MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 10005–10016
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 22
Homologues of the Caenorhabditis elegans Fox-1 Protein Are
Neuronal Splicing Regulators in Mammals†
Jason G. Underwood,1Paul L. Boutz,2Joseph D. Dougherty,3Peter Stoilov,2and
Douglas L. Black1,2,4*
Molecular Biology Institute,1Department of Microbiology, Immunology and Molecular Genetics,2Interdepartmental
Program in Neuroscience,3and Howard Hughes Medical Institute,4University of California, Los Angeles,
Los Angeles, California 90095-1662
Received 8 June 2005/Returned for modification 8 July 2005/Accepted 11 August 2005
A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element
GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a
splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse
Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle
and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from
multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large
family of proteins with variable N and C termini and internal deletions. We show that the overexpression of
both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing
activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox
protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large
family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function,
in addition to their apparent negative effects on splicing in vertebrate muscle and in worms.
Alternative splicing allows the production of multiple mRNAs
from a single pre-mRNA via selection of different splice sites.
Regulated exons are controlled by splicing enhancer and si-
lencer elements within the exon or in the adjacent introns.
These RNA sequences bind to specific regulatory proteins that
contribute to the tissue specificity of splicing. Most exons are
controlled by combinations of both positive and negative reg-
ulators, and how tissue specificity of splicing is achieved is
poorly understood (5, 44).
The N1 exon of the c-src gene serves as a model for an exon
under both positive and negative control. In nonneuronal cells,
the exon is repressed by the polypyrimidine tract binding pro-
tein (PTB) that binds to intronic splicing silencer elements
flanking the N1 exon (1, 7, 9). In neurons, PTB-mediated
repression is absent, and the exon is activated for splicing by an
intronic splicing enhancer (4, 38). The enhancer region down-
stream of the N1 exon is complex, with binding sites for many
proteins. However, the element most critical for enhancer ac-
tivity is the sequence UGCAUG, which is flanked by PTB
binding elements (4, 37, 38). Several proteins, including the
hnRNPs F and H, the neuronal homologue of PTB, and the
KH-type splicing regulatory protein, assemble onto this region
in splicing extracts (8, 30, 34, 35). Immunodepletion and anti-
body inhibition experiments have indicated a role for these
proteins in the splicing of N1 in vitro. However, none of these
proteins specifically recognizes the UGCAUG element, and
they do not positively affect an exon controlled by just a UG
CAUG element in vivo (J. G. Underwood and D. L. Black,
unpublished observations). Thus, they do not seem to mediate
the function of the strongest enhancer element. Their function
may be related to preventing PTB-mediated repression in neu-
rons rather than true positive control of splicing. The proteins
responsible for the UGCAUG-dependent enhancer activity
are not known.
The UGCAUG hexanucleotide has been identified as con-
trolling many alternative exons in addition to src N1 (11, 12, 18,
20, 24). This element has been studied extensively as a regu-
lator of fibronectin EIIIB exon splicing, which is highly depen-
dent on a group of UGCAUG elements dispersed throughout
the downstream intron (29). Interestingly, these elements act
at some distance from the upstream, activated exon, and their
wide spacing is conserved between vertebrate species. Simi-
larly, the UGCAUG element is found downstream of the c-src
N1 exon in all vertebrates (4, 36, 45). These elements also play
an important role in regulating the splicing of a neuron-specific
exon in nonmuscle myosin heavy chain, as well as a neuronal
pattern of processing in the calcitonin/calcitonin gene-related
peptide (CGRP) transcript (18, 24, 39). The element UG
CAUG was also identified in a computational study as the most
common hexanucleotide found in the introns downstream of a
set of neuron-specific exons (6). Thus, this element is a hall-
mark of many systems of neuronal splicing regulation.
Recently, several groups identified vertebrate homologues
of the Caenorhabditis elegans protein Fox-1 (22, 46). The Fem-
inizing locus on X (Fox-1) gene encodes a protein with a single
RNA recognition motif (RRM)-type RNA binding domain
and is a numerator element in reading the X-to-autosome ratio
in C. elegans sex determination (19, 32, 33, 40, 43). Fox-1
protein controls expression of the Xol-1 gene (XO lethal), a
key switch in determining male-versus-hermaphrodite devel-
* Corresponding author. Mailing address: Howard Hughes Medical
Institute, University of California, Los Angeles, Los Angeles, CA
90095-1662. Phone: (310) 794-7644. Fax: (310) 206-8623. E-mail:
† Supplemental material for this article may be found at http://mcb
opment. Jin et al. identified homologues of Fox-1 in zebra fish
and mouse and showed that they specifically recognize the
element GCAUG (22). The zebrafish Fox-1 mRNA was spe-
cifically expressed in muscle, whereas the mouse mRNA was
abundant in muscle, heart, and particularly brain. It was shown
in cotransfection assays that this protein functioned as a re-
pressor of certain exons in muscle but also enhanced the splic-
ing of the fibronectin EIIIB exon (22).
We examined the role of Fox proteins in mammalian neu-
ron-specific splicing, and in c-src N1 exon splicing in particular.
We found several homologous genes for these proteins in
mammals that each give rise to a large family of proteins
through extensive alternative splicing. Within the brain, these
proteins are specifically expressed in neurons and not glia.
Through both loss-of-function and gain-of-function experi-
ments, we show that both of these proteins are the mediators
of UGCAUG-mediated splicing enhancement in neurons.
MATERIALS AND METHODS
Northern blot analysis. A commercial mouse poly(A)?Northern blot (Am-
bion) was probed according to the manufacturer’s instructions. Northern probes
were sequences from the first constitutive exon (?100 to 125 nucleotides [nt]) of
each Fox gene PCR amplified with a T7 promoter in the antisense orientation.
cRNA probes were synthesized by T7 transcription using the Ambion Strip-EZ
T7 kit and [?-32P]UTP. ?-Actin levels were assayed with the template provided
with the blot.
