U1 snRNA Directly Interacts with Polypyrimidine
Tract-Binding Protein during Splicing Repression
Shalini Sharma,1Christophe Maris,3Fre ´de ´ric H.-T. Allain,3and Douglas L. Black1,2,*
1Howard Hughes Medical Institute
2Department of Microbiology, Immunology, and Molecular Genetics
University of California, Los Angeles, Los Angeles, CA 90095, USA
3Institute of Molecular Biology and Biophysics, ETH Zu ¨rich, CH-8093 Zu ¨rich, Switzerland
Splicing of the c-src N1 exon is repressed by the
PTBP1). During exon repression, the U1 snRNP
binds properly to the N1 exon 50splice site but is
made inactive by the presence of PTB. Examining
the patterns of nuclease protection at this 50splice
site, we find that the interaction of U1 is altered by
tifies a direct contact between the pre-mRNA-bound
internal loop of U1 snRNA stem loop 4. The PTB/U1
interaction prevents further assembly of the U1
snRNP with spliceosomal components downstream.
This precise interaction between a splicing regulator
and an snRNA component of the spliceosome points
to a range of different mechanisms for splicing
Most mammalian genes produce multiple mRNA and protein
products through alternative patterns of pre-mRNA splicing
(Nilsen and Graveley, 2010). Numerous pre-mRNA binding
proteins have been identified that can alter splicing choices
(Black, 2003; Chen and Manley, 2009; Matlin et al., 2005).
However, the mechanisms by which these proteins act are not
well understood. Precise contacts between regulatory proteins
and components of the core splicing machinery have only rarely
The spliceosome assembles onto each intron through the
sequential binding of the five small ribonucleoprotein particles
(snRNPs) U1, U2, U4, U5, and U6 and multiple auxiliary proteins
to form discreet RNP complexes termed E, A, B, and C (Wahl
et al., 2009). Splicing catalysis takes place in the spliceosomal
C complex, but a key question in the regulation of alternative
splicing regards how the choice is made of which splice sites
to pair within that later complex. Cross-intron contacts between
the U1 snRNP complex at the 50splice site and the U2 snRNP
complex at the branchpoint and 30splice site are crucial in deter-
mining splice site pairing. On a simple single-intron pre-mRNA
studied in vitro, such contacts are seen in the ATP-independent
E complex. Commitment to splice site pairing isthought to occur
during the formation of the subsequent A complex with the ATP-
dependent base pairing of the U2 snRNA to the branchpoint
(Donmez et al., 2007; Kotlajich et al., 2009; Lim and Hertel,
2004; Michaud and Reed, 1993). However, a typical metazoan
pre-mRNA containing multiple exons and introns will undergo
a process of exon definition, allowing U1 and U2 to bind on
each exon prior to their interaction across introns. The molecular
nature of the cross-intron contacts between defined exons is not
known, but they have been identified as targets for regulating
splice-site pairing choices (Bonnal et al., 2008; House and
Lynch, 2006; Sharma et al., 2008).
The polypyrimidine tract binding protein (PTB) is a widely
expressed splicing regulator controlling many alternative exons
(Auweter and Allain, 2008; Spellman et al., 2005; Spellman and
Smith, 2006). Earlier studies recognized PTB mainly as a splicing
repressor, but recent genomewide identification of PTB target
exons using microarray and CLIP-Seq methods show that PTB
can also enhance inclusion of some exons (Boutz et al., 2007;
Llorian et al., 2010; Xue et al., 2009). Exons repressed by PTB
often contain multiple CU-rich binding elements: a high-affinity
site in the upstream intron and a second sometimes weaker
site either within the exon or the downstream intron. In one
well-studied example, PTB represses splicing of the N1 exon
of the c-src pre-mRNA (Chan and Black, 1997). This regulation
has been reconstructed in vitro using extracts from HeLa cell
nuclei containing high levels of PTB that inhibit N1 exon splicing
(Chou et al., 2000; Markovtsov et al., 2000). In contrast, extracts
from WERI retinoblastoma cells have reduced PTB, and contain
the neuronal PTB homolog (nPTB, brPTB, PTBP2). In these
extracts, the N1 exon is efficiently spliced. We previously
showed that PTB binding to these CU elements does not inter-
fere with the recognition of the N1 exon 50splice site by the U1
snRNP (Sharma et al., 2005). Instead, PTB inhibits splice site
pairing by blocking interaction of the U1 snRNP bound at the
N1 exon 50splice site with the U2 snRNP complex at the down-
stream 30splice site (Sharma et al., 2008). This block to intron
definition interactions prevents binding of the U4/U6-U5
tri-snRNP and the formation of an intronic spliceosomal B
complex. The multiple PTB molecules assembled in the N1
exon complex interact, and are thought to create a RNA loop
(Amir-Ahmady et al., 2005; Lamichhane et al., 2010; Spellman
Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc. 579
and Smith, 2006). In other systems, PTB also interacts with the
corepressor protein Raver 1 to inhibit splicing (Rideau et al.,
2006). However, in spite of the presence of snRNPs assembled
on the repressed exons, direct interactions between PTB and
the spliceosome have not been described.
We now examine how PTB prevents spliceosome assembly
by the adjacent U1 snRNP. We find that the pre-mRNA-bound
PTB directly contacts the U1 snRNA. This contact changes
the interaction of the bound U1 with the 50splice site, prevent-
ing its productive interaction with downstream spliceosomal
U1 snRNP/50Splice Site Interactions Are Altered during
In previous work, we found that when splicing of the c-src N1
exon is repressed by PTB, the U1 snRNP is still bound to the
N1 50splice site (Sharma et al., 2005). However, this U1 fails to
stably interact with the exon 4 complex assembled downstream
and progress into a higher-order intronic spliceosome (Sharma
et al., 2008). We used the PTB-dependent in vitro splicing
system to examine how PTB was preventing this cross-intron
interaction. We analyzed an RNA substrate containing the N1
exon and its flanking regulatory elements as well as the down-
stream exon 4 (BS713; Figure 1A). As seen for similar RNAs
tested previously, this RNA was spliced in WERI extract (lanes
Figure 1. U1 snRNP/50Splice Site Interac-
tion Is Altered during Splicing Repression
(A) Schematic maps of the wild-type and mutant
N1 exon containing pre-mRNAs.
for splicing repression. In vitro splicing of the wild-
type and mutant BS713 transcripts was carried
out in HeLa (lanes 1–3) and WERI-1 (lanes 4–6)
extract. The RNA splicing products and intermedi-
ates are shown to the right.
