transgene. In contrast, human
tau pre-mRNA in the transgenic mice was shown to undergo
exon 10 alternative splicing similar to that observed in Human brain.
This result is consistent with cis-elements in the human gene (such as
the stem-loop) regulating alternative splicing of this exon.
mRNA was not detected by
RT-PCR analysis of whole brain RNA, in agreement with previous reports of the Tau isoform composition in adult mouse brain (18). The large
difference in Tau isoform composition between adult human and mouse
brains (and other rodents) is likely to reflect, if not underlie, some
fundamental, undetermined differences in the functions and
characteristics of neurons from these species.
mRNA were detected in the brains of transgenic mice
expressing the human tau gene (Fig. 6C). This demonstrated
that human-like alternative splicing of exon 10 was occurring in
pre-RNA generated from the human transgene. Control animals gave no
RT-PCR product with the human-specific primers. Western blot analysis
of Tau protein in the transgenic mouse brains also demonstrated the
presence of human Tau isoforms, generated by alternative splicing of
pre-mRNA, that are absent in endogenous mouse Tau protein (not shown).
ratio is somewhat altered in
these mice) clearly suggests that cis-acting sequence elements specific
to the human gene regulate alternative splicing of exon 10 (Fig.
6C). This is obviously consistent with the hypothesis that
the stem-loop sequence in the 5' splice site of human exon 10, which is
absent in the mouse gene, is at least partially involved in the
regulation of alternative splicing of exon 10.
14 to
22 and +13 to +21). The
significance of this repeated sequence is unclear however its position
on either side of the splice site would suggest that it may be involved
in the regulation of alternative splicing of exon 10 in some manner
possibly as a binding site for a splicing factor. The FTDP-17 mutations
+13, +14, and +16 will alter this repeat sequence, and thus it is
possible that this might be an additional mechanism by which these
mutations affect the alternative splicing of exon 10 beyond the
disruption of the predicted stem-loop structure that is the subject of
this study.
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Fig. 7.
RNA secondary structure predictions for the
tau exon 10 5' splice site. Proposed structures
for the tau exon 10 pre-mRNA 5' splice site. The minimal
6-bp stem-loop in addition to alternative structures 1 and 2 predicted
using RNAFOLD (28) are shown. Structure 1 ( G =
11.9 Kcal/mol) is similar to that proposed in Ref. 3 with additional
base pairing in the lower stem region; structure 2 (
G =
12.8 Kcal/mol) is similar in stability to
structure 1 but with an alternative lateral stem-loop. A repeated
ACACGUCCC sequence equidistant from the splice site (
14 to
22 and
+13 to +21, indicated by boxes) allows for the formation of
either structure 1 or 2 with similar stability.
RNA ratio in the exon trapping studies, compared with
the +13 and +14 mutations (Figs. 2 and 3). The extended (10 bp) stem
RNA migrated as a single band however this product displayed marginally
slower migration compared with the major wild-type product consistent with the presence of a more stable stem-loop creating a less flexible molecule. The data from this study are completely consistent with the
presence of a dynamic stem-loop structure in the exon 10 RNAs that is
disrupted by the FTDP-17 splice site mutants but which is stabilized by
the lengthening of the predicted 6-bp stem, to 10 bp.
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Fig. 8.
RNA secondary structure analysis of the
stem-loop region. A, RNA gel migration.
Analysis was performed on wild-type (WT) RNA; FTDP-17 +13,
+14, and +16 mutant RNAs; and extended 10-bp stem-loop (EXT)
RNA. The upper panel gives the results from a typical
nondenaturing agarose gel, while the lower panel gives
results from a denaturing agarose/formaldehyde gel. The different RNAs
all migrated at identical rates in the denaturing gel, since they are
of identical size. In contrast, different migration rates were observed
for the different tau exon 10 RNAs in nondenaturing gels
consistent with predicted differences in secondary structure.