Antibodies. Rabbit polyclonal antibodies (Alpha Diagnostics, Inc.) were raised
to an N-terminal peptide of each protein shared by all known isoforms. (Fox-1
NT peptide, MAQPYASAQFAPPQN, and Fox-2 NT peptide, TTTPDAMVQ
PFTTIP). No cross-reactivity was observed on Western blots with recombinant
proteins. Specific antibodies were purified using the cognate peptide coupled to
Sulfo-Link resin (Pierce). Briefly, serum was mixed 1:1 with Tris-buffered saline
(TBS), passed through the peptide column, and washed with 5 column volumes
of TBS and 5 column volumes of TBS plus 0.05% sodium dodecyl sulfate (SDS)
and 0.1% Triton X-100. After buffer strength was reduced with 5 volumes of 20
mM Tris-HCl, pH 7.5, specific immunoglobulin G (IgG) was eluted with 100 mM
glycine-HCl and fractions were neutralized with 1/10 volume of 1 M Tris-HCl,
Tissue culture. All cell lines were grown according to ATCC-recommended
protocols. Neuronal N2A and N1E-115 cells were differentiated in Dulbecco’s
modified Eagle’s medium (DMEM) plus 1% fetal bovine serum and 2% dimethyl
sulfoxide (DMSO) for 5 days (17). Myoblast C2C12 cells were differentiated with
DMEM in low sodium bicarbonate and 2% horse serum for 5 days (27).
Extract preparation and cross-linking. Preparation of HeLa nuclear extracts,
site-specific labeling, and cross-linking were performed as described previously
Western blot analysis. Cells were lysed in RIPA buffer containing Complete
protease inhibitors (Roche) and 30 U/ml benzonase (Sigma) and incubated on
ice for 10 min to allow lysis and nucleic acid fragmentation. Protein concentra-
tions were normalized using a Bradford assay. Normalized protein samples were
added to SDS loading buffer and heated to 90°C for 10 min. For the cell line
Western analysis, proteins were resolved on 4 to 20% Tris-glycine gels. All other
protein gels were 10% NuPage Bis-Tris gels (Novex). After transfer to nitrocel-
lulose, Western blots were carried out with Fox-1 NT (1:200), Fox-2 NT (1:250),
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Covance; 1:400,000),
and FLAG (Sigma; 1:3,000) antibodies. Detection used horseradish peroxidase-
conjugated secondary antibodies (Amersham Pharmacia) with ECL or Femto
Supersignal reagents (Pierce).
Immunohistochemistry. Adult CD1 mice were perfused transcardially with
ice-cold phosphate-buffered saline (PBS), followed by ice-cold 4% paraformal-
dehyde in PBS. The brains were removed, fixed in 4% paraformaldehyde over-
night, sunk in 20% sucrose-PBS, frozen in 4-methyl-butane, and stored at ?80°C
until use. Forty-micrometer sections were cut on a cryostat and stored in PBS
plus 0.1% azide at 4°C until use. Free-floating sections were incubated overnight
in 24-well plates on a rotator at room temperature in the presence of 0.1% azide,
0.25% Triton X-100, and 5% normal goat serum in 500 ?l PBS and primary
antibody at the following concentrations: Fox-1 NT, 1:100; Fox-2 NT, 1:500;
GFAP, 1:1,000 (Chemicon AB1540); NeuN, 1:100 (Chemicon MAB377). Sec-
ondary antibodies were diluted 1:1,000 and included Cy3-conjugated anti-rabbit
IgG and Cy5-conjugated anti-guinea pig IgG (Jackson Immunoresearch) and
Alexa 488-conjugated anti-mouse IgG (Molecular Probes) antibodies. Nuclei
were counterstained with Hoechst dye (Molecular Probes) and mounted with a
Prolong AntiFade kit (Molecular Probes). In all cases, controls with no primary
antibody yielded no labeling. For Fox-1 and Fox-2 double labeling, sections were
incubated overnight with Fox-1 antibody and then for 1 h with Cy3 anti-rabbit
IgG and blocked for 30 min in normal rabbit serum and then for 20 min in 1:50
donkey anti-rabbit Fab fragment (Jackson Immunoresearch), followed by 5 h of
incubation with Fox-2 antibody and 1 h of incubation with Alexa goat anti-rabbit
IgG (Molecular Probes). Nuclei were counterstained with ToPro 3 iodide (Mo-
lecular Probes) and mounted with a Prolong AntiFade kit (Molecular Probes).
Lack of cross-reaction was confirmed by identification of nuclei in the cerebellum
and olfactory bulb that labeled exclusively with either Fox-1 or Fox-2. Confocal
microscopy was performed on a Zeiss LSM 510 META confocal microscope, and
pseudocolored images were overlaid with Zeiss software or Adobe Photoshop.
Three-dimensional reconstructions were used to confirm overlap of signals. In-
frared wavelengths were most often pseudocolored blue.
Plasmids. The DUP reporter plasmids were described previously (37, 38).
Additional plasmids were constructed by similar methods. The synthetic en-
hancer constructs (see Fig. 5) were constructed by ligating phosphorylated an-
nealed oligonucleotides into the unique Bgl2 site in the second intron of DUP
4-1. This placed the test enhancer 45 nucleotides away from the alternative exon
5? splice site. The Fox cDNA expression plasmids were constructed in pcDNA3.1
(Invitrogen). Each carried an N-terminal FLAG tag.