(C) C-src transcripts containing the N1 exon were
splice site. The labeled wild-type and PTB binding
site mutant RNAs were incubated in Buffer DG
(lanes 1–3 and 10–12), HeLa extract (lanes 5–7
and 13–15), and WERI extract (lanes 7–9 and
16–18). After incubation, reactions were treated
with MNase (lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15,
17, and 18). Extracts were either mock treated or
preincubated with oligonucleotide U11-15prior to
addition of the labeled N1 RNA. The protected
fragments were extracted using PCA, ethanol
precipitated, separated using urea-PAGE, and
visualized by phosphorimaging. The presence of
a downstream 30splice site did not alter the
MNaseprotection patterninHeLaextract (seeFig-
32P labeled at the N1 exon 50
4–6) but repressed in HeLa extract (lanes
1–3). Mutating either PTB binding site
allowed splicing in HeLa extract (Fig-
ure 1B). Using this substrate, we exam-
ined whether the U1 interaction at the N1 50splice site differed
between the active prespliceosome assembled in WERI extract
and the PTB-repressed complex assembled in HeLa extract. In
other studies, the presence of silencer elements was found to
affect the extent of RNA surrounding the splice site that is pro-
tected by U1 snRNP from nuclease digestion (Yu et al., 2008).
These differences in U1 interactions with the 50splice site were
shown to correlate with the ability of the U1 snRNP to progress
in spliceosome assembly. An RNA containing the N1 exon and
its flanking CU elements was specifically labeled with32P at
the 50splice site and assembled into RNP complexes in HeLa
and WERI extracts. The assembled complexes were treated
with micrococcal nuclease (MNase) as described (Maroney
et al., 2000). The RNA fragments remaining after MNase treat-
ment were extracted and visualized by urea-PAGE. Both
a wild-type RNA and a mutant RNA with mutations in the
upstream PTB binding site were nearly completely degraded
by MNase in buffer (Figure 1C, lanes 2, 3, 11, and 12). However,
after incubation in HeLa or WERI extract, specific RNA frag-
ments encompassing the 50splice site were protected from
MNase digestion (lanes 5 and 8). This protection was dependent
on the U1 snRNP, as preincubation with an oligonucleotide
complementary to the 50end of U1 snRNA led to loss of pro-
tected fragments (lanes 6, 9, 15, and 18). Interestingly, the
pattern of protected fragments in HeLa extract was different
from that in WERI extract, indicating that the U1/50splice site
interaction was different under the two conditions. Fragments
PTB Contacts U1 snRNA during Splicing Repression
580 Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc.
of approximately 55, 35, and 13 nucleotides were more strongly
protected in HeLa extract than in WERI extract. Fragments
common to both protection patterns included major bands at
38, 19, 18, and 15 nucleotides and some less prominent bands.
In WERI extract, the protected fragments included a series of
closely spaced bands between 45 and 50 nucleotides that
were not seen in HeLa extract on the wild-type substrate.
However, these bands varied in intensity between experiments
(see lane 17) and are likely nuclease digestion intermediates.
Significantly, an RNA carrying a mutation in the upstream PTB
binding site altered the nuclease protection pattern in HeLa
extract to that seen in WERI extract (lane 14). Protection of the
HeLa-specific 55, 35, and 13 nucleotide fragments was lost
with the mutant RNA, but the 38, 19, 18, and 15 nucleotide frag-
ments common to both extracts were still generated. With this
mutant RNA, the sporadic group of fragments between 45 and
50 nucleotides was now seen in HeLa extract (lane 14), but not
in WERI (lane 17). Thus, in the absence of PTB binding to the
upstream site, the pattern of protected bands was very similar
betweenthe two extracts. Alongerspliceable transcript contain-
ing the downstream exon 4 yielded a protection pattern in HeLa
that complex at the N1 50splice site was not affected by the
downstream 30splice site (see Figure S1 available online). We
conclude that during splicing repression, PTB binding alters
the interaction of the U1 snRNP with the 50splice site.
To map the RNA fragments protected by the U1 snRNP, we
phoresis, the RNA fragments remaining after MNase treatment
were further incubated with RNase H and DNA oligos comple-
mentary to the pre-mRNA. RNase H cleavage sites within
indicates that it is complementary to atleast a portion of the RNA
(Lapham et al., 1997). Since the RNA is labeled at a specific
phosphate in the 50splice site, the size of the cleaved fragments
can be used to determine the extent of protection surrounding
this site. The 55, 38, and 35 nt fragments were all cleaved by
the oligos complementary to RNA nucleotides 146–160 (Fig-
ure 2A, lane 4), generating products either 8 or 12 nucleotides
shorter. This indicates that these three fragments each contain
a 50end within the same region, which is at least 8 or 12 nucleo-
tides from the 50end of the DNA oligo or between nucleotides
149 and 153 (Figure 2B). All the fragments were cleaved by the
DNA oligo that encompasses the labeled phosphate at the 50
splice site (nucleotides 161–175; Figure 2A, lane 5). Given its
length and start site, the 38 nt fragment should end between
nucleotides 183 and 190. The lack of cleavage by the 176–190
oligo could result from the unpaired 50end of the DNA oligo,
which can be a favored position for RNase H cleavage (lane 6).
The 55 nt fragment is cleaved by two downstream oligos
lanes 6 and 7). Measuring 55 nucleotides from its possible start
sites, this long HeLa-specific fragment must end between nucle-
otides200and203.Inagreement with this,cleavagebythe most
downstream oligo generates a fragment approximately 50 nt
long. RNase H mapping with other oligonucleotides, whose
complementary regions are offset from this group, gave equiva-
lent results (Figure S2).
The mixed cleavage patterns produced by RNase H prevent
mapping of the RNA fragment ends precisely to the nucleotide
(Lapham et al., 1997). Nevertheless, from these data it is clear
that the 50ends of the two major protected RNA fragments
Figure 2. U1 snRNP Protects a Much Longer Region of the
Pre-mRNA under PTB-Dependent Repression
(A) The extent of protection of the labeled c-src RNAs was determined using
oligonucleotide-mediated RNase H cleavage. Site-specifically labeled wild-
type RNAs were incubated in HeLa extract (lanes 1–8) and WERI extract (lanes
9–16). After incubation, reactions were treated with MNase (lanes 2–8 and
10–16). The protected fragments were extracted using PCA and ethanol
precipitated. The protected RNAs were then subject to RNase H cleavage in
presence of 15 nt long oligonucleotides as indicated. After RNase H treatment,
the RNAs were separated using urea-PAGE and visualized by phosphorimag-
ing. Oligos with complimentarity offset from those shown here gave equivalent
results in both HeLa and WERI extracts (see Figure S2).
(B) Sequence of the N1 exon containing pre-mRNA. Positions of DNA oligonu-
cleotides used for RNase H cleavage are indicated above the sequence. The
lines below the sequence indicate the boundaries of nuclease protection in
HeLa and WERI extracts.