B, RNA secondary structure mapping (20). RNAs
in vitro transcribed from wild-type (WT), FTDP-17
+14 mutant (+14), and the extended 10-bp stem-loop (EXT)
constructs were analyzed. Digestion sites for V1 (double
strand-specific) and T1 (single strand-specific) RNases were mapped by
primer extension. Control samples (C) were treated
identically to RNase digestion but the enzyme was omitted. The location
and sequence of the predicted stem regions are indicated both for the
6-bp and artificial 10-bp stem-loops by vertical bars.
3 to +3) in the 5' side of the stem.
Wild-type RNA also gave significant V1 signals in the predicted double
strand region, on both the 5' and 3' sides of the stem-loop, although the shorter stem was reflected in the absence of bands at +17 and +18.
In contrast, markedly weaker V1 signals were observed from the +14
mutant RNA in the predicted stem region. This is consistent with the
proposed disruption of the wild-type secondary structure in this region
by the +14 mutation. Interestingly, however, additional V1 products
that are specific to the +14 mutant RNA are observed in the predicted
loop region (+9, +10), suggesting that this mutation may cause the
formation of an alternative, and presumably less stable, secondary
structure feature in this region. Digestion with T1 enzyme gave little
obvious difference between each of the RNAs (strong bands in the 10-bp
stem construct at +19 to +21 are also present in the control and
reflect a cDNA polymerization stop). The lack of digestion in the
terminal loop region with the T1 RNase may reflect the base specificity
of this enzyme, cutting after a guanine residue, and the fact that the G residue at +5 in the loop is predicted to base pair to the uracil residue at +10 (Fig. 7). A strong stop in cDNA polymerization is
observed in the control lane for the extended stem (10 bp) RNA just
before the predicted stem-loop structure. This stop is specific for
this RNA and is consistent with the introduction of a more stable
structure at this position. While loading of RNA samples was equalized,
and enzyme conditions were designed to generate one cleavage per
molecule, some artifactual strengthening of V1 signals in the 10-bp
stem RNA 3' of the stem-loop region is likely due to secondary cutting
caused by the strength of digestion at the
3 to +3 positions. However
this artifact is still a reflection of the stabilized stem-loop that is
present in this RNA.
mRNAs is largely determined by the stability of this
structure. Stem-loop formation at the 5' splice site of exon 10 is
likely to compete with the binding of specific factors required for the
early stages of spliceosome assembly, most likely the U1 snRNP, after
transcription has occurred. Inserted stem-loops were shown previously
in vivo to block U1 snRNP binding to a 5' splice site in the
yeast RP51A gene (27). Formation of the stem-loop at the 5'
splice site of tau exon 10 will therefore block exon 10 definition and result in skipping of exon 10 in tau
mRNA. Further experiments are needed to confirm the role of the U1
snRNP in this mechanism; however, sequence changes in the 5' splice
site, which are predicted to alter the stability of U1 snRNP binding,
result in altered exon 10 splicing in vitro independent of
the of stem-loop stability. The exact structure of the stem-loop region
has yet to be determined with two different confirmations predicted by
RNAFOLD (28) analysis (Fig. 7). It is thus clear that the stem-loop
structure plays a major role in the regulation of tau exon
10 alternative splicing however additional levels of splicing control
may also be mediated through the 5' splice site. In particular, a
9-base region that is repeated at equidistant positions from the splice
site may also be involved in splicing regulation possibly by providing a binding site for a splicing factor.
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ACKNOWLEDGEMENTS |
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We acknowledge Sarah Lincoln and Nitin Mehta for cell culture assistance and Eileen McGowan for helpful discussions.
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FOOTNOTES |
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* This work was supported by NINDS Project Grant RO1 NS37143-01, by the Smith Scholar program, and by the Mayo Foundation (all to M. H.).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: Mayo Clinic
Jacksonville, 4500 San Pablo Rd., FL 32224. Tel.: 904-953-0159; Fax: 904-953-7370; E-mail: hutton.michael{at}mayo.edu.
2 K. Duff, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: FTDP-17, fronto-temporal dementia and parkinsonism linked to chromosome 17; RT-PCR, reverse transcriptase-polymerase chain reaction; snRNP, small nuclear ribonucleoprotein particle; MOPS, 3-(N-morpholino)propanesulfonic acid; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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