siRNAs. The small interfering RNAs (siRNAs) were designed against target
sequences that were identical in the human and mouse Fox genes and used the
following established protocols (http://www.rockefeller.edu/labheads/tuschl/sirna
.html; 1). siRNAs were synthesized from DNA oligonucleotide templates con-
taining a T7 promoter and encoding one strand of the desired siRNA after a
G-rich 5? flap sequence. Each siRNA strand was separately transcribed with T7
RNA polymerase and then annealed to the cognate strand. Treatment with
DNase I removed the template DNA, and treatment with RNase T1 removed the
unhybridized G-rich flap to yield the siRNA with correct ends. The siRNAs were
purified on G-25 resin and quantified by UV absorbance and/or serial dilutions
on 4% agarose gels. Template oligonucleotides and product siRNAs for Fox-1
and Fox-2 were as follows: Fox-1 DNA template for the sense strand of siRNA,
5?-AATTAGTATGGAGCAAAACGGCCTGTCT; Fox-1 DNA template for
the antisense strand of siRNA, 5?-AACCGTTTTGCTCCATACTAACCTG
TCT; product strands of Fox-1 siRNA, CCGUUUUGCUCCAUACUAAUU
and UUGGCAAAACGAGGUAUGAUU; Fox-2 DNA template for the sense
strand of siRNA, 5?-AAAAATATTGCAATAGCCAGGCCTGTCT; Fox-2
DNA template for the antisense strand of siRNA, 5?-GGCCTGGCTATTGCA
ATATTTCCTGTC; product strands of Fox-2 siRNA, CCUGGCUAUUGCAA
UAUUUUU and CCGGACCGAUAACGUUAUAAA; T7 promoter primer,
Transfections. DNA and siRNA transfections were performed with Lipo-
fectamine 2000 (Invitrogen). Briefly, 1 ?g of plasmid DNA was diluted in 50 ?l
of Optimem and complexed with 2 ?l of Lipofectamine 2000 in 50 ?l of Opti-
mem for 20 min. Trypsinized suspensions of N2A or HeLa cells (1.5 ? 105cells
in 500 ?l in DMEM plus 10% fetal bovine serum) were added to the lipid
complex and rotated manually for 10 min. The mix was then aliquoted into
24-well plates, and the medium was changed after 4 h. For splicing reporter
assays, 100 ng of reporter and 900 ng of total pcDNA plasmid were used. For
siRNA transfections, the siRNA was added to 1 ?g of pUC plasmid DNA during
the lipid formulation phase. For siRNA reporter assays, 100 ng of reporter and
900 ng of pUC were cotransfected. The siRNA titration (see Fig. 6) was per-
formed with 5, 10, and 20 pmol of siRNA. All other siRNA experiments utilized
20 pmol of siRNA.
Reverse transcription (RT)-PCR. Total RNA was isolated from cells with TRI
reagent (MRC) and reverse transcribed with random hexamers and Superscript
II (Invitrogen). Splice products were PCR amplified (22 cycles) using an end-
labeled primer in the 3? constitutive exon and a cold primer in the 5? constitutive
exon. Primers for c-src and EWS were as published (17, 31). Products were
separated by urea-polyacrylamide gel electrophoresis (PAGE) and visualized by
phosphorimager. Exon inclusion was quantified using NIH ImageJ software.
Protein accession numbers. The protein accession numbers of the isoforms
used for expression are NP_067452 for Fox-1 and AAL67150 for Fox-2.
Mouse Fox-1 and Fox-2 are both expressed in neurons. To
test whether a vertebrate Fox-1 homologue was responsible for
10006 UNDERWOOD ET AL.MOL. CELL. BIOL.
mediating the action of the N1 exon splicing enhancer, we
isolated cDNAs encoding these proteins and also obtained
plasmids from other laboratories and the Riken DNA Bank.
There are several homologues in the mouse and human ge-
nomes to C. elegans Fox-1. In addition to human Fox-1
(hFox-1) (gene name, A2bp1; chromosome 16) (42), there are
additional paralogous genes in the mammalian genomes. One
gene we called Fox-2 (gene name, RBM9; also called fxh and
RTA; human chromosome 22) (28, 41). There is also a third
paralog (gene name, HRNbp3; chromosome 17) (46). How-
ever, we did not initially find evidence for expression of this
gene in the cells used for our experiments (data not shown),
and it was not studied further. Alignments of the cDNA, ex-
pressed sequence tag (EST), and genomic databases indicate
that both the fox-1 and fox-2 genes are large in the mouse
(mFox-1, 1.5 Mb, and mFox-2, 220 kb) and larger in humans
(hFox-1, 2.5 Mb, and hFox-2, 280 kb). Both genes have mul-
tiple alternative promoters and multiple internal cassette exons
that are variably included in the mRNA (Fig. 1A; see Table S1
in the supplemental material). Thus, alternative splicing gives
rise to a large family of proteins from each gene.
The RRMs for Fox-1 and Fox-2 are identical over their
entire lengths in both human and mouse and presumably rec-
ognize the same sequence (see Fig. S1 in the supplemental
material). The RRM of mFox-3 is slightly more divergent,
differing from Fox-1 and Fox-2 at 4 of 72 residues. In addition
to the C. elegans Fox-1, there is another homologous gene on
worm chromosome 3. Within the RRM domains, these two
FIG. 1. Mammalian Fox proteins are expressed in neuronal tissues and cell lines. (A) Human Fox genes are represented with first exons in
yellow, untranslated regions in red, and coding exons in blue. Exons showing clear homology in the mouse are shaded. The exons are numbered
to allow their identification in the table of genomic coordinates (see Table S1 in the supplemental material). This numbering does not necessarily
correspond to previously reported exon numbers. The exon used for probing the Northern blot in panel B is marked with a green bar, and the
peptides from the same exon used for antibody production are indicated. The exons encoding the RRM are marked with a gray bar. Both genes
have multiple promoters and numerous alternative splicing events. The coding alternative exons shown all conform to splice site consensus rules
and are conserved between human and mouse. Nearly all are predicted from at least two ESTs, and some were confirmed experimentally by
RT-PCR (data not shown). Alternative splicing of exon 19 of Fox-1 and exon 13 of Fox-2 gives rise to two C-terminal peptide sequences. These
two splice variants of Fox-1, Fox-2, and Fox-3 from human and mouse are aligned in Fig. S1 in the supplemental material, along with two worm
homologues. (B) Northern blot analysis of mouse tissues. A blot (Ambion) carrying 2 ?g of poly(A)?RNA from each of the indicated mouse
tissues was probed sequentially with Fox-1, Fox-2, and ?-actin cRNAs. MWM, molecular weight markers; 14d, 14 days. (C) Western blot analysis
of human and mouse cell lines. Protein (50 ?g) from whole-cell lysates was separated by 4 to 20% gradient SDS-PAGE, transferred to
nitrocellulose, and probed with the indicated antibodies. GAPDH is shown as a loading control. The top and middle panels are different exposures
of the same blot. For the longer exposure, the GAPDH portion of the blot was cut off. The cell lines are HeLa, HEK293, 3T3, C2C12 without and
with myotube differentiation (diff), mouse N2A and N1E-115 neuroblastomas without and with neuronal differentiation, human neuroblastoma
LA-N-5, and human retinoblastoma WERI-1. Differentiated samples are marked with a red dash.