PTB Contacts U1 snRNA during Splicing Repression
Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc. 581
(55 and 38 nucleotides long) are nearly the same and are within
nucleotides 149–153 of the N1 RNA (Figure 2B). The 30end is
between nucleotides 183 and 190 for the 38 nt fragment and
between nucleotides 200 and 203 for the 55 nt fragment. The
38 nucleotide fragments produced in both HeLa and WERI
extract give identical patterns of cleavage. We conclude that
the pattern of 50splice site protection by U1 is altered under
PTB repression to produce a larger protected region within the
PTB Interacts with the U1 snRNA
We showed previously that the U1 snRNA is correctly base
paired to the N1 exon 50splice site in both HeLa and WERI
extract (Sharma et al., 2005). Thus, the extended protection of
RNA seen in HeLa extract is not due to a shift in the U1 snRNA
base pairing to a different position. The extended protection
downstream of the 50splice site isalso not likely due to PTB itself
directly contacting that segment of RNA, as the nearest PTB
binding site is approximately 20 nt downstream from the end
of the longest protected fragment. Instead, PTB could recruit
additional factors to the protected region, or possibly a direct
interaction of PTB with the U1 snRNP could alter its interaction
with the 50splice site, as seen with a synthetic non-PTB-depen-
dent repressor element (Yu et al., 2008). To examine whether
additional factors were being recruited to this region in HeLa
extract, we performed site-specific labeling and UV-crosslinking
experiments using32P-labeled phosphates placed at nucleo-
tides 198 and 208. No proteins were found to crosslink to posi-
tion 198 (data not shown). The proteins crosslinking to position
208 included PTB and hnRNP H in HeLa extract and PTB,
nPTB, and hnRNP H in WERI extract (Figure S3). These proteins
are known to assemble at positions 218–231 (Chou et al., 2000;
Markovtsov et al., 2000). Importantly, there was no difference in
the crosslinking patterns between HeLa and WERI extracts, and
none of the U1 snRNP-specific proteins crosslinked in either
extract, indicating that the extended protection seen in HeLa
extract is not due to recruitment of new proteins to that region.
To examine whether PTB was interacting with the U1 snRNA
and possibly changing its interaction with the 50splice site
region, we UV crosslinked N1 exon complexes purified from
HeLa extract using a MS2 affinity tag (Figure 3A). The complex
formed on the wild-type RNA was compared to that assembled
on a mutant RNAlacking the PTB binding site downstream of the
50splice site. This mutation eliminates PTB-dependent repres-
sion in HeLa extract, but not PTB binding to the high-affinity
site upstream of the exon. Immunoblot analysis of complexes
assembled on the wild-type RNA and mutant RNAs showed
the presence of PTB and the U1 snRNP-specific protein U1C
in both complexes (Figure 3B). The purified complexes were
exposed to 254nm UV light to crosslink proteins to RNA and
the PTB within the complexes was isolated by immunoprecipita-
tion (Figure 3A). After Proteinase K digestion, total RNA was
extracted, labeled with
PAGE (Figure 3C). The uniformly labeled wild-type and mutant
RNAs were both efficiently immunoprecipitated after crosslink-
ing, relative to no UV and no antibody controls, indicating that
PTB was binding and crosslinking to the upstream binding site
in the mutant RNA (compare lanes 4 and 10 to lanes 2 and 3
32P-pCp, and visualized using urea-
or lanes 8 and 9). Most interestingly, labeling the RNA with
32P-pCp showed that in complexes containing the wild-type
N1 RNA, the U1 snRNA was also crosslinked to PTB (lane 7).
Moreover, mutation of the downstream PTB binding site nearly
eliminated the crosslinking of PTB to U1 (lane 13). Thus, PTB
directly contacts with the U1 snRNA in the repressed HeLa
complex, and mutation of the PTB binding site downstream
can disrupt this interaction.
Purified PTB Interacts with U1 snRNA Stem Loop 4
The U1 snRNP consists of the U1 snRNA, seven Sm core
proteins, and three U1-specific proteins, U1 70k, U1C, and
U1A. In the structures of the U1 snRNP determined by both
cryoelectron microscopy and by X-ray crystallography, stem
loop 4 (SL4) of the U1 snRNA is not bound by any U1-specific
Figure 3. PTB Interacts with the U1 snRNA during Repression
(A) Schematic outline of the method used for studying U1 snRNA/PTB interac-
tion. The MS2 hairpin-tagged N1 exon RNAs were incubated in HeLa extract.
The assembled complexes were purified using the MS2 affinity tag method.
The purified complexes were exposed to UV-254 nm and immunoprecipitated
using anti-PTB antibody, PTB-NT that was prebound to gamma-bind
Sepharose. The immunoprecipitated complexes were then treated with
SDS/Proteinase K while still bound to the gamma bind beads, and total RNA
was extracted, ethanol precipitated, labeled with
(B) Western blot analysis of the proteins from the purified complexes. Proteins
from complexes purified on wild-type RNA and mutant RNAs containing muta-
tions in the downstrem PTB binding sites were separated using SDS-PAGE
and probed using anti-PTB and anti-U1C antibodies.
(C) Analysis ofRNA from purified complexes.RNA fromcomplexesassembled
and purified on wild-type (lanes 2–7) and mutant RNAs (lanes 8–13) were
exposed toUV-254 nm (lanes 2, 4,5,7, 8,10, 11, and 13) and subject toimmu-
noprecipitation using beads alone (lanes 2,5, 8,and 11)or withbeads contain-
ing the anti-PTB antibody, PTB-NT (lanes 3, 4, 6, 7, 9, 10, 12, and 13). In HeLa
extract, no additional proteins were recruited to the region between the N1
exon 50splice site and the PTB binding site (see Figure S3).
32P-pCp, and visualized
PTB Contacts U1 snRNA during Splicing Repression
582 Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc.
protein (Pomeranz Krummel et al., 2009; Stark et al., 2001). SL4
is a very stable GC-rich helix with a pyrimidine-rich internal loop
capped by a stable UUCG tetraloop. Although PTB recognizes
single-stranded RNA sequences, several reports indicate that
PTB can also bind bulged pyrimidine-nucleotides embedded in
RNA duplexes or stem loops, as seen in viral or cellular internal
ribosome entry sites (IRESs) (Bushell et al., 2006; Kafasla et al.,
2009; Mitchell et al., 2005). We thus hypothesized that SL4 could
be the binding site of PTB in U1 snRNA. To test this, we per-
formed electrophoretic mobility shift assays (EMSA) of in vitro
concentrations of purified 6x-His tagged PTB (Figure 4A). The
32P-labeled SL4 RNA incubated with increasing
Figure 4. Recombinant PTB Binds to U1-SL4 RNA
(A) EMSA to analyze binding of PTB to U1-SL4 RNA. Wild-type and mutant U1-SL4 RNAs at 50 nM were incubated with increasing concentrations of His-PTB
(0, 0.1, 0.5, 0.75, 1.0, 2.5, and 5.0 mM). The complexes were separated on 8% native-PAGE.
at 30?C. The substrate at the lowest concentration is in the calorimeter cell. The upper panel displays the raw electrical power trace of the binding titration (base-
line set at 0 mcal/s). The lower panel plots the integrated and normalized heat signal for each injection in the binding titration against the stoichiometry.
