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.
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.