VOL. 25, 2005 Fox-1 HOMOLOGUES REGULATE NEURONAL SPLICING10007
proteins are remarkably similar to the mammalian homo-
logues, differing from the mammalian proteins at 17 and 23 out
of 72 residues, respectively. There is also a Drosophila gene
encoding a nearly identical RRM, which apparently produces a
much larger protein with extended N- and C-terminal domains
(CG32062; chromosome 3L) (data not shown). All three mam-
malian proteins have homologous alternative C termini and N
termini arising from alternative exons and promoters. Using
similar termini, mammalian Fox-1 and Fox-2 share 66% iden-
tity over their entire lengths (see Fig. S1 in the supplemental
material). The worm proteins are much more divergent in the
N- and C-terminal domains. However, the extreme C termini
of the worm proteins (RFTPY and RFAPY) clearly match one
of the C termini of each of the mammalian homologues (see
Fig. S1 in the supplemental material).
Alternatively spliced internal exons create additional
changes in the protein. One exon-skipping event deletes essen-
tial amino acids from the RRM and presumably eliminates
RNA binding. EST expression data and recent work from the
Kawamoto laboratory indicate that some of these transcripts
are expressed in a tissue-specific manner (reference 39 and
data not shown). In the face of this complexity, we decided to
characterize an equivalent isoform from each gene and chose
those forms that have C termini similar to those of the worm
protein (see Fig. S1 in the supplemental material).
To confirm the expression pattern of mFox-1 and determine
that of mFox-2, we generated Northern blot probes from the
first common exon of all the transcripts from each gene (Fig.
1B). When applied to a mouse tissue Northern blot, the ob-
served pattern of mFox-1 mRNA expression was the same as
seen previously (22, 26). Fox-1 expression was high in brain
and heart, with lower levels in embryos. (Skeletal-muscle RNA
was not on this Northern blot, but the expression of Fox-1 was
observed in muscle cell lines.) Fox-2 showed higher expression
than Fox-1 in the embryo relative to the adult, and expression
in the ovary, but otherwise showed a pattern of expression very
similar to that of Fox-1 (Fig. 1B).
To examine the expression of the Fox proteins in cell lines
that we would use in later experiments, we raised antibodies to
mFox-1 and mFox-2. Antibodies were raised to peptides within
the first common exon of Fox-1 and Fox-2 (Fig. 1A). These
antibodies reacted well with recombinant target protein but
did not cross-react with the opposite homologue (data not
shown). When applied to immunoblots, the Fox-2 antibody
gave two prominent bands of approximately 48 and 40 kDa
that presumably resulted from two of the many splice variants
of the Fox-2 mRNA (Fig. 1C). These Fox-2 proteins were most
highly expressed in the mouse neuroblastoma cell line N1E-
115, which had been induced to differentiate with DMSO and
low serum, and in the human neuroblastoma LA-N-5 (Fig. 1C,
lanes 9 and 10). On longer exposure, these same Fox-2 proteins
were seen in other cell lines, although the relative abundances
of the two forms vary. In a second neuroblastoma, N2A, Fox-2
was also induced by the differentiation treatment (Fig. 1C,
lanes 6 and 7). The Fox-1 antibody was reactive with one
prominent band of about 43 kDa, as well as other less abun-
dant species. Fox-1 expression was more limited than that of
Fox-2. It was seen in the mouse neuroblastomas N1E-115 and
N2A, but not in LA-N-5. Interestingly, the differentiation of
the neuroblastomas into neurons again stimulated expression
of Fox-1 (Fig. 1C, lanes 6 to 9). Differentiation of the muscle
cell line C2C12 into myotubes also increased Fox-1 expression,
but in this case it decreased Fox-2 expression (Fig. 1C, lanes 4
To examine what cell types in the brain express Fox proteins,
we performed immunofluorescence staining with the Fox an-
tibodies on sections of brain tissue. Both proteins were broadly
expressed throughout the brain in largely, but not exactly,
overlapping patterns. Both proteins show strong staining in the
neurons of the dentate gyrus and CA3 region of the hippocam-
pus (Fig. 2A and B). This staining is specific to neurons;
costaining for glia with anti-GFAP showed no overlap with the
Fox proteins, whereas staining with anti-NeuN gave a com-
pletely coincident pattern in both the hippocampus and the
cortex (Fig. 2A and data not shown). As seen in the staining of
cortical sections, both Fox-1 and Fox-2 are predominantly nu-
clear, as expected, but Fox-2 shows some additional cytoplas-
mic staining (Fig. 2C). Staining other regions of the brain gave
similar results. Interestingly, there were some neurons in both
the cerebellum and the olfactory bulb that expressed only
Fox-1 or Fox-2, but not both (data not shown). In all experi-
ments, these proteins were specific to neurons and were not
seen in glia (Fig. 2A and 2C).
Fox-1 and Fox-2 activate splicing of neuronal exons through
binding to downstream UGCAUG elements. We previously
observed that splicing of the c-src N1 exon increases about
twofold in mouse neuroblastoma cells when they are induced
to differentiate, correlating with the observed increase in Fox
protein expression (Fig. 1C, lanes 6 to 9) (17). To test whether
other exons were spliced in parallel with Fox protein expres-
sion, we tested these cells for splicing of the neuron-specific 4?
exon in the EWS gene, which has regulatory elements similar
to those of c-src N1 (31). Both genes have UGCAUG elements
in the downstream intron which are conserved between mul-
tiple mammalian species (36). As seen previously for c-src N1,
induction of N2A cells with DMSO and low serum caused an
increase in EWS exon 4? splicing, in parallel with the change in
Fox expression (Fig. 3A).
Since an increase in Fox protein expression during neuro-
blastoma differentiation correlates with splicing of neuronal
exons in both c-src and EWS, we next tested whether either
protein expressed alone could induce splicing of these exons.
The Fox proteins were transiently expressed in N2A cells (un-
treated for differentiation), and splicing of the c-src and EWS
exons was assayed by RT-PCR (Fig. 3B and C). The expression
of the Fox proteins was monitored by immunoblotting for the
Flag epitope tag on each construct (Fig. 3D). N2A cells trans-
fected with empty expression vector included the N1 exon in
14% of the c-src mRNA and included the 4? exon in 19% of the
EWS mRNA (Fig. 3B and C, lanes 1). Overexpression of either
Fox-1 or Fox-2 approximately doubled the splicing of each
exon (Fig. 3B and C, lanes 2 to 5); splicing of c-src N1 went up
to 27 to 40%, and splicing of EWS 4? went up to 43%. Note
that these assays were performed on the endogenous c-src and
EWS mRNAs. Since a large fraction of the cells (30 to 40%) do
not take up DNA in these transfections, the induction of neu-
ral exon splicing by Fox proteins is likely to be larger than can
be measured in this experiment (data not shown).