(C) On the left panel, overlay of1H-1H TOCSY spectra of U1-SL4 RNA26 (26 nt) free (blue peaks) and bound (red peaks) to PTB RRM2 at 40?C at a ratio 1 to 1
recorded on the Bruker Avance 900 MHz spectrometer and on the right panel, secondary structure of U1-SL4 RNA26 that highlights in green the cytidine and in
blue the uridines that are the most shifted upon binding of PTB RRM2.
PTB Contacts U1 snRNA during Splicing Repression
Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc. 583
wild-type SL4 RNA assembled into a PTB complex that
increased with increasing protein concentration. Mutation of
the pyrimidines in the internal loop and the UUCG tetraloop
regions (M3) led to a significant loss of PTB binding, indicating
that PTB can bind specifically to SL4 of U1, and that binding
requires interactions with pyrimidines. The binding of PTB to
SL4 RNA did not go to completion, and thus did not appear
to be of high affinity. Even at low affinity, this interaction is
likely to occur more efficiently when the U1 snRNP and PTB
are bound to adjacent sites in the pre-mRNA.
To examine the interaction of PTB with SL4 more quantita-
tively, we used isothermal titration calorimetry (ITC) and NMR
spectroscopy with isolated RRM1 and RRM2 domains of PTB.
These two domains were reported to prefer structured RNA
over single-stranded RNA (Clerte and Hall, 2009). Both RRM1
and RRM2 show clear ITC binding curves to U1 SL4 (Figure 4B).
The Kds of SL4 for RRM 1 and 2 were 0.85 and 0.39 mM, respec-
tively. Remarkably, the affinity of these domains for SL4 is much
higher than the affinity of RRM1 for a short UCUCU single-
stranded RNA (Kdof 24.7 mM). Thus, the two N-terminal RRMs
of PTB bind to U1 SL4 with higher affinity than to a typical
pyrimidine element found within splicing repressor elements of
To map the interactions of RRM2 onto the SL4 RNA structure,
we examined how the pyrimidines of SL4 RNA are affected
upon binding of RRM2 using1H-1H TOCSY NMR spectrometry
(Figure 4C). The H5-H6 cross-peaks corresponding to C144,
U145, U156, U157, and U158 within the pyrimidine internal
loop of SL4 all show clear shifts upon the binding of RRM2 at
a 1 to 1 stochiometric ratio. In contrast, the cross-peaks corre-
sponding to pyrimidines in the tetraloop or in the base-paired
portions of the stem were unaffected by RRM2 binding. Thus,
PTB RRM2 binds specifically to the pyrimidine-rich internal
loop of SL4 (with the integrity of the stem being maintained)
rather than to the terminal loop or to the nine pyrimidine-tract
(155–163) at the 30end of the SL4. This mode of RNA binding
by the protein is likely to be different than RRM2 binding to
single-stranded RNA (Oberstrass et al., 2005).
Excess U1-SL4 Prevents the PTB-Induced Change
in the Pre-mRNA/U1 Interaction
We next wanted to examine whether the alteration of U1 binding
with the interaction of PTB with U1 in the repressed HeLa
complex. To test this, we titrated SL4 RNA into U1 assembly
reactions (Figure 5). HeLa and WERI extracts were preincubated
with the SL4 RNA prior to addition of N1 exon RNA labeled at the
50splice site. These reactions were treated with MNase and the
protected fragments analyzed as described above. Increasing
tion of the 55 nt protected RNA fragment that is specific to the
repressed complex from HeLa extract (lanes 5–7). At higher
concentrations of SL4 RNA, the 38 and 35 nucleotide fragments
were also reduced. In WERI extract, the pattern of 50splice site
protection was largely unchanged by SL4 RNA (lanes 9–12).
Similarly, the mutant SL4 RNA (M3) that does not bind PTB
and did not significantly alter the pattern of protected 50splice
site fragments in HeLa (lanes 14–17) or WERI (18-22) extract.
Thus, in the presence of excess SL4 RNA, PTB does not alter
the interaction of U1 snRNP with the 50splice site.
We find that PTB bound to a pre-mRNA directly contacts the U1
snRNA within an exon complex that is repressed for splicing.
PTB specifically interacts with the pyrimidine-rich internal loop
We also find that when splicing is repressed by PTB, a pattern of
nuclease protection is observed at the 50splice site that is
different from that seen when PTB is absent and splicing is
active. Competition experiments with excess SL4 RNA indicate
that the PTB interaction with U1 is apparently required for the
extended protection of the 50splice site region seen in a PTB
repressed exon complex. Our previous investigations showed
that binding of PTB to the pre-mRNA does not prevent 50splice
site binding by the U1 snRNP but alters the ability of this U1 to
progress further in spliceosome assembly (Sharma et al.,
2008). The contact of PTB with SL4 could force the U1 into
a conformation that is incompatible with further assembly. This
is similar to what has been proposed for the action of splicing
silencer elements identified by in vitro selection (Yu et al.,
2008). Alternatively, a specific contact between SL4 and another
spliceosomal component may be required for later assembly
steps and be blocked by PTB binding. For example, SL4 may
mediate interactions between the U1 and U2 snRNPs during
splice site pairing. A role for U1 SL4 during spliceosome
Figure 5. Competition with Free U1-SL4 RNA Shifts the Interaction
between the U1 snRNP and the Pre-mRNA to that Seen in the
Absence of PTB
The c-src N1 exon-containing transcripts were site-specifically labeled at the
N1 exon 50splice site. Prior to addition of the labeled transcript to HeLa and
WERI extracts, the extracts were preincubated with increasing concentrations
of free wild-type (lanes 1–12) and mutant (lanes 13–22) U1-SL4 RNAs at 0, 2.5,
5.0, and 10 mM. After preincubation, the site-specifically labeled transcript
was added and incubation continued. The reactions were then treated with
MNase. The protected fragments were extracted and visualized as described
in Figure 1.