To examine the roles of particular RNA elements in the Fox
protein activation of N1 splicing, we cotransfected N1 exon
10008UNDERWOOD ET AL.MOL. CELL. BIOL.
minigenes with the Fox expression plasmids (Fig. 4A). For this,
we used HeLa cells, which have low levels of Fox-2 and no
Fox-1 (Fig. 1C). As shown previously, a minigene carrying the
N1 exon and its flanking intronic regulatory elements showed
nearly complete (99%) skipping of the N1 exon in HeLa cells
(pDUP4-5) (Fig. 4B, lane 1) (37). When the pDUP4-5 plasmid
is coexpressed with either mFox-1 or mFox-2, the exon is
activated 10- to 12-fold and is spliced into about 10 to 12% of
the product mRNA (Fig. 4B, lanes 2 to 6). This level of N1
splicing in HeLa cells with Fox coexpression is similar to that
seen with the reporter transfected into a mouse neuroblastoma
cell line (37). This confirms that the N1 exon is strongly af-
fected by either Fox protein and demonstrates that introducing
a Fox protein into a nonneuronal cell line is sufficient to stim-
ulate N1 inclusion.
The N1 exon is controlled by multiple regulatory proteins
(5). Most notably, in HeLa cells, the exon is strongly repressed
by PTB (1, 7, 9). To examine the enhancer activity of Fox
protein in the absence of PTB repression and to confirm that
the Fox stimulation required the enhancer sequence, we used
a test exon that is activated by the N1 enhancer but is missing
many of the other N1 regulatory sequences, including essential
upstream PTB sites (pDup4-28) (Fig. 4C) (38). This enhancer
contains PTB binding elements, but they are insufficient to
mediate splicing repression without the two high-affinity PTB
sites upstream of the alternative exon (1, 7). The enhancer
fragment contains one UGCAUG element and two elements
that match at five of six positions. This enhancer activates the
test exon in the pDUP4-28 plasmid to 18 to 25% inclusion in
HeLa cells (Fig. 4D, lane 1). Splicing enhancement is depen-
dent on the three elements, as their mutation lowers test exon
splicing to 2% (Fig. 4D, lane 4). When the enhancer-contain-
ing minigene is coexpressed with either Fox protein, test exon
splicing is strongly stimulated to 70 to 75% inclusion (Fig. 4D,
lanes 2 and 3). The construct with mutations in the UGCAU
G-like elements did not respond to Fox expression (Fig. 4D,
lanes 5 and 6). Thus, the stimulation of splicing by the Fox
proteins is mediated by the enhancer sequences and is inde-
pendent of splicing repression by PTB.
In previous work, it was shown that Fox-1 binds to the
FIG. 2. Fox-1 and Fox-2 are coexpressed in the nuclei of most neurons of the adult mouse brain but excluded from glia. (A) Immunostaining
of Fox proteins in hippocampus. Fox-1 expression (red) overlaps completely with neuronal marker NeuN (green) but shows no overlap with glial
marker GFAP (blue). Individual channels are shown in small panels on the right, and overlay images are shown in larger panels on the left. The
boxed region in the left panel is shown in the right panels. (B) In the hippocampus, Fox-1 expression (red) also overlaps completely with Fox-2
expression (green). (C) High-power confocal microscopy in the cortex reveals that both Fox-1 (red) and Fox-2 (green) are expressed primarily in
nuclei (blue). Some small nuclei (arrowheads), likely glia, contain no Fox labeling.
VOL. 25, 2005 Fox-1 HOMOLOGUES REGULATE NEURONAL SPLICING10009
element GCAUG with high specificity (22, 46). To confirm that
the Fox proteins were binding to this element in the context of
the N1 exon enhancer, we performed site-specific labeling and
cross-linking experiments. For this, we used HeLa nuclear ex-
tract, which contains low levels of Fox-2 and no Fox-1 but
which is known to be active for splicing. The BS27 RNA was
chosen for analysis because it lacks the upstream binding sites
that are needed for repression of N1 splicing by PTB. Thus,
PTB binds only weakly to the downstream enhancer region,
and BS27 RNA is active for splicing in HeLa extract (7, 9). By
reducing PTB binding to the enhancer, we hoped to increase
Fox-2 binding, given its low level of expression in HeLa cells.
To localize the observed protein binding to the enhancer ele-
ment, BS27 RNA containing a single32P label within the UG
CAUG element of the N1 enhancer was synthesized (Fig. 5A).
This was compared to a mutant BS27 RNA in which the UG
CAUG was changed to UGACUG but which carried the label
in the same position (Fig. 5A).
These RNAs were incubated in HeLa extract, irradiated
with 254-nm UV light, and treated with RNase to leave a small
oligonucleotide tag on the cross-linked proteins. For the wild-
type RNA, a prominent set of cross-linked proteins was ob-
served migrating at about 50 kDa on SDS-PAGE (Fig. 5B, lane
2). To confirm that these bands contained Fox-2 protein, the
cross-linked proteins were immunoprecipitated with anti-
Fox-2 antibodies. One 50-kDa protein and other minor bands
were clearly precipitable with the Fox-2 antibodies (lane 4).
The specificity of the immunoprecipitation was confirmed with
preimmune serum and with Fox-2 antibodies preblocked with
the antigenic peptide, neither of which reacted with any of the
cross-linked proteins (Fig. 5B, lanes 3 and 5). The requirement
for the UGCAUG element in the Fox-2 interaction was dem-
onstrated by the mutant BS27 RNA. Cross-linking to this RNA
yielded a different distribution of ?50-kDa proteins, and none
of these were brought down by the Fox-2 antibody (Fig. 5C).
Thus, Fox-2 protein very specifically binds to the UGCAUG
element within the context of the N1 enhancer.
To demonstrate that the UGCAUG hexanucleotide alone
was sufficient for Fox activation of splicing, we tested several
minigenes carrying short synthetic enhancers containing this
element (Fig. 6). As shown previously, a fragment carrying
three copies of the element separated by 10-nucleotide spacer
sequences from ?-globin intron 1 activates the test exon weakly
in HeLa cells but is more potent in neuronal cell lines. The
background enhancer activity in HeLa cells is presumably due
to a low level of endogenous Fox-2 (Fig. 1C). If the pDUP4-
108 construct is coexpressed with either Fox-1 or Fox-2, splic-
ing is stimulated from 7 to 9% to 24 to 35% (Fig. 6A). A
similar fragment with the spacer sequences reversed (pDUP4-
108Rev) was also activated by Fox proteins (Fig. 6B). In con-
trast, mutating 2 nucleotides within each element to UG
ACUG in the pDUP4-108Mut reporter eliminated splicing
enhancement altogether, indicating that enhancement is in-
deed dependent on the UGCAUG element (Fig. 6C). A frag-
FIG. 3. Fox proteins enhance neuron-specific splicing patterns in mouse N2A mRNAs. (A) EWS neuron-specific splicing is induced during
neuronal differentiation of N2A cells. Cells were grown in standard media or differentiation media for 5 days, and RNA was harvested. EWS exon
4? splicing was assayed by RT-PCR and quantified. The percent exon inclusion is indicated. std. dev., standard deviation. (B) Inclusion of the N1
exon in the endogenous c-src mRNA is enhanced by Fox overexpression. Each Fox plasmid (100 ng or 1 ?g) was transfected into N2A cells, and
total RNA was harvested after 60 h. RNA was assayed by RT-PCR and quantified. (C) Inclusion of the 4? exon in the endogenous EWS mRNA
is enhanced by Fox overexpression; similar to panel B, except assayed for EWS splicing by RT-PCR using an end-labeled primer in EWS exon 4.
(D) Immunoblot confirming overexpression of Fox-1 and Fox-2 proteins in N2A cells. Whole-cell lysates (50 ?g) were separated by 10% NuPage
and subjected to immunoblotting with FLAG and GAPDH antibodies.
10010 UNDERWOOD ET AL.MOL. CELL. BIOL.
ment carrying only one element did not enhance splicing, with
or without expressed Fox protein. Similarly, a fragment carry-
ing three directly abutted hexanucleotide elements without
spacers showed much weaker activity than three elements
spaced by 10 nucleotides (data not shown). These experiments
indicate that the number and spacing of the elements are
important for their activity.
Loss of Fox proteins leads to loss of UGCAUG enhancer
function. In addition to assessing the effect of Fox protein
overexpression, we examined whether splicing was altered
when the Fox proteins were eliminated using RNA interfer-
ence (RNAi). Several siRNAs were tested in HeLa cells using
transfected Fox cDNAs as targets (data not shown and refer-
ence 13). siRNAs that led to the specific reduction in Fox-1 or
Fox-2 were identified (data not shown). HeLa cells exhibit
efficient RNAi but contain no Fox-1 protein and very little
Fox-2 protein. To assess whether this small amount of Fox-2
was responsible for the low enhancer activity seen in HeLa
cells, we used the pDUP4-183 reporter, which is enhanced for
basal exon inclusion by the addition of a binding site for ASF/
SF2 within the alternative exon (Fig. 6D) (37, 41a). The com-
bination of this exonic splicing enhancer with the triple UG
CAUG yields about 30% exon inclusion (Fig. 6D, lane 1).
Cotransfection with an siRNA against Fox-1 had no effect on
splicing (Fig. 6D, lane 2). However, the siRNA against Fox-2
reduced exon inclusion to about 8% (lane 3). Thus, the small
amount of Fox-2 in HeLa cells drives the basal activity of the
Measuring alterations in endogenous c-src splicing by RNAi
presented technical difficulties. Neuronal cell lines that exhib-
ited high levels of N1 inclusion proved inefficient for siRNA
transfection (data not shown). Conversely, efficient RNAi-me-
FIG. 4. Fox enhances splicing through the c-src intronic splicing enhancer. (A) A chimeric minigene, pDUP4-5, with ?-globin exons 1 and 2
flanking the c-src N1 exon and its intronic regulatory regions. This is completely repressed in HeLa cells. (B) Fox expression enhances N1 splicing.
Splicing reporter (100 ng) was cotransfected with empty expression vector (lane 1) or 100 ng (lanes 2 and 5) or 900 ng (lanes 3 and 6) of Fox
expression plasmid. Lanes 2 and 3 show increasing Fox-1, and lanes 5 and 6 are increasing Fox-2. RNA was harvested after 48 h, assayed by
RT-PCR, and quantified. (C) A minigene carrying ?-globin exons 1 and 2 with a 33-nt generic internal exon composed of ?-globin sequence. The
wild-type (WT) (pDUP4-28) or mutant (MUT) (pDUP4-28Mut) c-src intronic enhancer sequence is inserted in the second intron (38). With the
wild-type enhancer, the internal exon is partially included without Fox expression. The putative Fox sites are shown below, with the mutations made
for disrupting enhancer activity. (D) Fox expression enhances splicing through sequences in the c-src enhancer. Cotransfections and analysis were
as for panel B, except with pDUP4-28 and pDUP4-28Mut. (E) Immunoblot confirming expression of the Fox proteins in HeLa cells. Whole-cell
lysates (50 ?g) were separated by 10% NuPAGE, transferred to nitrocellulose, and probed with FLAG and GADPH antibodies.
VOL. 25, 2005 Fox-1 HOMOLOGUES REGULATE NEURONAL SPLICING10011
diated knockdown of Fox-2 was observed in N2A cells, but
these cells exhibit only low levels of N1 inclusion, making it
difficult to observe a large loss of splicing for N1. The N2A cells
showed a consistent reduction of N1 inclusion, dropping from
18% with a control siRNA to 13% with Fox-2 siRNA (Fig. 7A,
lanes 1 and 3). The low splicing of N1 in these cells is in part
due to PTB repression; when PTB was targeted with an
siRNA, N1 exon splicing increased to 59% (Fig. 7A, lane 2).
Consistent with its near absence in N2A cells, a Fox-1 siRNA
showed no effect on c-src splicing (data not shown). EWS 4?
splicing paralleled the regulation of c-src N1 (Fig. 7B). In this
case, the siRNA against PTB increased exon 4? inclusion from
9% to 43% and the siRNA against Fox-2 decreased inclusion
to 2% (Fig. 7B, lanes 1 to 3). Thus, both the c-src and EWS
neuron-specific exons share regulation by the repressor pro-
tein, PTB, and the enhancer protein, Fox-2.