PTB Contacts U1 snRNA during Splicing Repression
584 Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc.
assembly has not been described. Crystal and cryo-EM struc-
tures of purified or in vitro assembled U1 snRNP do not show
interaction of the SL4 with any snRNP protein (Pomeranz Krum-
mel et al., 2009; Stark et al., 2001). However, a previous study
found that interactions of the phosphate backbone of the SL4
stabilize the association of the Sm ring with the U1 snRNA
(McConnell et al., 2003). Blocking these interactions could alter
the conformation of RNA-bound U1 or alter its interactions with
other spliceosomal factors. A model for PTB mediated splicing
repression is shown in Figure 6.
0.85 mM for RRM1) is higher than that measured for a short
pyrimidine oligonucleotide (UCUCU) found in typical splicing
repressor elements (Kdof 24.7 mM for RRM1) (Auweter et al.,
2007; Oberstrass et al., 2005). SL4 must contain specific struc-
tural features that are recognized by PTB RRM1 and RRM2.
This indicates that PTB can engage in modes of binding that
differ from those reported for single-stranded RNA (Oberstrass
et al., 2005). On the other hand, the affinity of the individual
PTB RRMs for the U1 SL4 is lower than the affinity of full-length
PTB for longer splicing silencer elements. It is likely that free PTB
in nuclear extract does not bind to the U1 snRNP, but binding of
simultaneous binding of PTB and U1 snRNP to the same regula-
tory region has been reported. In fact, exonic binding of PTB has
been shown to stimulate U1 binding to the Fas exon 6 50splice
site (Izquierdo et al., 2005). During repression of exon 6 of
b-Tropomyosin, U1 snRNP also binds in the vicinity of the PTB
binding site (Sauliere et al., 2006). Similarly, in CT/CGRP pre-
stream of exon 4 to activate polyadenylation (Lou et al., 1999).
The regulation of pre-mRNA processing in these systems may
also involve direct contacts between PTB and the U1 snRNP.
Studies indicate that splicing repression by PTB requires
multiple binding sites and that multiple PTB molecules are
present in RNA/PTB complexes (Amir-Ahmady et al., 2005;
Cherny et al., 2010; Chou et al., 2000; Clerte and Hall, 2009;
Kafasla et al., 2009). After assembly with the pre-mRNA, there
are likely free RRM domains available to interact with U1-SL4.
Analysis of PTB binding to the encephalomyocarditis virus
(ECMV) IRES showed that the individual RRMs bind to separate
stem-loop domains to stabilize the IRES structure (Kafasla et al.,
2009). We find that the PTB binding site downstream of U1 is
required for PTB/U1 crosslinking and that both RRM1 and
RRM2 can bind U1-SL4 with similar affinity. These two RRMs
are connected by flexible linkers with the rest of the protein
and could adopt a variety of conformations to contact a nearby
snRNA. It needs to be determined which of these two RRMs
contacts U1 in the repressed N1 exon/PTB complex and
whether this RRM must be placed downstream of the 50splice
site. RRM3 and RRM4 presumably bind the pre-mRNA rather
RNA (Clerte and Hall, 2009). If both of these domains engage the
pre-mRNA, they will generate an RNA loop through their interdo-
main interaction. One of these domains will likely anchor the
protein in position downstream from U1, while the other may
interact with an adjacent element or with a portion of the
repressor element upstream of the exon (Lamichhane et al.,
2010; Oberstrass et al., 2005).
It will also be interesting to investigate whether this interaction
between PTB and U1-SL4 also plays a role in enhancement of
exon inclusion by PTB. Tethering experiments with chimeric
PTB-MS2 proteins indicate that recruiting RRM2 along with its
adjacent linker to the pre-mRNA is sufficient for both repression
et al., 2006; Robinson and Smith, 2006). Similar studies may
allow identification of the PTB domains important for interaction
Other splicing repressors are also found not to interfere with
the recognition of splice sites but to prevent later steps in spli-
ceosome assembly. Studies of CD45 exons 4 and 5 showed
that hnRNP L binding to an exonic splicing silencer does not
prevent recognition of the 50and 30splice sites by the U1 and
U2 snRNPs (House and Lynch, 2006). However, the resulting
exon definition complex is inactive and does not bind the
U4/U6-U5 tri-snRNP. Repression of Fas exon 6 by RBM5 and
PTB also results in the formation of a nonfunctional exon defini-
tion complex (Bonnal et al., 2008; Izquierdo et al., 2005). Direct
interactions of hnRNP L and RBM5 with a U1 or U2 snRNP
component have not been identified. However, as seen here,
these exon complexes must either have a conformation that is
not optimal for further spliceosome assembly, or be blocked
for a specific interaction needed in the next assembly step.
Many proteins have been identified that bind to pre-mRNA
and control alternative splicing patterns. However, few direct
interactions are known between splicing regulators and the spli-
ceosome, and these regulatory interactions are with protein
components of the spliceosome. In the classic example, SR
proteins promote 50and 30splice site use through interactions
with the SR domains of the U1 70k and U2AF proteins (Graveley,
2000; Kohtz et al., 1994; Wu and Maniatis, 1993). Similarly, the
TIA1 protein can help recruit the U1 snRNP to a 50splice site
via interactions with the U1C protein (Forch et al., 2002).
Figure 6. Model for N1 Exon Repression by PTB
Binding of PTB to the CU-rich elements flanking the N1 exon allow its interac-
tion with SL4 of U1 snRNA.This prevents U1 from interacting withthe complex
at the downstream 30splice site.
PTB Contacts U1 snRNA during Splicing Repression
Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc. 585
Potential regulatory interactions with snRNAs are also known.
The hnRNP C protein was found to bind a uridine containing
tetraloop in the 50stem loop of U2 snRNA (Temsamani and
Pederson, 1996). Another study showed that PSF and p54nrb
associate with the U4/U6-U5 tri-snRNP via an interaction with
the U5 snRNA stem Ib (Peng et al., 2002). In these cases,
a role for the protein/snRNA interaction in splicing regulation
has not yet been demonstrated. However, from the results pre-
sented here, it is clear that a relatively small contact on the
snRNA can have a significant effect on a choice of splicing
pattern. Most known splicing regulators contain multiple RNA-
binding domains that recognize short RNA elements potentially
found in one of the spliceosomal snRNAs. The structural flexi-
bility of RNA would allow it to adopt conformations providing
protein contacts that might not occur in normal assembly and
function. Such protein interactions stabilizing RNA conforma-
tions that are nonproductive for further spliceosome assembly
can have dramatic effects on the competition between two
splice site choices. Thus, it seems likely that snRNAs will prove
to be targets of other splicing regulators.