To measure a stronger effect from the Fox siRNA on an
endogenous transcript, we needed an exon that exhibited a
higher starting level of exon inclusion. For this, we used the
fibronectin EIIIB exon. Although not a neuron-specific exon,
EIIIB is known to be dependent on UGCAUG enhancer ele-
ments, and overexpression of Fox-1 was shown to increase its
inclusion in minigene transcripts (20, 22, 29). N2A cells show
high levels of EIIIB inclusion (91%) (Fig. 7C, lane 1). N2A
cells make little Fox-1 protein, and an siRNA targeting the
Fox-1 transcript had little effect on EIIIB splicing (lanes 2 to
4). In contrast, transfection of the Fox-2 siRNA led to a large
decrease in EIIIB splicing (39% exon inclusion) (lanes 5 to 7).
The progressive loss of Fox-2 protein with higher doses of
Fox-2 siRNA correlates with the loss of EIIIB splicing (Fig. 7C
and D). Thus, the splicing of UGCAUG-dependent exons is
very sensitive to the loss of endogenous Fox protein.
The loss of Fox-2 affected both endogenous N1 exon splicing
and the activity of the triple UGCAUG enhancer. To examine
whether the loss of endogenous Fox-2 protein specifically af-
fected the activity of the src N1 enhancer, we cotransfected
siRNAs with the pDUP4-28 minigene into N2A cells in which
the N1 enhancer was active. Treatment with Fox-2 siRNA
reduced enhancer activity by 50% (data not shown). Taken
together, these data indicate that an endogenous Fox protein is
needed for activity of the c-src intronic splicing enhancer. In
N2A cells, the active family member is Fox-2.
Many neuronally regulated exons are controlled by intronic
splicing enhancers containing the element UGCAUG (4, 18,
24). We show that mammalian Fox-1 and its homologue Fox-2
are specifically expressed in neurons and that both proteins
activate neuron-specific exons through UGCAUG-dependent
enhancers. Recently, it was demonstrated that Fox proteins
could also enhance splicing of a neuron-specific exon from the
nonmuscle myosin heavy chain pre-mRNA (39). Thus, the Fox
proteins are likely key regulators of splicing in the nervous
In addition to neurons, the Fox proteins also play roles in
other tissues. Fibronectin exon EIIIB, whose inclusion is not
neuron specific, is also dependent upon Fox proteins. The
proteins are also expressed in heart and muscle (22). In muscle
cells, Fox-1 was shown to act as a splicing repressor, inhibiting
the splicing of exons in actinin and ATP synthase transcripts.
These exons are normally skipped in muscle but used else-
where (Fig. 8A). Interestingly, the GCAUG regulatory ele-
ments needed for this repression are upstream of the skipped
exons. So far, all of the UGCAUG enhancer elements known
to activate splicing are found downstream of the regulated
exon (36) (Fig. 8B). Thus, the Fox proteins can apparently act
either positively or negatively, depending on where they bind
relative to the affected exon. Similar location dependence is
seen in the SR proteins that activate splicing when bound in
exons but inhibit splicing when bound to intronic elements (14,
In addition to the location of the Fox protein binding site,
the particular Fox isoform expressed is likely to affect its ac-
tivity. In mammals, Fox-1 and Fox-2 are members of an ex-
tended family of proteins arising from multiple genes and
complex alternative splicing. Recent work demonstrated that
the expression of certain spliced isoforms is specific to partic-
ular tissues and that their enhancer activities are variable (39).
It will thus be important to characterize which forms are ex-
pressed in which cell types and to determine whether particular
isoforms are needed for the regulation of specific transcripts.
The Fox proteins have been identified in other contexts. In
C. elegans, the original Fox gene is required for proper assess-
ment of the X-to-autosome ratio for dosage compensation
FIG. 5. Fox-2 cross-links specifically to the UGCAUG element
within the N1 exon intronic splicing enhancer. (A) Location of the
labeled phosphate within the wild-type (WT) and mutant (MUT) BS27
RNAs. The labeled phosphates are indicated by asterisks. The wild-
type and mutant UGCAUG elements are underlined. (B) Cross-link-
ing to the wild-type UGCAUG element. (C) Cross-linking to the
mutant UGACUG element (MUT). Lane 1 contains molecular weight
markers and lane 2 the total cross-linking reaction (20% of the input
to the immunoprecipitations in lanes 3 to 5). Lanes 3 to 5 are immu-
noprecipitations with the following antibodies: preimmune serum
(lane 3); affinity purified anti-Fox-2 NT (Lane 4); anti-Fox-2 NT pre-
blocked with the cognate Fox-2 NT peptide (lanes 5).
10012UNDERWOOD ET AL.MOL. CELL. BIOL.
during sexual development (19, 33, 40, 43). Interestingly, this
worm protein controls a downstream transcript, Xol-1, which
exhibits alternative splicing patterns. This worm system may
show an effect on splicing different from that seen in mammals.
The sixth intron of Xol-1 is more efficiently spliced in the
absence of Fox-1, which may cause its retention (Fig. 8C) (43).
This intron contains two copies of the pentamer GCAUG, but
whether this is the target sequence for the worm protein has
not been reported. The RRM of the worm Fox protein is 75%
identical to the mouse homologues, and its auxiliary domains
have similarities to mammalian splice variants. Worm Fox-1
also functions in the adult, where it is expressed in some neu-
rons and other cells. Thus, Fox-1 may function in the adult
worm in a manner similar to its role in mammalian cells.
Conversely, there may be additional embryonic functions for
the mammalian protein.
The mammalian Fox proteins have also been identified in
other studies. Fox-1 was identified in yeast two-hybrid screens
as interacting with ataxin-2 and was called A2bp1 (42). Trinu-
cleotide expansions in the ataxin-2 gene cause spinocerebellar
ataxia type 2 (21). Interestingly, ataxin-2 also has homologues
in worms and other distantly related species and is also thought
to be an RNA binding protein (10, 25). A2bp1 has been re-
ported to localize to the Golgi apparatus (26, 42). We see
predominantly the nuclear localization expected for a splicing
regulator. However, we cannot rule out the possibility that the
cytoplasmic population is Golgi associated. It also appears that
different isoforms of the protein show differential localization
Interestingly, human mutations in A2bp1 were identified in
a limited number of patients with an inherited epilepsy and
mental retardation disorder (3). The Fox1/A2bp1 gene also lies
FIG. 6. Fox enhances splicing through tandem UGCAUG sequences in HeLa cells. Test enhancers were inserted into the second intron of
pDUP4-1 ?-globin splicing reporter. (A) An enhancer construct with three copies of UGCAUG element separated by 10-nt globin intron spacers.
pDUP4-108 (100 ng) was cotransfected with empty expression vector (lane 1) or 100 ng or 900 ng of Fox-1 or Fox-2 expression plasmid. Lanes 2
and 3 have increasing Fox-1, and lanes 5 and 6 have increasing Fox-2. RNA was harvested after 48 h, and inclusion was assayed by RT-PCR.