Site-specifically32P-labeled c-src RNAs were made as described previously
(Reed and Chiara, 1999). Transcription templates for the 50and 30RNA
fragments were generated by PCR. The RNA fragments were synthesized
in standard T7 RNA polymerase transcription reactions. The 50fragment
reaction contained the 50CAP nucleotide analog, G(50)ppp(50)G, whereas the
reaction for the 30fragment did not. Fifteen pmols of the 30RNA was first
dephosphorylated using shrimp alkaline phosphatase (USB). The dephos-
phorylated RNA was extracted with phenol:chloroform:isoamyl alcohol
(PCA) and ethanol precipitated.32P labeling was carried out in a 15 ml reaction
containing the 1 ml of [g-32P]ATP (6000 Ci/mmol), 1.5 ml of T4 polynucleotide
kinase (10,000 units/ml), and 13 T4 PNK buffer (NEB). After labeling, the
RNA was extracted with PCA and 15 pmols of the 50RNA and the 40-mer
bridging oligonucleotide were added. This mix was then ethanol precipitated.
The precipitate was then resuspended in 7 ml of water and denatured. Ligation
was carried out at 25?C for 3–12 hr in a 15 ml reaction containing 1.5 ml of T4
DNA ligase (400,000 units/ml), 0.5 ml of RNAguard, and 13 T4 DNA ligase
buffer (NEB). Ligated RNA was separated and extracted using urea-PAGE.
MNase Protection Assay
The MNase protection assay was carried out as described by Maroney et al.
(Maroney et al., 2000). The site-specifically32P-labeled RNA (10,000 cpm)
was incubated at 30?C for 30 min in a 25 ml splicing reaction that contained
15 ml of HeLa or WERI nuclear extract, 0.4 mM ATP, 20 mM creatine phos-
phate, 2.2 mM MgCL2, and 10 units of RNAguard. MNase (NEB) and CaCl2
were then added to a final concentration of 5 units/ml and 5 mM, respectively.
MNase digestion wascarried outat25?Cfor 20min.Digestion wasstoppedby
adding EDTA to a final concentration of 20 mM. The reactions were then
treated with SDS and Proteinase K. Total RNA was extracted using PCA and
ethanol precipitated. The protected RNA fragments were separated on
a 10% urea-PAGE gel and visualized using Phophorimager. When treated
with the U11-15oligonucleotide, the reactions were preincubated with 2 pmols
of the oligo and 10 units of RNase H (Ambion) for 30 min at 30?C prior to addi-
tion of the site-specifically labeled RNA.
Electrophoretic Mobility Shift Assay
Recombinant 6x-His tagged PTB was purified using Ni-NTA agarose (Invitro-
gen) according to the manufacturer’s instructions. The purified protein was
dialyzed and stored in Buffer DG (20 mM HEPES-KOH [pH 7.9], 80 mM K.
glutamate, 20% glycerol, 2.2 mM MgCl2, 1 mM DTT, and 0.1 mM PMSF).
The wild-type and mutant U1SL4 RNAs were transcribed in vitro using T7
RNA polymerase. For EMSA, the binding reactions were 10 ml and contained
50 nM RNA, 2.2 mM MgCL2, and increasing concentrations of His-PTB
(0, 0.1, 0.5, 0.75, 1.0, 2.5, and 5.0 mM) and sufficient Buffer DG to bring its
volume to 60% of the reaction volume. After incubation at room temperature
for 20 min, the complexes were separated on an 8% native-PAGE gel using
25 mM Tris-glycine buffer at 4?C.
Purification and Analysis of N1 RNA Complexes
100 nM MS2-MBP fusion protein, 0.4 M ATP, 20 mM CP, 2.2 mM MgCl2, 10U
RNAguard, and 300 ml of nuclear extract (Sharma et al., 2005). The MS2-
tagged RNAs were preincubated with MS2-MBP prior to the addition of
nuclear extract. The reactions were incubated at 30?C for 30 min, and the
assembled complexes were purified using amylose resin as described previ-
ously (Sharma et al., 2005). The purified complexes were then UV crosslinked
using the Stratalinker to total energy of 1600 mJoules. After crosslinking, the
complexes were subject to immunoprecipitation using anti-PTB antibody
(PTB-NT) coupled to gamma-bind Sepharose. The immunoprecipitated
complexes were digested with SDS and Proteinase K. Total RNA was then ex-
tracted using PCA, ethanol precipitated, and 30end labeled using 50 32P-pCp
and T4 RNA ligase (NEB). The labeled RNA was separated on 8% urea-PAGE
gels and visualized by Phosphorimager. Total RNA from HeLa extracts was
also labeled with 50 32P-pCp.
Sample Preparation for ITC Measurements and NMR Spectroscopy
The RRM1 (residues 41–163) and RRM2 (residues 172–316) domains of PTB
(accession number X62006) were subcloned in pTYB11 vector (N-terminal
fusion vector), whichispartof IMPACT-CN systemfromNew England Biolabs,
Chitin column. The subcloned vectors were transformed into E. coli strain
BL21 (DE3) codonPlus-RIL. The expression was carried out in M9 minimal
medium containing15N isotopically labeled NH4Cl and 100 mg/ml of ampicillin
at 37?C until induction. At OD600?0.6, expression was induced with 1 mM
according to the IMPACT-CN manual of New England Biolabs, Inc. To elimi-
(Ambion) and Protease Inhibitor Cocktail (Roche) after sample concentration.
In order to eliminate the peptide generated from self-cleavage reaction, the
sample wasthenpurifiedby size-exclusion chromatography usinga Superdex
75 10/300 GL column.
The RNA was synthesized by in vitro transcription using T7 RNA polymerase
and syntheticDNA templates.The oligonucleotideproductswerethenpurified
by anion exchange chromatography in denaturing conditions (6M urea at
pH 8.0 and 85?C). The purified RNA was precipitated by butanol extraction
to eliminate urea and salts. The precipitate was resuspended in 3 ml of deion-
was repeated three times before lyophilization overnight. The RNA samples
were then dissolved in aqueous solution containing 20 mM NaCl and 10 mM
NaH2PO4adjusted at pH 6.5.
ITC Measurement and Analysis
ITC experiments were performed using a VP-ITC (Microcal, Inc.), and normal-
ized heat signals were calculated using the bundled Origin software. Prior to
each experiment, both the PTB RRM1 and RRM2 domains and the RNA
constructs were dialyzed overnight against 5 L of 20 mM NaCl and 10 mM
NaH2PO4adjusted at pH 6.5. For the ITC experiments, the titrant at a concen-
tration from 30 to 410 mM was injected with an injection size of 10 ml into a cell
containing 2.5–10mMof thesubstrate. Forallexperiments, aninjectioninterval
of 3 min was set to allow for complete equilibration. The RNA and protein
concentrations were measured, respectively, by UV spectroscopy and NMR
spectroscopy using 1D PULCON experiment (Wider and Dreier, 2006). All
ITC data were analyzed and plotted in Origin software using a 1-to-1 binding
PTB Contacts U1 snRNA during Splicing Repression
586 Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc.
Titration of U1-SL4 RNA with PTB RRM2 was carried out on the 900 MHz
Bruker Avance spectrometer at 40?C. We acquired a series of 2D
1H-1H TOCSY (50 ms mixing time) to monitor proton chemical shift changes
upon protein binding. The pyrimidine resonances of the U1-SL4 RNA were as-
signed in the free state based on a 2D1H-1H NOESY, a natural abundance
1H-13C HSQC and 2D1H-1H TOCSY.
Supplemental Information includes three figures and can be found with this
article at doi:10.1016/j.molcel.2011.02.012.
We thank Tim Nilsen and members of the Black lab for helpful discussion and
comments. This work was supported by grants from the Swiss National
Science Foundation (3100ab-133134) and the SNF-NCCR Structural Biology
(to F.H.-T.A.), and by National Institutes of Health grant RO1:GM49662 (to
D.L.B.). D.L.B. is an investigator of the Howard Hughes Medical Institute.
Received: September 2, 2010
Revised: December 8, 2010
Accepted: January 7, 2011
Published: March 3, 2011
Amir-Ahmady, B., Boutz, P.L., Markovtsov, V., Phillips, M.L., and Black, D.L.
(2005). Exon repression by polypyrimidine tract binding protein. RNA 11,
Auweter, S.D., and Allain, F.H. (2008). Structure-function relationships of the
polypyrimidine tract binding protein. Cell. Mol. Life Sci. 65, 516–527.
Auweter, S.D., Oberstrass, F.C., and Allain, F.H. (2007). Solving the structure
of PTB in complex with pyrimidine tracts: an NMR study of protein-RNA
complexes of weak affinities. J. Mol. Biol. 367, 174–186.
Black, D.L. (2003). Mechanisms of alternative pre-messenger RNA splicing.
Annu. Rev. Biochem. 72, 291–336.
Bonnal, S., Martinez, C., Forch, P., Bachi, A., Wilm, M., and Valcarcel, J.
(2008). RBM5/Luca-15/H37 regulates Fas alternative splice site pairing after
exon definition. Mol. Cell 32, 81–95.
M., Jr., and Black, D.L. (2007). A post-transcriptional regulatory switch in poly-
pyrimidine tract-binding proteinsreprograms alternative splicing indeveloping
neurons. Genes Dev. 21, 1636–1652.
Bushell, M., Stoneley, M., Kong, Y.W., Hamilton, T.L., Spriggs, K.A., Dobbyn,
H.C., Qin, X., Sarnow, P., and Willis, A.E. (2006). Polypyrimidine tract binding
protein regulates IRES-mediated gene expression during apoptosis. Mol. Cell
Chan, R.C., and Black, D.L. (1997). The polypyrimidine tract binding protein
binds upstream of neural cell-specific c-src exon N1 to repress the splicing
of the intron downstream. Mol. Cell. Biol. 17, 4667–4676.
Chen, M., and Manley, J.L. (2009). Mechanisms of alternative splicing regula-
tion: insights from molecular and genomics approaches. Nat. Rev. Mol. Cell
Biol. 10, 741–754.
Cherny, D., Gooding, C., Eperon, G.E., Coelho, M.B., Bagshaw, C.R., Smith,
C.W., and Eperon, I.C. (2010). Stoichiometry of a regulatory splicing complex
revealed by single-molecule analyses. EMBO J. 29, 2161–2172.
Chou, M.Y., Underwood, J.G., Nikolic, J., Luu, M.H., and Black, D.L. (2000).
Multisite RNA binding and release of polypyrimidine tract binding protein
during the regulation of c-src neural-specific splicing. Mol. Cell 5, 949–957.
Clerte, C., and Hall, K.B. (2009). The domains of polypyrimidine tract binding
Donmez, G., Hartmuth, K., Kastner, B., Will, C.L., and Luhrmann, R. (2007).
The 50end of U2 snRNA is in close proximity to U1 and functional sites of
the pre-mRNA in early spliceosomal complexes. Mol. Cell 25, 399–411.
Forch, P., Puig, O., Martinez, C., Seraphin, B., and Valcarcel, J. (2002). The
splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment
to 50splice sites. EMBO J. 21, 6882–6892.
Graveley, B.R. (2000). Sorting out the complexity of SR protein functions. RNA
House, A.E., and Lynch, K.W. (2006). An exonic splicing silencer represses
spliceosome assembly after ATP-dependent exon recognition. Nat. Struct.
Mol. Biol. 13, 937–944.
Izquierdo, J.M., Majos, N., Bonnal, S., Martinez, C., Castelo, R., Guigo, R.,
Bilbao, D., and Valcarcel, J. (2005). Regulation of Fas alternative splicing by
antagonistic effects of TIA-1 and PTB on exon definition. Mol. Cell 19,
Kafasla, P., Morgner, N., Poyry, T.A., Curry, S., Robinson, C.V., and Jackson,
R.J. (2009). Polypyrimidine tract binding protein stabilizes the encephalomyo-
carditis virus IRES structure via binding multiple sites in a unique orientation.
Mol. Cell 34, 556–568.
Kohtz, J.D., Jamison, S.F., Will, C.L., Zuo, P., Luhrmann, R., Garcia-Blanco,
M.A., and Manley, J.L. (1994). Protein-protein interactions and 50-splice-site
recognition in mammalian mRNA precursors. Nature 368, 119–124.
Kotlajich, M.V., Crabb, T.L., and Hertel, K.J. (2009). Spliceosome assembly
pathways for different types of alternative splicing converge during commit-
ment to splice site pairing in the A complex. Mol. Cell. Biol. 29, 1072–1082.
Lamichhane, R., Daubner, G.M., Thomas-Crusells, J., Auweter, S.D.,
Manatschal, C., Austin, K.S., Valniuk, O., Allain, F.H., and Rueda, D. (2010).
RNA looping by PTB: evidence using FRET and NMR spectroscopy for
a role in splicing repression. Proc. Natl. Acad. Sci. USA 107, 4105–4110.
Lapham, J., Yu, Y.T., Shu, M.D., Steitz, J.A., and Crothers, D.M. (1997). The
position of site-directed cleavage of RNA using RNase H and 20-O-methyl
oligonucleotides is dependent on the enzyme source. RNA 3, 950–951.
Lim, S.R., and Hertel, K.J. (2004). Commitment to splice site pairing coincides
with A complex formation. Mol. Cell 15, 477–483.