(B) Conditions similar to those for panel A but using the pDUP4-108Rev reporter plasmid, which has the spacer sequences reversed. (C) Con-
ditions similar to those for panel A but with the pDUP4-108Mut reporter plasmid carrying three copies of UGACUG. (D) Knockdown of Fox-2
in HeLa cells results in loss of enhancer activity. The pDUP4-183 reporter plasmid is similar to that in panel A but carries the 5? half of the c-src
N1 exon, a known ASF/SF2-dependent exonic splicing enhancer (ESE) (41a). HeLa cells were cotransfected with 100 ng of pDUP4-183, 900 ng
pUC carrier DNA, and the indicated siRNAs. RNA was harvested after 60 h, and exon inclusion was assayed by RT-PCR.
VOL. 25, 2005 Fox-1 HOMOLOGUES REGULATE NEURONAL SPLICING10013
within an autism susceptibility locus on chromosome 16 (2).
Given its function described here, these mutations in Fox-1 will
lead to changes in the neuronal regulation of splicing. Thus, it
will be very interesting to test for changes in the splicing of
UGCAUG-dependent exons in multiple forms of neurological
disease, including ataxias, epilepsy, and autism. The recent
implication of splicing defects in other neurological disorders,
such as frontotemporal dementia with Parkinsonism linked to
chromosome 17 (FTDP-17) and myotonic dystrophy, provides
an interesting precedent for this idea (15, 16).
Studies of Fox-2 (RBM9) identified it as a gene upregulated
by androgens and as a repressor of tamoxifen activation of the
estrogen receptor (28, 41). There are splice variants of Fox-2
that, judging from EST frequency data, appear specific to
breast, ovary, and other estrogen-sensitive tissues (data not
shown). Thus, hormone signaling may regulate alternative
splicing through changes in Fox transcription or isoform ratios.
Analysis of downstream Fox targets in these tissues is likely to
uncover another important role for Fox proteins.
The mechanism of splicing activation by the Fox proteins
and their interaction with other splicing regulators are also
interesting directions for future studies. Fox-regulated exons
often show repression by PTB, and PTB binding elements are
often found adjacent to Fox sites. It will be interesting to
examine whether these proteins bind in a common regulatory
complex or perhaps antagonize each other’s binding.
The roles of the conserved N- and C-terminal Fox domains
are also unknown. Understanding their interactions with the
general splicing machinery will be important in determining
the mechanism of splicing regulation. Most intronic regulatory
elements studied to date cluster near 5? or 3? splice sites and
often overlap with them (5). The neuronal exons studied here
have a UGCAUG element within 100 nucleotides of the alter-
native 5? splice site. Many other neuron-specific exons have at
least one conserved UGCAUG in this region (6). However,
fibronectin exon EIIIB and calcitonin/CGRP carry important
UGCAUG elements at a distance of several hundred nucleo-
tides from the affected splice sites (18, 29). Splice site proximity
and enhancer location relative to the exon are additional fea-
tures that must be understood to predict Fox-regulated exons
and whether Fox will be a repressor or enhancer.
UGCAUG is among the most precisely conserved splicing
FIG. 7. RNAi of Fox-2 affects splicing of endogenous mRNAs in N2A cells. (A) Splicing of the c-src N1 exon after siRNA treatment. Cells were
transfected with 1 ?g of pUC plasmid and 20 pmols of the siRNA indicated at the top. After 60 h, RNA was harvested and assayed by RT-PCR.
(B) Splicing of EWS 4? exon after siRNA treatment. Similar to the conditions in panel A but assayed for EWS exon 4?. (C) Splicing of fibronectin
EIIIB after siRNA treatment. The same procedure as in panel A, except with 5, 10, or 20 pmol of siRNA and with fibronectin primers. Note that
an siRNA directed against Fox-1 had no effect (lanes 2 to 4), but the Fox-2 siRNA strongly increased skipping of fibronectin EIIIB (lanes 5 to 7;
products labeled to the right). (D) Immunoblot of N2A whole-cell lysates after siRNA treatment. Carrier pUC DNA and siRNA were
cotransfected, and whole-cell lysates were harvested after 60 h. Protein (50 ?g) was separated by 10% NuPAGE, transferred to nitrocellulose, and
probed with Fox-2 NT and GAPDH antibodies. The protein in lane 1 is from cells transfected with pUC plasmid only. Lanes 2 to 4 contain 1 ?g
pUC and 5 pmol, 10 pmol, or 20 pmol of Fox-2 siRNA, respectively. An immunoblot for Fox-1 showed no detectible expression in this experiment.
GADPH is shown as a loading control.
10014 UNDERWOOD ET AL.MOL. CELL. BIOL.
regulatory elements. The presence of these elements in groups
of commonly regulated exons is a clue to the biological role of
regulation by Fox proteins (36). This biological role is likely
conserved across diverse species. Genetic analyses in the
worm, fish, and mouse will yield information on their common
function in the lives of these different organisms.
We thank Dan Geschwind, in whose laboratory the immunostaining
was performed, and John Winkelmann and Donald McDonnell for
providing Fox cDNAs. Thanks are given to members of the Black
laboratory for advice, discussions, and manuscript review. We are
grateful to Brent Graveley and Jiuyong Xie for comments on the
J.G.U. was supported by a USPHS National Research Service
Award (GM07185). J.D.D. was supported by a Howard Hughes Med-
ical Institute predoctoral fellowship. This work was supported by NIH
grant RO1 GM49662 to D.L.B., who is an investigator of the Howard
Hughes Medical Institute.
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FIG. 8. Fox proteins bind to introns to influence the splicing of
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transcript is spliced poorly in the presence of C. elegans Fox-1 (43). The
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