Llorian, M., Schwartz, S., Clark, T.A., Hollander, D., Tan, L.Y., Spellman, R.,
Gordon, A., Schweitzer, A.C., de la Grange, P., Ast, G., and Smith, C.W.
(2010). Position-dependent alternative splicing activity revealed by global
profiling of alternative splicing events regulated by PTB. Nat. Struct. Mol.
Biol. 17, 1114–1123.
Lou, H., Helfman, D.M., Gagel, R.F., and Berget, S.M. (1999). Polypyrimidine
tract-binding protein positively regulates inclusion of an alternative 30-terminal
exon. Mol. Cell. Biol. 19, 78–85.
Markovtsov, V., Nikolic, J.M., Goldman, J.A., Turck, C.W., Chou, M.Y., and
Black, D.L. (2000). Cooperative assembly of an hnRNP complex induced by
a tissue-specific homolog of polypyrimidine tract binding protein. Mol. Cell.
Biol. 20, 7463–7479.
Maroney, P.A., Romfo, C.M., and Nilsen, T.W. (2000). Nuclease protection of
RNAs containing site-specific labels: arapid method for mapping RNA-protein
interactions. RNA 6, 1905–1909.
Matlin, A.J., Clark, F., and Smith, C.W. (2005). Understanding alternative
splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6, 386–398.
McConnell, T.S., Lokken, R.P., and Steitz, J.A. (2003). Assembly of the U1
snRNP involves interactions with the backbone of the terminal stem of U1
snRNA. RNA 9, 193–201.
Michaud, S., and Reed, R. (1993). A functional association between the 50and
30splice site is established in the earliest prespliceosome complex (E) in
mammals. Genes Dev. 7, 1008–1020.
J.P., Spriggs, R.V., and Willis, A.E. (2005). Identification of a motif that medi-
ates polypyrimidine tract-binding protein-dependent internal ribosome entry.
Genes Dev. 19, 1556–1571.
Nilsen, T.W., and Graveley, B.R. (2010). Expansion of the eukaryotic proteome
by alternative splicing. Nature 463, 457–463.
PTB Contacts U1 snRNA during Splicing Repression
Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc. 587
Oberstrass,F.C.,Auweter,S.D.,Erat,M.,Hargous,Y.,Henning,A.,Wenter,P., Download full-text
Reymond, L., Amir-Ahmady, B., Pitsch, S., Black, D.L., and Allain, F.H. (2005).
Structure of PTB bound to RNA: specific binding and implications for splicing
regulation. Science 309, 2054–2057.
Peng, R., Dye, B.T., Perez, I., Barnard, D.C., Thompson, A.B., and Patton, J.G.
(2002). PSF and p54nrb bind a conserved stem in U5 snRNA. RNA 8,
Pomeranz Krummel, D.A., Oubridge, C., Leung, A.K., Li, J., and Nagai, K.
(2009). Crystal structure of human spliceosomal U1 snRNP at 5.5 A˚resolution.
Nature 458, 475–480.
Reed, R., and Chiara, M.D. (1999). Identification of RNA-protein contacts
within functional ribonucleoprotein complexes by RNA site-specific labeling
and UV crosslinking. Methods 18, 3–12.
Rideau, A.P., Gooding, C., Simpson, P.J., Monie, T.P., Lorenz, M.,
Huttelmaier, S., Singer, R.H., Matthews, S., Curry, S., and Smith, C.W.
(2006). A peptide motif in Raver1 mediates splicing repression by interaction
with the PTB RRM2 domain. Nat. Struct. Mol. Biol. 13, 839–848.
Robinson, F., and Smith, C.W. (2006). A splicing repressor domain in polypyr-
imidine tract-binding protein. J. Biol. Chem. 281, 800–806.
Sauliere, J., Sureau, A., Expert-Bezancon, A., and Marie, J. (2006). The
polypyrimidine tract binding protein (PTB) represses splicing of exon 6B
from the beta-tropomyosin pre-mRNA by directly interfering with the binding
of the U2AF65 subunit. Mol. Cell. Biol. 26, 8755–8769.
Sharma, S., Falick, A.M., and Black, D.L. (2005). Polypyrimidine tract binding
protein blocks the 50splice site-dependent assembly of U2AF and the prespli-
ceosomal E complex. Mol. Cell 19, 485–496.
Sharma, S., Kohlstaedt, L.A., Damianov, A., Rio, D.C., and Black, D.L. (2008).
Polypyrimidine tractbindingproteincontrolsthe transition fromexon definition
to an intron defined spliceosome. Nat. Struct. Mol. Biol. 15, 183–191.
Spellman, R., and Smith, C.W. (2006). Novel modes of splicing repression by
PTB. Trends Biochem. Sci. 31, 73–76.
Spellman, R., Rideau, A., Matlin, A., Gooding, C., Robinson, F., McGlincy, N.,
Grellscheid, S.N., Southby, J., Wollerton, M., and Smith, C.W. (2005).
Regulation of alternative splicing by PTB and associated factors. Biochem.
Soc. Trans. 33, 457–460.
Stark,H., Dube,P.,Luhrmann, R.,and Kastner, B.(2001).ArrangementofRNA
and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle.
Nature 409, 539–542.
Temsamani, J., and Pederson, T. (1996). The C-group heterogeneous nuclear
ribonucleoprotein proteins bind to the 50stem-loop of the U2 small nuclear
ribonucleoprotein particle. J. Biol. Chem. 271, 24922–24926.
Wahl, M.C., Will, C.L., and Luhrmann, R. (2009). The spliceosome:design prin-
ciples of a dynamic RNP machine. Cell 136, 701–718.
Wider, G., and Dreier, L. (2006). Measuring protein concentrations by NMR
spectroscopy. J. Am. Chem. Soc. 128, 2571–2576.
Wu, J.Y., and Maniatis, T. (1993). Specific interactions between proteins
implicated in splice site selection and regulated alternative splicing. Cell 75,
Xue, Y., Zhou, Y., Wu, T., Zhu, T., Ji, X., Kwon, Y.S., Zhang, C., Yeo, G., Black,
D.L., Sun, H., et al. (2009). Genome-wide analysis of PTB-RNA interactions
reveals a strategy used by the general splicing repressor to modulate exon
inclusion or skipping. Mol. Cell 36, 996–1006.
Yu, Y., Maroney, P.A., Denker, J.A., Zhang, X.H., Dybkov, O., Luhrmann, R.,
Jankowsky, E., Chasin, L.A., and Nilsen, T.W. (2008). Dynamic regulation of
alternative splicing by silencers that modulate 50splice site competition. Cell
PTB Contacts U1 snRNA during Splicing Repression
588 Molecular Cell 41, 579–588, March 4, 2011 ª2011 Elsevier Inc.